RESPUESTAS ECOFISIOLÓGICAS DE ANGIOSPERMAS MARINAS MEDITERRÁNEAS ( POSIDONIA OCEANICA Y CYMODOCEA NODOSA) FRENTE A CONDICIONES DE ESTRÉS HIPERSALINO. ECOPHYSIOLOGICAL RESPONSES OF MEDITERRANEAN SEAGRASSES (POSIDONIA OCEANICA AND CYMODOCEA NODOSA) UNDER HYPERSALINE STRESS CONDITIONS

José Miguel Sandoval

José Miguel Sandoval Gil

Tesis Doctoral

RESPUESTAS ECOFISIOLÓGICAS DE

ANGIOSPERMAS MARINAS MEDITERRÁNEAS

(POSIDONIA OCEANICA Y CYMODOCEA NODOSA)

FRENTE A CONDICIONES DE ESTRÉS HIPERSALINO

ECOPHYSIOLOGICAL RESPONSES OF

MEDITERRANEAN SEAGRASSES

(POSIDONIA OCEANICA AND CYMODOCEA NODOSA)

UNDER HYPERSALINE STRESS CONDITIONS

DIRECCIÓN

Juan Manuel Ruiz Fernández Grupo de Ecología de Angiospermas Marinas. Instituto Español de Oceanografía, Centro Oceanográfico de Murcia

CO-DIRECCIÓN Lázaro Marín-Guirao Grupo de Ecología de Angiospermas Marinas. Instituto Español de Oceanografía, Centro Oceanográfico de Murcia Jose Luis Sánchez-Lizaso Dept. Ciencias del Mar y Biología Aplicada. Universidad de Alicante

RESPUESTAS ECOFISIOLÓGICAS DE ANGIOSPERMAS MARINAS MEDITERRÁNEAS

( POSIDONIA OCEANICA Y CYMODOCEA NODOSA) FRENTE A CONDICIONES DE ESTRÉS HIPERSALINO

ECOPHYSIOLOGICAL RESPONSES OF THE MEDITERRANEAN SEAGRASSES

(POSIDONIA OCEANICA AND CYMODOCEA NODOSA) UNDER HYPERSALINE STRESS CONDITIONS

José Miguel Sandoval Gil

Tesis Doctoral -PhD Thesis

Instituto Español de OceanografíaCentro Oceanográfico de MurciaGrupo de Ecología de Angiospermas MarinasSeagrass Ecology Group

Universidad de AlicanteDepartamento de Ciencias del Mar y Biología Aplicada

A las mayores, tita y abuela

A la pequeña, Adriana

Cuando uno tiene tanto que agradecer a tantos, no debería resultar sorprendente que, a

pesar de todo el esfuerzo y el trabajo que han supuesto el desarrollo y finalización de esta

tesis, una de las cosas más difícles de afrontar sea la redacción de los agradecimientos.

En primer lugar, debo confesar que nunca podré compensar a mi familia todo lo que han

hecho por mí todos estos años. Una vez más, son ellos los que han supuesto ese empuje incon-

dicional necesario cuando los vientos no siempre soplan a tu favor o con la fuerza necesaria.

Quiero dar las gracias a mis padres y a mi hermana por respetar, apoyar y admirar lo que hago,

a mi abuela y a mi “tita”, sencillamente por todo lo que son y suponen para mí, y por supuesto

a mi pequeña y primera sobrina Adriana, que me ha brindado ese primer año inolvidable, ale-

gre y fresco. Gracias de nuevo, “papá” y “mamá” por haberme inculcado los valores que me

permiten ser lo que soy ahora, con mis múltiples defectos y mis pocas virtudes. Y gracias a tí

María, que has estado a mi lado todos estos años, a todas horas, siempre, sincera y cariñosa.

Como suele ocurrir con las cosas que terminan siendo importantes en la vida de uno,

cabe resaltar que mi incursión en la biología marina, y sobre todo, la realización de esta te-

sis, nunca fueron del todo premeditadas. No fue premeditado mi primer trabajo relacio-

nado con el mundo marino en el laboratorio de acuariología de la Universidad de Mur-

cia, no fueron planeadas mis primeras prácticas en el Instituto Español de Oceanografía,

del mismo modo que no fue prevista mi participación en el proyecto OSMOGRASS. Pero

el caso es que todo ocurrió así, de manera sencilla, felizmente. De este modo, ha sido du-

rante mis años de trabajo en el IEO, dentro del Grupo de Ecología de Angiospermas Ma-

rinas (GEAM), donde he tenido la gran fortuna de compartir mi vida con grandes gran-

des personas, no sólo excepcionales en el ámbito laboral, sino inigualables en lo personal.

Creo que lo que pueda escribir aquí de poco servirá para hacer entender la grati-

tud que siento hacia Juan Manuel Ruiz. A Juanma, y después de siete años desde que nos

conocimos, tengo que agradecer, de forma especial, la confianza que siempre ha mos-

trado hacia mí. No sólo ha ejercido de dedicado y riguroso director de la presente Te-

sis Doctoral, sino de gran maestro y verdadero amigo, con el que espero seguir compar-

tiendo momentos y proyectos algún día, quién sabe, en el futuro. Me gustaría pensar que

he correspondido su confianza, al menos en parte, con mi trabajo, respeto y admiración.

Muchas gracias Lázaro, amigo y hermano, por todo lo que me has dado y enseña-

do. Contadas son las personas que han supuesto tanto para mí, y ya sabes que en el futu-

ro, tal vez en la distancia, siempre podrás contar con alguien que te admira infinitamente.

Muchas gracias Rocío, por estar siempre presente en cada cambio importan-

te; tú estuviste ahí cuando comencé con la Red de Posidonia oceanica, y estuviste tam-

bién al otro lado de la línea telefónica al comienzo del proyecto. Tras una amistad cur-

tida con los años, sólo puedo tener palabras hacia tí de halago y gratitud sinceros.

Muchas gracias Jaime, gran persona y mejor doctorando, por permitir nuestra gran amistad,

por aquellos días de congreso tunecino, y por las incontables horas compartidas de despacho,

enmarcadas en un contexto de música instrumental minimalista, sutilmente apreciada por pocos.

Muchas gracias Arantxa, porque aunque no hemos tenido demasiado tiempo en co-

nocernos, siempre te has mostrado como la mejor de las amigas y compañeras de trabajo.

Muchas gracias también a tí, Aurora, que durante tu estancia en el grupo, ofreciste una gran

ayuda tanto en trabajos de campo como de laboratorio.

Infinitas gracias las que merece Jose Luis, por haberme dado la oportunidad de continuar mi

trabajo en estos últimos años, y por tantas ayudas y consejos prestados en el desarrollo del mismo.

Ha sido también gracias a tí, que he podido desarrollar mi estancia en la Universidade do Algarve

(CCMAR), donde he conocido a personas excepcionales dentro del grupo de investigación

ALGAE (Rui, João, Isabel, Monya, Mafalda, Laura, Irene, Bruno, Ana, Silvia, Begoña, Gianmi).

“Muito muito obrigado miudos e miudas, verdadeiramente foi impressionante compartilhar vivên-

cias e horas de laboratório convosco; sento-me muito agradecido e eternamente em dívida com to-

dos. Nunca nunca esquecerei meu pequeno trabalho no grupo, e sempre levarei comigo às pessoas

que conheci ali. Bom, e nunca se sabe, ¡talvez nossos caminhos voltem a se encontrar num dia!.”

Si hay algo que pretendo reflejar con este apartado de agradecimientos, es que la reali-

zación de esta Tesis no hubiera sido posible sin vosotros. Hemos recorrido los mismos sen-

deros juntos desde el principio, y ahora, cuando parece que nuestros caminos comienzan

a distanciarse, es cuando más afortunado y orgulloso me siento de haber podido compartir

todo esto con vosotros, todos estos años. Enhorabuena a todos por este trabajo, sólo vuestro.

Cuando llegados a este punto vuelves tu mirada hacia atrás, y observas y recapaci-

tas sobre los años de estechas vivencias compartidas con personas tan especiales, sencilla-

mente comprendes que uno mismo no es nadie sin las personas importantes que lo rodean,

y que son precisamente las relaciones de amistad, hermandad y confianza, las que orien-

tan tu destino, éxitos y fracasos. Los trabajos pueden ser desarrollados, escritos y publica-

dos con la misma rapidez con que pueden caer en el olvido, pero son las relaciones que has

cultivado en el transcurso de los mismos las que prevalecen y te acompañan para siempre.

Por último, también quisiera agradecer a todos aquellos que no confían o compren-

den demasiado el camino que muchos como yo emprendimos hace algunos años, el ha-

ber impulsado aún más si cabe, mis deseos de continuar por él, para lo bueno y lo malo.

ÍNDICE

1 INTRODUCCIÓN GENERAL

27 RESPONSES OF THE MEDITERRANEAN SEAGRASS POSIDONIA OCEANICA TO

IN SITU SIMULATED SALINITY INCREASE CHAPTER 1

57 PHOTOSYNTHESIS, GROWTH AND SURVIVAL OF THE MEDITERRANEAN

SEAGRASS POSIDONIA OCEANICA IN RESPONSE TO SIMULATED

SALINITY INCREASES IN A LABORATORY MESOCOSM SYSTEM CHAPTER 2

85 THE EFFECT OF SALINITY INCREASE ON THE PHOTOSYNTHESIS,

GROWTH AND SURVIVAL OF THE MEDITERRANEAN SEAGRASS

CYMODOCEA NODOSA CHAPTER 3

115 TOLERANCE OF MEDITERRANEAN SEAGRASSES (POSIDONIA OCEANICA

AND CYMODOCEA NODOSA) TO HYPERSALINE STRESS: WATER RELATIONS

AND OSMOLYTE CONCENTRATIONS CHAPTER 4

139 ANALYSIS OF THE ECOPHYSIOLOGICAL PLASTICITY OF PLANTS FROM

SHALLOW AND DEEP MEADOWS OF THE MEDITERRANEAN SEAGRASSES

(POSIDONIA OCEANICA AND CYMODOCEA NODOSA) IN RESPONSE TO

EXPERIMENTAL SIMULATION OF CHRONIC HYPERSALINE STRESS CHAPTER 5

177 DISCUSIÓN GENERAL

201 CONCLUSIONES

207 BIBLIOGRAFÍA

ANEXOS

1 EVOLUCIÓN ESTACIONAL DE DESCRIPTORES FISIOLÓGICOS Y

VEGETATIVOS EN PRADERAS DE REFERENCIA DE POSIDONIA OCEANICA

Y CYMODOCEA NODOSA

2 DISEÑO Y DESCRIPCIÓN TÉCNICA DEL SISTEMA DE MESOCOSMOS

1INTRO

DUCC

IÓNGE

NERA

L

2

3 LA SALINIDAD COMO FACTOR CLAVE EN LA EVOLUCIÓN

Y ECOLOGÍA DE ANGIOSPERMAS MARINAS

4 ALTERACIONES ANTROPOGÉNICAS DEL RÉGIMEN

DE SALINIDAD EN PRADERAS MARINAS

7 LA HIPERSALINIDAD COMO CONDICIÓN DE ESTRÉS

9 EFECTOS ECO-FISIOLÓGICOS DEL INCREMENTO DE

SALINIDAD EN ANGIOSPERMAS MARINAS

17 TOLERANCIA DE ANGIOSPERMAS MARINAS

MEDITERRÁNEAS (POSIDONIA OCEANICA Y

CYMODOCEA NODOSA)

AL INCREMENTO DE LA SALINIDAD

22 OBJETIVOS Y ESTRUCTURA DE LA TESIS

3

1. LA SALINIDAD COMO FACTOR CLAVE EN LA EVOLUCIÓN Y ECOLOGÍA DE ANGIOSPERMAS MARINAS

Las angiospermas marinas (Subclase Alismastidae) son plantas clonales, posiblemente descendientes de hidró-

fitos acuáticos y plantas de ambientes costeros (Larkum y den Hartog 1989), que han colonizado con éxito los fon-

dos marinos infralitorales de costas tropicales y templadas de todo el mundo formando extensas praderas mari-

nas (Green y Short 2003). Estas praderas representan uno de los hábitats marinos de mayor relevancia ecológica,

ya que su elevada producción primaria y la persistencia de su estructura, les confieren una serie de funciones y

servicios considerados clave para al ecosistema marino costero, su funcionamiento y configuración actual. Como

ejemplos de ello, podrían resaltarse el mantenimiento de la biodiversidad y las pesquerías locales, las redes tróficas

propias y de ecosistemas adyacentes, la protección de la costa frente la erosión, el almacenamiento de carbono y

nutrientes (ciclos biogeoquímicos), la sedimentación de partículas, y el mantenimiento de la transparencia y cali-

dad del agua, sin olvidar funciones de otro tipo relacionadas con sus propios valores estéticos y culturales. Algu-

nos estudios han estimado que además del valor ecológico, estas funciones/servicios pueden revertir en impactos

socio-económicos considerables para la propia población humana (p.e. Constanza et al. 1997; Orth et al. 2006).

Las angiospermas marinas representan un grupo ecológico de plantas que han evolucionado y se han adaptado

para completar sus ciclos de vida completamente sumergidas en ambientes marinos y estuáricos (Hemminga y Duarte

2000; Kuo y den Hartog 2000; den Hartog y Kuo 2006). Desde el punto de vista taxonómico, es un grupo polifilético

constituido por unas 60 especies, en el que las diferentes trayectorias evolutivas convergen en una serie de adapta-

ciones particulares relacionadas con el nicho ecológico que ocupan (Spalding et al. 2003). De entre las propiedades

postuladas por Arber (1920) indispensables para el conjunto de plantas marinas existentes, sólo aquellas relacionadas

con la “adaptabilidad a la vida en un medio salino” diferencian realmente a las angiospermas marinas de los grupos

de plantas acuáticas. Las especies de angiospermas consideradas estrictamente marinas son todas las pertenecientes

a las familias Zosteraceae, Cymodoceae y Posidoniaceae y a tres géneros de la familia Hydrocaritaceae (los otros 14

géneros de esta familia son propios de aguas dulces). No obstante, el grupo de las denominadas eurisalinas (Familias

Ruppiaceae, Zannichelliaceae y Potamogetonaceae) no ocurren en ambientes marinos, pero comparten los criterios

de Arber y han desarrollado una notable tolerancia a la salinidad (Kuo y den Hartog 2000, den Hartog y Kuo 2006).

3 LA SALINIDAD COMO FACTOR CLAVE EN LA EVOLUCIÓN

Y ECOLOGÍA DE ANGIOSPERMAS MARINAS

4 ALTERACIONES ANTROPOGÉNICAS DEL RÉGIMEN

DE SALINIDAD EN PRADERAS MARINAS

7 LA HIPERSALINIDAD COMO CONDICIÓN DE ESTRÉS

9 EFECTOS ECO-FISIOLÓGICOS DEL INCREMENTO DE

SALINIDAD EN ANGIOSPERMAS MARINAS

17 TOLERANCIA DE ANGIOSPERMAS MARINAS

MEDITERRÁNEAS (POSIDONIA OCEANICA Y

CYMODOCEA NODOSA)

AL INCREMENTO DE LA SALINIDAD

22 OBJETIVOS Y ESTRUCTURA DE LA TESIS

4

Así pues, las angiospermas marinas presentan ciertas propiedades generales compartidas con otros grupos de

plantas acuáticas; entre ellas, la polinización hidrófila o el desarrollo de un sistema de anclaje, les permitirán completar

sus ciclos biológicos en condiciones de inmersión. Sin embargo, las angiospermas marinas también pueden exhibir

ciertas características únicas, sólo compartidas con especies de macrófitos adaptados a ambientes marinos, y que

serán responsables de la mayor o menor tolerancia a las altas concentraciones salinas de este medio, y sus varia-

ciones espaciales (entre diferentes ambientes costeros) y temporales. En efecto, ciertas propiedades identificadas

a nivel fisiológico (p.e. potenciales hídricos muy negativos), ultraestructural (p.e. adelgazamiento de la cutícula y

libre intercambio de agua y ciertos solutos) y morfológico (p.e. protección de meristemos por escamas peciolares),

parecen estar relacionadas con adaptaciones de las distintas especies de angiospermas marinas a desarrollarse en am-

bientes salinos (Jagels 1983; Tyerman 1989; Jagels y Barnabas 1989; Iyer y Barnabas 1993; Kuo y den Hartog 2006).

Las diferentes especies de angiospermas marinas ocupan una amplia gama de ambientes costeros, caracterizados

por muy diferentes regímenes de salinidad: desde aguas abiertas de salinidad estable, a lagunas costeras hipersalinas

y estuarios con salinidad muy inestable. Esto sugiere, por un lado, la existencia de diferentes capacidades adaptativas

y aclimatativas entre especies y, por otra parte, que la salinidad, además de otros factores clave que también varían

entre estos ambientes (p.e. luz, nutrientes), debe haber ejercido una presión selectiva determinante para explicar las

variaciones inter- e intra- específicas de dichas capacidades. Sin embargo, y a pesar del presumible papel central de la

salinidad en la ecología y evolución de las angiospermas marinas, el nivel de conocimiento básico sobre las adaptacio-

nes mencionadas y de los diferentes mecanismos de ajuste a los cambios de la salinidad del medio, es en la actualidad

bastante escaso en relación a otros aspectos de la biología de las mismas (Larkum et al. 2006b; Ruiz et al 2009a).

2. ALTERACIONES ANTROPOGÉNICAS DEL RÉGIMEN DE SALINIDAD EN PRADERAS MARINAS

Las praderas de angiospermas marinas son particularmente vulnerables a las perturbaciones de las condi-

ciones ambientales causadas por la actividad humana, principales responsables de una aceleración de la tasa de

pérdida de la superficie y funcionalidad de este hábitat en las últimas décadas (Ralph et al. 2006; Boudouresque

et al. 2009). La destrucción directa de las praderas marinas debido a la construcción de infraestructuras coste-

ras (puertos, playas artificiales, etc.), al desarrollo de determinadas prácticas pesqueras (pesca de arrastre, maris-

queo, etc.), así como por los efectos directos e indirectos del deterioro de la calidad de las aguas costeras por

contaminación (metales pesados, hidrocarburos, pesticidas, etc.) y aportes de nutrientes (eutrofización), son las

5

principales causas habitualmente consideradas para explicar las pérdidas de este tipo de hábitat (Pergent-Mar-

tini y Pasqualini 2000; Ruiz et al. 2001; Ruiz y Romero 2003; González-Correa et al. 2005; González-Correa

et al. 2008). En este sentido, gran parte de la investigación en este campo ha sido enfocada a estudiar la ca-

pacidad de aclimatación y tolerancia de las angiospermas marinas a efectos estresantes como los de la eutrofi-

zación, la hiper-sedimentación o la reducción de la luz, mientras que menor atención se ha prestado a otros ti-

pos de perturbaciones antrópicas menos frecuentes o más recientes, como los directamente relacionados con

el cambio climático (calentamiento, acidificación, etc.), la introducción de especies invasoras o los cambios en

la salinidad del hábitat (Short y Neckles 1999; Williams 2007; Bernardeau-Esteller et al. 2011; Ruiz et al 2011).

Como se ha explicado, las angiospermas marinas están particularmente adaptadas a un medio salino, y la

composición y distribución de las diferentes especies pueden presentarse muy condicionadas por las variacio-

nes espaciales y temporales de este factor entre los diferentes ambientes costeros (McMillan y Moseley 1967;

Zieman 1974; Walker y McComb 1990; Vermaat et al. 2000). Sin embargo, las alteraciones de la salinidad cau-

sadas por la actividad humana pueden superar la capacidad de respuesta de las angiospermas marinas a las va-

riaciones naturales de este factor, y de este modo, pueden suponer una amenaza potencial para la conservación

de las mismas (McIvor et al. 1994; Ruiz 2005; Sánchez-Lizaso et al. 2008). En este sentido se han documenta-

do alteraciones del régimen de salinidad de ambientes someros marinos, a consecuencia de modificaciones ar-

tificiales del régimen hidrológico local o de planes de regulación hídrica a nivel de cuenca, y que han sido rela-

cionadas con el deterioro del estado y distribución de las praderas marinas durante las últimas décadas. Los casos

documentados al sur de Florida (Fourqurean et al. 2003), en la laguna hipersalina del Mar Menor (Murcia, Te-

rrados y Ros 1991) y en el Mar de Wadden (costas holandesas, van Katwijk et al. 1999), son algunos ejemplos.

En la actualidad, una de las causas más frecuentes de incremento de la salinidad sobre las praderas de angios-

permas marinas (y otros tipos de comunidades bentónicas) son los vertidos hipersalinos resultantes de la desalación

de agua de mar (Morton et al. 1996; Castriota et al. 2001; del-Pilar-Ruso et al. 2007, 2008, 2009; Riera et al. 2011,

2012). El desarrollo reciente de la industria de la desalación, está siendo especialmente notable en regiones semi-

áridas del planeta, de clima tipo mediterráneo, como el sureste de España o el suroeste de Australia (Lattemann y

Höpner 2008; Palomar y Losada 2010). Precisamente en estas zonas de aguas templadas, la praderas marinas son

el hábitat infralitoral dominante y, por tanto, es donde se han identificado mayores conflictos medioambientales

potenciales con los vertidos de las plantas desalinizadoras. Este tipo de conflictos también han sido identificados en

otras zonas costeras azotadas por déficits hídricos, y caracterizadas por una industria desalinizadora establecida desde

6

hace ya varias décadas, como es el caso de las Islas Canarias, cuyas aguas subtropicales infralitorales se encuentran

también dominadas por extensas praderas marinas (Pérez-Talavera y Quesada-Ruiz 2001; Portillo et al. 2012a, b).

El procedimiento de desalación más ampliamente extendido es el de “ósmosis inversa”, caracterizado por una efi-

ciencia del orden del 45%; esto significa que además de la obtención de agua destinada al uso por la población huma-

na, se produce un residuo hipersalino o salmuera que puede alcanzar salinidades comprendidas entre 40-60 ups (i.e.

unidades prácticas de salinidad) que es vertido de nuevo al medio marino. Estas aguas hipersalinas (o de rechazo)

de elevada densidad, pueden avanzar como estrechas capas de agua (o plumas) que apenas se mezclan con el agua

circundante y que por tanto, son capaces de recorrer extensas distancias y afectar amplias superficies de fondo marino

siguiendo las líneas de máxima pendiente, dependiendo del sistema de vertido y difusión (Fernández-Torquemada

et al. 2005a; Antequera y Jáuregui 2001; Ruiz-Mateo 2007; Lattemann y Höpner 2008; Portillo et al. 2012 a, b). Las

comunidades bentónicas presentes en esta zona de influencia no se encuentran adaptadas a tolerar estas salinida-

des y/o cambios bruscos y repentinos de la salinidad, y experimentan alteraciones significativas de su composición,

abundancia y distribución (Chesher 1975; Castriota et al. 2001; Fernández-Torquemada et al. 2005a; Portillo et al.

2012b). Pero además de la salinidad, otros factores asociados a este tipo de vertidos y que pueden potencialmente

afectar el desarrollo y supervivencia de los organismos bentónicos son el pH, la temperatura, la composición iónica

o la presencia de productos químicos que se añaden al proceso de desalación con diferentes propósitos (desinfec-

tantes, desincrustantes, etc) (Morton et al. 1996). El alcance final de sus efectos en los hábitat bentónicos puede

ser muy variable dependiendo de las características del vertido (volumen, salinidad, composición química, sistema de

dilución y difusión, etc), del medio receptor (corrientes, salinidad, profundidad, clima, etc.) y de las comunidades bio-

lógicas afectadas (tipos, crecimiento, plasticidad, límites de tolerancia, etc.) (Ruiz 2005; Sánchez-Lizaso et al. 2008).

La preocupación suscitada por las consecuencias ecológicas del impacto de los vertidos de salmuera en hábitats

vulnerables y ecológicamente relevantes como las praderas marinas, ha motivado un creciente esfuerzo por deter-

minar los límites de tolerancia del crecimiento y supervivencia de las angiospermas marinas frente al incremento de

la salinidad. De este modo, estudios que relacionen estos aspectos han sido demandados a la comunidad científica

por parte de la administración pública responsable de la gestión y conservación de los ecosistemas marinos coste-

ros y la explotación de sus recursos, con el fin de basar la gestión y control medioambiental de estos vertidos en

criterios científicos objetivos. Sin embargo, desde el punto de vista científico, si bien ya se comienza a tener cierta

información fiable y coherente la sensibilidad de algunas especies, apenas se dispone del conocimiento básico sobre

las características (y mecanismos) genéticos, fisiológicos, morfológicos y poblacionales que determinan la tolerancia

al incremento de la salinidad por las diferentes especies de angiospermas marinas, ecotipos, genotipos y poblaciones.

7

3. LA HIPERSALINIDAD COMO CONDICIÓN DE ESTRÉS

Antes de avanzar en aspectos más específicos de esta tesis, parece necesario y conveniente aclarar qué se en-

tiende por estrés, y en qué situaciones las alteraciones de salinidad del medio pueden ser tratadas como factores

capaces de causar estrés. El concepto de estrés se encuentra bien definido y desarrollado en plantas terrestres, ya

que la capacidad de ciertas plantas cultivadas de resistir o tolerar condiciones de estrés hídrico o salino tiene impor-

tantes consecuencias socio-económicas en todo el planeta (Kahn y Weber 2006). En la Fig. 1 se muestra un modelo

conceptual del síndrome de estrés en plantas terrestres, en el que se indican las diferentes fases que lo componen

a medida que el factor de estrés persiste, y que será empleado a continuación como marco teórico de referencia.

Figura 1. Modelo secuencial que comprende las distintas fases de respuesta generales de las plantas frente a la aparición de una determinada condición de estrés. Una vez aparece el estrés y tras reducirse el estado fisiológico óptimo, se activan mecanismos de aclimatación en la planta que le permiten alcanzar un nuevo estado fisiológico de resistencia. Si la condición de estrés persiste o aumenta su severidad, los recursos de la planta pueden llegar a un estado de agotamiento tal, que puede resultar en efectos deletéreos a nivel de vitalidad. (Modificado de: Lichtenthaler 1996, Tadeo y Gómez-Cadenas 2008)

8

Las características fisiológicas y tolerancias particulares de cada especie, confieren a las plantas la capacidad

de aclimatarse y adaptarse al régimen de condiciones ambientales característico del hábitat que ocupan, y man-

tener así un estado fisiológico óptimo en el que el crecimiento y el resto de funciones biológicas son adecuados.

En este estado, las plantas pueden responder con ajustes eficaces y reversibles de sus flujos metabólicos, a cam-

bios cotidianos del ambiente, y repetidos a diferentes escalas espaciales y temporales. Así por ejemplo, las an-

giospermas marinas pueden experimentar diariamente fluctuaciones más o menos extremas de factores como

la luz (p.e. debido a ciclos diarios, a reflejos instantáneos de las olas o “sunflecks”, al paso de una nube o al en-

turbiamiento repentino de la columna de agua; Pearcy 2007), la temperatura (p.e. por el ascenso de aguas frías

profundas) y la salinidad (p.e. fluctuaciones debidas a la pluviosidad o a la existencia de ramblas cercanas, etc).

Sin embargo, hay situaciones de cambio ambiental que por su grado (intensidad, frecuencia) o su naturaleza

(bióticos-abióticos, natural-antrópico) conllevan una alteración del medio más allá de los rangos de fluctuación na-

tural. Este tipo de alteraciones pueden ser consideradas como causantes de estrés, ya que requieren de mayores

respuestas de aclimatación (denominadas de tensión), y pueden derivar en efectos deletéreos a distintos niveles me-

tabólicos, y provocar así la desviación de los niveles óptimos fisiológicos de la planta. En angiospermas marinas, estas

situaciones potencialmente estresantes pueden darse de forma natural, por ejemplo, en los límites de distribución

geográfica y vertical (profundidad), o por la influencia de eventos climáticos u oceanográficos extremos y poco fre-

cuentes. No obstante, las perturbaciones antrópicas del medio marino representan en la actualidad una de las fuentes

más importantes de perturbación y estrés, responsables del deterioro y regresión de estos hábitats (Ralph et al. 2006).

Siguiendo el modelo conceptual general ilustrado en la Fig. 1, los casos de incrementos más o menos persis-

tentes de la salinidad más allá de sus límites naturales de variación, pueden suponer una situación de estrés que

a su vez, inducirá la activación de distintos tipos de respuestas de aclimatación en la planta. En una fase inicial o

“fase de alarma” tras la exposición a la condición estresante, las plantas reaccionan ralentizando sus funciones me-

tabólicas más básicas, reduciendo incluso su vitalidad. Durante esta fase también pueden activarse mecanismos

de aclimatación y/o acomodación fisiológica a las nuevas condiciones osmóticas del medio que, en función de

las capacidades inherentes a cada especie, ecotipo o genotipo, permiten a la planta alcanzar un nuevo óptimo fi-

siológico (o de máxima resistencia), y que da nombre y caracteriza la denominada “fase de resistencia” al estrés.

Pero si el estrés se mantiene en el tiempo, y/o se incrementa la intensidad del mismo, los recursos metabólicos y

la vitalidad de la planta pueden verse considerablemente reducidos. Esta fase de estrés severo, conocida como de

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“fase de agotamiento o extenuación”, puede derivar en un aumento de los procesos de senescencia y muerte celu-

lar, si el factor estresante persiste; si, por el contrario, el estrés desaparece en esta fase, la recuperación al estado

original es poco probable, aunque la planta puede alcanzar un nuevo estado fisiológico o “fase de regeneración”.

Los estudios que componen la presente Tesis Doctoral, se centran en las respuestas del síndrome de estrés

desarrolladas por especies de angiospermas marinas mediterráneas bajo la acción persistente (crónica) de estrés

hipersalino, en sus fases de alarma, resistencia o incluso de agotamiento, según las capacidades aclimatativas pro-

pias de las especies objeto de estudio (Posidonia oceanica y Cymodocea nodosa). No obstante, cabe destacar aquí

que la naturaleza clonal de las angiospermas marinas puede complicar o confundir las fases definidas en este mo-

delo general; por ejemplo, la translocación de recursos entre haces (ramets) o tejidos de un mismo clon (Marbá

et al. 2002; Romero et al. 2006) podría resultar en la aparición simultánea de mecanismos de aclimatación, esta-

dos de resistencia y estados de extenuación (senescencia o muerte) entre los distintos individuos interconecta-

dos por la misma estructura clonal. Precisamente, esta posibilidad de integrar la respuesta al estrés a nivel clonal

se ha sugerido como el mecanismo principal de aclimatación a gradientes ambientales (p.e. reducción de la luz

con la profundidad), y explica la escasa plasticidad fisiológica a nivel de haces individuales observada en algunos

casos (Olesen et al. 2002; Collier et al 2008). El estudio de la fase de regeneración y por tanto, de la capacidad

de recuperación tras la desaparición del factor estresante, se sitúa fuera de los objetivos que plantea esta Tesis.

4. EFECTOS ECOFISIOLÓGICOS DEL INCREMENTO DE SALINIDAD EN ANGIOSPERMAS MARINAS

Tanto el estrés hídrico de plantas terrestres, como el debido a la hipersalinidad en macrófitos bentónicos marinos

(macroalgas y angiospermas marinas), deben sus más importantes efectos negativos a dos tipos de factores: el factor

osmótico y el factor iónico (Kramer y Boyer 1995; Bisson y Kirst 1995; Tyerman 1989). En macrófitos marinos, el factor

osmótico se relaciona con la dificultad del mantenimiento de un balance hídrico positivo desde el medio hacia los teji-

dos. El factor iónico, por su parte, hace referencia a la toxicidad iónica que el incremento de sales puede causar a distin-

tos niveles metabólicos (Kirst 1989). Ambos factores, componentes principales de una condición de estrés hipersalino,

pueden inducir una serie de alteraciones a diferentes niveles fisiológicos y compartimentos metabólicos, cuya expresión

se mostrará estrechamente relacionada con las tolerancias y capacidades propias de cada especie (Touchette 2007).

Sin embargo, en comparación con los notables avances alcanzados en plantas terrestres sobre campos relacionados

(estrés hídrico y salino; Parida y Das 2005; Farooq et al. 2009), el conocimiento actual de las respuestas fisiológicas de

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macrófitos marinos al estrés hipersalino es relativamente escaso y limitado a cierto de especies y grupos taxonómicos.

En concreto, el conocimiento relativo a las angiospermas marinas es aún más reducido (Touchette 2007), y los resul-

tados obtenidos hasta nuestros días se resumen en la Tabla 1 y en los apartados que se desarrollan a continuación.

Tabla 1. Resumen de los efectos fisiológicos del incremento de salinidad (bajo condiciones experimentales o debidas a rangos naturales de salinidad) observados en distintas especies de angiosper-mas marinas. En la columna correspondiente a los niveles/rangos salinos, se incluyen entre paréntesis los niveles empleados como salinidad control, o los correspondientes a las condiciones naturales de las especies, de acuerdo con la infor-mación provista en cada estudio. Dichos datos no se muestran en el caso de aquellos estudios que no los facilitan, o que simplemente describen las respuestas fisiológicas a lo largo de un rango salino.

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4.1. ALTERACIÓN DE LAS RELACIONES HÍDRICAS

Las angiospermas marinas son plantas sumergidas cuyos procesos fisiológicos se hayan íntimamente rela-

cionados con el medio salino circundante. Esto implica que, ante cambios de la salinidad del medio, las respues-

tas de las relaciones hídricas constituirán la base de las estrategias ecofisiológicas y metabólicas que las plantas

podrán adoptar para resistir condiciones de estrés hipersalino. Por tanto, el conocimiento de las relaciones hí-

dricas de estas plantas y sus relaciones con los cambios de salinidad es fundamental. No obstante, las relaciones

hídricas per se y la forma en que son alteradas por los cambios externos en salinidad, figuran entre los aspectos

menos estudiados en estas plantas (Tyerman et al. 1984; Tyerman 1989; Murphy et al. 2003; Koch et al. 2007b).

En general, las macroalgas (Kirst 1989) y las angiospermas marinas (Tyerman 1989) se encuentran os-

móticamente adaptadas (osmoadaptadas, sensu Kirst 1989) al régimen salino que ocupan, a través del man-

tenimiento de potenciales hídricos (Ψw) y osmóticos (Ψπ) foliares más negativos que los valores del me-

dio (Tyerman 1989; Kirst 1989). Esta diferencia supone un balance hídrico positivo a favor de los tejidos y, en

consecuencia, el mantenimiento de valores óptimos de turgescencia (presión de turgor, Ψp), condición a su

vez imprescindible para el metabolismo, crecimiento y conformación estructural de las células vegetales (Zim-

mermann 1978; Kramer y Boyer 1995). Al aumentar la salinidad, el potencial hídrico del medio puede disminuir

hasta valores más próximos, o incluso más negativos, que el de los tejidos. Este nuevo escenario osmótico re-

sulta en la disminución o interrupción del balance hídrico, lo que puede ocasionar distintas alteraciones a nivel

metabólico y la activación de ciertas estrategias fisiológicas tendentes a restablecer el balance original. A con-

tinuación se explican con más detalle dichas estrategias, y se representan esquemáticamente en la Fig. 2.

4.1.1. ESTRATEGIAS DE EVITACIÓN DE DESHIDRATACIÓN

Bajo ciertas condiciones del medio (p.e. salinidad, sequía) capaces de ocasionar la ruptura del balance hídri-

co entre éste y los tejidos, las plantas pueden alcanzar grados de deshidratación severos. En este estado, un gru-

po selecto de plantas denominadas poiquilohídricas (p.e. líquenes, musgos, helechos, plantas de zonas desérticas,

etc) pueden desarrollar estrategias de «tolerancia a la deshidratación», manteniendo sus capacidades metabólicas

intactas (en ocasiones en estado de letargo) hasta la aparición de situaciones más favorables (Oliver et al. 2000;

Sánchez-Díaz y Aguirreolea 2000). Sin embargo, la mayoría de las plantas terrestres, acuáticas y marinas son

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homeohídricas, lo que implica que deben activar ciertos mecanismos con el fin de evitar la pérdida del agua ce-

lular. Dichos mecanismos, estudiados en el contexto de estrategias de evitación de la deshidratación (sensu Vers-

lues et al. 2006), se basan en la reducción del Ψw de los tejidos para permitir el restablecimiento del balance

hídrico inicial previo a la aparición del estrés. A lo largo de la escasa bibliografía disponible, no se han encontra-

do evidencias de las activación de estrategias de tolerancia a la desecación en angiospermas marinas, y aunque

su funcionamiento no deba ser descartado en especies adaptadas a cambios bruscos del régimen hídrico o sali-

no (p.e. especies intermareales), nos centraremos aquí en los mecanismos de evitación de deshidratación (Fig.

2), ya puestos de manifiesto para algunas especies de angiospermas marinas sujetas a incrementos de salinidad.

Figura 2. Esquema representativo de las distintas etapas y diferentes estrategias aclimatación conocidas como de “evitación de deshidratación” (i.e. osmorregulación, procesos de endurecimiento de pared celular) que las angiospermas marinas podrían desarrollar frente a condiciones de incremento de salinidad. Los paneles de la izquierda representan la sucesión de dichos procesos a nivel celular en tejido foliar (Ψext=potencial hídrico del medio;Ψw= Potencial hídrico foliar; Ψπ= Potencial osmótico;Ψp= Potencial de turgor; ∆V= variación de volumen celular). Adicionalmente se aporta en los paneles de la derecha, un hipotético ejemplo del desarrollo de ambas estrategias en respuesta a un hipotético incremento de salinidad de 37 a 43 ups (i.e. unidades prácticas de salinidad), con valores numéricos de los distintos potenciales expresados en unida-des de presión (i.e. Megaspascales, MPa).

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Los principales mecanismos relacionados con estrategias de evitación son dos: a) los relacionados con proce-

sos de osmorregulación y ajuste osmótico, y b) los relacionados con las características de la pared celular (Fig. 2).

Los procesos de osmorregulación o ajuste osmótico (Kramer y Boyer 1995) se basan en la disminución del Ψw a

través de una reducción de Ψπ, que ocurre como consecuencia directa del aumento de la concentración de solu-

tos intracelulares. Por otra parte, y de acuerdo con lo descrito en plantas terrestres y algas marinas (Khan y We-

ber 2006; Bisson y Kirst 1995; Kramer y Boyer 1995; Verslues et al. 2006), una estrategia alternativa de reducción

de Ψw consiste en la reducción de Ψp, relacionada ésta con procesos de reajustes volumétricos entre citoplasma,

membrana y pared celular. En este sentido, y de forma general, en células de paredes celulares elásticas (i.e. con

elevada capacidad de ajuste a distintos volúmenes celulares), la magnitud en la pérdida del turgor celular viene de-

terminada por la pérdida de agua intracelular o simplástica. Sin embargo, plantas que presentan paredes celulares

relativamente inelásticas, o que muestran la capacidad de alterar el grado de endurecimiento de las mismas (p.e.

a través de reorganización o engrosamiento de fibras de celulosa; Verslues et al. 2006), pueden presentar seve-

ras reducciones del turgor celular (y así de Ψw) con insignificantes pérdidas hídricas; en estas situaciones, Ψπ se

mantiene constante por la ausencia de efectos de deshidratación y acumulación pasiva de solutos. En angiospermas

marinas sólo se han descrito mecanismos de tipo osmorregulatorio en respuesta a incrementos de la salinidad del

medio, aunque existe alguna evidencia puntual (i.e. a través de mediciones del módulo de elasticidad de las pare-

des celulares, ε) de que ambos tipos de estrategias pueden operar en estas plantas marinas, dependiendo de las

estrategias ecológicas de cada especie y su capacidad de adaptarse a distintos regímenes salinos (Tyerman 1982).

4.1.2. ACUMULACIÓN DE OSMOLITOS

En el proceso de osmorregulación, la concentración de solutos osmóticamante activos (también deno-

minados osmolitos) en los principales compartimentos intracelulares (citoplasma y vacuola), vendrá deter-

minada por la naturaleza de los solutos (inorgánicos/orgánicos) y otros factores como el tiempo de expo-

sición al estrés o el estado fisiológico y nutricional de la planta (Hsiao 1973; Wyn Jones y Gorham 1983).

En general, tras un incremento de salinidad, tiene lugar la acumulación inmediata de sales e iones (Na+ y Cl- en su

mayoría) en el citoplasma, en su mayoría por transporte a favor de gradiente desde el medio externo. Dicho proceso

se presenta como la respuesta osmorreguladora más rápida (minutos-horas), que tiene lugar con el fin de restituir el

balance hídrico celular (Hasegawa et al. 2000). Si las condiciones de hipersalinidad persisten o aumentan, la acumu-

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lación de estos osmolitos inorgánicos puede continuar hasta el punto en que sus concentraciones pueden resultar

tóxicas para el metabolismo celular; de hecho, altas concentraciones iónicas pueden interferir con la actividad de

diversas enzimas citoplasmáticas, alterar potenciales de membrana (tanto celular como de organelas) o incluso, da-

ñar la estructura de diferentes complejos proteínicos como los de cadenas de transporte electrónico de cloroplastos

y mitocondrias (Flowers et al. 1977; Hasegawa et al. 2000; Zhu 2003; Munns 2002). De este modo y para evitar su

acumulación excesiva, dichos osmolitos inorgánicos pueden ser excluídos de nuevo al medio externo, transportados

a otros tejidos, o compartimentalizados en ciertos orgánulos especializados (i.e. vacuola) (Niu et al. 1995; Zhu 2003),

al mismo tiempo que son reemplazados en el citoplasma por solutos orgánicos también osmóticamente activos. La

acumulación de estos solutos se ha sugerido como un proceso más lento, que tiene lugar en fases más tardías de

la respuesta de osmorregulación (i.e. días-semanas), lo que hace de estos compuestos excelentes indicadores de

procesos de ajuste osmótico bajo exposiciones prolongadas o crónicas a condiciones de incremento de salinidad.

La importancia de dichos solutos no radica únicamente en la función osmótica que suponen, sino también en el

papel de osmoprotección que desempeñan en procesos de ajuste osmótico (e.g. estabilización de membranas y

complejos enzimáticos), así como por su reducida toxicidad metabólica a elevadas concentraciones, cualidad ésta

última por la cual reciben el nombre de solutos compatibles (Munns 2002). El aumento de concentración solutos

orgánicos en tejidos foliares (sobre todo carbohidratos no estructurales y aminoácidos libres-prolina en especial) ha

sido demostrado en algunas especies de angiospermas marinas (Stewart y Lee 1974; Brock 1981; Pulich 1986; Tyer-

man 1989; Murphy et al. 2003; Adams y Bates 1994a; Koch et al. 2007b); sin embargo, sólo a partir de los trabajos

realizados por Tyerman y colaboradores en la década de los 80 (Tyerman et al. 1984;Tyerman 1989), se ha obtenido

evidencia directa de la acumulación de iones respecto a tratamientos experimentales de incremento de salinidad.

4.2. ALTERACIONES A NIVEL DE FOTOSÍNTESIS Y PIGMENTOS FOTOSINTÉTICOS

La fotosíntesis es un proceso fisiológico clave para el crecimiento y productividad de las plantas, por lo que las

reducciones de las tasas fotosintéticas debidas a una condición estresante pueden ser consideradas como uno de

los efectos más importantes a nivel metabólico (Kramer y Boyer 1995; Huchzermeyer y Koyro 2005). Bajo con-

diciones de estrés hipersalino, se ha observado una reducción de la actividad fotosintética de plantas terrestres y

macrófitos marinos (Kirst 1989; Huchzermeyer y Koyro 2005), como consecuencia de numerosos factores: i) re-

ducción del Ψw y sus consecuencias fisiológicas (e.g. gasto energético, acumulación de solutos a concentracio-

nes tóxicas), ii) alteraciones a nivel de ultraestructura, número y disposición de cloroplastos, iii) modificaciones del

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aparato fotosintético a nivel de tilacoides , iv) inhibición de reacciones del metabolismo del carbono mediadas por

enzimas y v) reducciones en la concentración de pigmentos fotosintéticos (clorofila a, clorofila b y carotenoides).

En angiospermas marinas, los estudios disponibles aportan evidencias de alteración sobre cualquiera de es-

tos niveles en respuesta al estrés hipersalino. Sin embargo, este conocimiento es bastante escaso y se resume en:

reducciones de las tasas fotosintéticas máximas (Ogata y Matsui 1964; Biebl y McRoy 1971; Drew 1978a; Kerr y

Strother 1985; Koch y Dawes 1991; Berns 2003; Fernández-Torquemada et al. 2005b; Koch et al. 2007b; Shafer

et al. 2011), disminuciones o incrementos en la eficiencia fotosintética (Koch y Dawes 1991; Berns 2003; Fernán-

dez-Torquemada et al. 2005b; Kahn y Durako 2006), reducciones a nivel de eficiencia fotoquímica del fotosis-

tema II (Fv/Fm, ΦPSII; Ralph 1998, 1999; Kamermans et al. 1999; Koch et al. 2007b; Pagés et al. 2010), dismi-

nuciones en la concentración de pigmentos fotosintéticos (McMillan Moseley 1967; Ralph 1998, 1999; Koch

y Dawes 1991; Trevathan et al. 2011), e inhibición de la actividad de enzimas carboxilativas (RuBisCo y PEPcar-

boxilasa, Beer et al. 1980a). No obstante, cabe destacar que todas estas evidencias disponibles, si bien aportan

un conjunto de síntomas de alteración de la estructura y funcionamiento de la maquinaria fotosintética, no per-

miten profundizar sobre los mecanismos implicados, los niveles a los que actúa el estrés y las relaciones entre

ellos, posiblemente a consecuencia del escaso conocimiento básico de la fotosíntesis en angiospermas marinas.

4.3. MODIFICACIONES EN LAS TASAS RESPIRATORIAS

Otro efecto importante del estrés salino es el derivado de la respuesta de la respiración mitocon-

drial, y en qué medida las alteraciones de la misma afectan a las tasas fotosintéticas (i.e. netas) y de pro-

ductividad de la planta. Las tasas respiratorias de los tejidos fotosintéticos son generalmente un orden de

magnitud inferiores respecto a las tasas fotosintéticas, pero su alteración puede resultar en consecuen-

cias funcionales importantes, debido a los desajustes del balance de carbono, resultantes en una reduc-

ción de los recursos disponibles para el crecimiento y supervivencia de la planta (Atkin y Macherel 2009).

En angiospermas marinas, la respiración no muestra un patrón de respuesta consistente ante el estrés hi-

persalino. Así, se ha observado que la respiración puede aumentar, disminuir o permanecer inalterada (o

incluso presentar un patrón bimodal), tanto entre especies diferentes como dentro de la misma especie,

bajo distintas condiciones experimentales y rangos de salinidad (Ogata y Takada 1968; Biebl y McRoy 1971;

Drew 1978a; Fernández-Torquemada et al. 2005b; Kahn y Durako 2006). Esta ausencia de una respuesta

17

general es consistente con lo descrito en plantas terrestres, y se atribuye a diversos factores como i) po-

sibles cambios en la actividad máxima de determinadas enzimas implicadas en distintos procesos respira-

torios (e.g. glucolisis, ciclo TCA, cadena de transporte electrónico mitocondrial), ii) cambios en la con-

centración de sustrato respiratorio (e.g. debido a la reducción de tasas fotosintéticas, y así, en la síntesis

de fotosintatos) e iii) incrementos en la demanda energética metabólica (p.e. para abastecer procesos de

osmorregulación, homeostasis iónica, etc) (Kramer y Boyer 1995; Munns 2002; Atkin y Macherel 2009).

5. TOLERANCIA DE ANGIOSPERMAS MARINAS MEDITERRÁNEAS (POSIDONIA OCEANICA Y CYMODOCEA NODOSA) AL INCREMENTO DE LA SALINIDAD

De las 5 especies que habitan en el Mediterráneo, P. oceanica y C. nodosa son las más abundantes

(Ruiz et al. 2009a); las praderas que forman representan uno de los hábitat infralitorales más extensos en-

tre 0 y 40 metros de profundidad y, al mismo tiempo, más vulnerables a los vertidos hipersalinos de la indus-

tria desaladora (Ruiz 2005; Boudouresque et al. 2009). Precisamente, el desarrollo de la desalinización de

agua de mar en la costa mediterránea española ha impulsado el conocimiento básico de la tolerancia de es-

tas especies al incremento de la salinidad, proceso que representa el antecedente principal en que se en-

marca esta tesis y a partir del cual surge la necesidad de avanzar en los objetivos y temas aquí planteados.

5.1. ASPECTOS GENERALES DE LA BIOLOGÍA Y ECOLOGÍA DE P. OCEANICA Y C. NODOSA

Posidonia oceanica L. (Delile) es una especie endémica presente en toda la cuenca mediterránea y cuyo límite se

sitúa en el sector más oriental (Líbano, Israel y Siria) (Gobert et al. 2006). De entre las especies mediterráneas, ésta

es la de mayor porte, y forma praderas monoespecíficas más o menos continuas con densidades poblacionales muy

elevadas (hasta > 1000 haces/m2 en poblaciones someras), que pueden dar lugar a doseles con índices de área foliar

comparables a los medidos en bosques terrestres (IAF = 13 m2 m-2; Romero 1986; Balestri et al. 2003). El crecimiento

de sus rizomas basales (verticales y horizontales) es de los más lentos observados en angiospermas marinas, y sus

P. oceanica y C. nodosa (Fig. 3) presentan características biológicas y estrategias ecológicas muy diferentes,

algunas de las cuales es necesario tener en cuenta para interpretar sus respectivas tolerancias tanto al estrés hiper-

salino, como a cualquier tipo de perturbación ambiental.

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poblaciones muestran una baja diversidad genética, propiedades que pueden limitar la capacidad de respuesta de

esta especie a las perturbaciones externas (Procaccini y Mazzella 1998; Procaccini y Piazzi 2001; Gobert et al. 2006).

Estas propiedades tienen que ver también con el carácter persistente de su biomasa, no solo a lo largo de su ciclo de

producción anual, sino también a largo plazo. Las praderas de esta especie representan la etapa clímax de la sucesión

ecológica de ambientes infralitorales mediterráneos (Borg et al. 2005; Gobert et al. 2006), y es considerada como

una especie estenobionte, cuyos requerimientos ecológicos son más estrictos que los de otras especies de distribución

más amplia; en este sentido, P. oceanica ocupa únicamente ambientes infralitorales de zonas costeras abiertas en los

que la salinidad es muy estable, las fluctuaciones de la temperatura son moderadas, las aguas son predominantemen-

te oligotróficas, sin influencia de aportes terrígenos importantes, de hidrodinamismo moderado-bajo y pendientes

suaves de plataforma (Boudouresque et al. 2009). No obstante, la presencia de praderas asentadas y estables de P.

oceanica ha sido documentada puntualmente en ambientes diferentes y más confinados, en los que la temperatura y

la salinidad pueden alcanzar valores mucho más extremos (Pergent y Zaouali 1992; Tomasello et al. 2009).

Cymodocea nodosa (Ucria) Ascherson es una especie de origen tropical, cuya distribución se haya actualmente

restringida al Mediterráneo y Atlántico nororiental desde las costas del sur de Portugal, hasta las de Senegal, inclu-

yendo las Islas Canarias y Madeira (Green y Short 2003). A diferencia de P. oceanica, esta especie puede formar

praderas tanto monoespecíficas como mixtas (con Zostera noltii), generalmente sobre fondos arenosos o fangosos.

En este caso, y también contrariamente a P. oceanica, se trata de una especie con atributos típicos de especies pio-

neras, de menor porte vegetativo, tasas de crecimiento mucho más elevadas y mayor diversidad genética (Procaccini

y Mazzella 1996; Cancemi et al. 2002), todas consideradas como características importantes en la capacidad de

desarrollar rápidas respuestas frente a los efectos de una posible perturbación (Olesen et al. 2002). De este modo,

C. nodosa es considerada como una especie eurobionte, con elevada plasticidad para aclimatarse y crecer en un

amplio espectro de condiciones ambientales (p.e. salinidad) (Drew 1978b), como zonas costeras abiertas (Enríquez

et al. 2004), zonas estuáricas (Pérez y Romero 1994) e incluso, lagunas costeras hipersalinas (Terrados y Ros 1991).

19

Figura 3. Praderas someras de angiospermas marinas mediterráneas: Posidonia oceanica y Cymodocea nodosa.

20

5.2. LA DESALINIZACIÓN DE AGUA DE MAR EN LA COSTA MEDITERRÁNEA ESPAÑOLA

La desalinización de agua de mar es uno de los principales recursos empleados para el abastecimiento hídrico de

regiones costeras áridas de todo el mundo. En la cuenca mediterránea, la industria de la desalinización ha experimen-

tado un notable crecimiento en las últimas décadas, y supone actualmente el 17% de la capacidad desaladora mundial

(Lattemann y Höpner 2008). Dicho crecimiento se ha mostrado especialmente significativo en España, la cual ocupa

posiciones a la cabeza de entre los países europeos que presentan una mayor instalación y actividad de plantas desalini-

zadoras, debido principalmente, a la irregularidad de precipitaciones y a ciertos impedimentos que restringen la explo-

tación de aguas subterráneas (p.e. contaminación agrícola, intrusiones de aguas marinas; Palomar y Losada 2010). En

este país la desalinización de agua de mar supone el 7% de la capacidad mundial (Medina San Juan 2001; Lattemann y

Höpner 2008). Los vertidos hipersalinos o salmueras resultantes de esta actividad industrial son vertidos al mar a través

de emisarios más o menos alejados de la línea de costa, en zonas en las que las praderas marinas de P. oceanica y C. nodosa

son particularmente abundantes (Fernández-Torquemada y Sánchez-Lizaso 2005. 2006; Ruiz 2005; Gacia et al. 2007).

5.3. ESTADO ACTUAL DE CONOCIMIENTO

Los primeros estudios realizados para determinar los límites de tolerancia al incremento salinidad de las principales

angiospermas marinas mediterráneas (Posidonia oceanica, Cymodocea nodosa y Zostera noltii), pusieron en evidencia

la sensibilidad diferencial de estas especies frente al estrés hipersalino, además de demostrar que tales diferencias eran

consistentes con sus diferentes estrategias ecológicas. En una primera aproximación basada en experimentos de labo-

ratorio de corta duración (10-15 días), se observó una reducción del crecimiento y la supervivencia de haces de P. oce-

anica a partir de 39-40 ups (100% de mortalidad a 50 ups), mientras que tales efectos sólo fueron aparentes en haces

de C. nodosa a partir de 41 ups (100% mortalidad a >56 ups) (Fernández-Torquemada y Sánchez-Lizaso 2005, 2011).

En una segunda aproximación, Gacia et al. (2007) estudiaron el efecto a largo plazo de un vertido de salmuera en una

pradera natural de P. oceanica; los resultados de estos autores apoyaron los límites de tolerancia propuestos en el primer

estudio, aunque no de forma inequívoca debido a posibles interacciones con otros factores ambientales alterados por

el vertido, como los nutrientes. Más tarde se realizó una tercera aproximación experimental, basada en la simulación

in situ de un incremento de la salinidad sobre una pradera de P. oceanica inalterada; sus resultados forman parte de la

presente tesis doctoral (capítulo 1) y, junto con los estudios anteriores, permitió establecer los criterios científicos para

el control del impacto de los vertidos de salmuera en este hábitat de las costas españolas (Sánchez-Lizaso et al. 2008).

21

Aunque el estudio de los rangos de tolerancia de C. nodosa y Z. noltii ha recibido menos atención que en el

caso de P. oceanica, algunos trabajos publicados han permitido establecer dichos límites para algunas poblaciones

de estas especies en el sureste peninsular (Pagés et al. 2010; Fernández-Torquemada y Sanchez-Lizaso 2011). Sin

embargo, en estas especies más plásticas, la sensibilidad al incremento puede estar condicionada no sólo por la

variabilidad geográfica, sino también por divergencias ecotípicas relacionadas con procesos adaptativos a los di-

ferentes ambientes que ocupan, como se ha demostrado entre poblaciones de una misma especie adaptadas a re-

gímenes diferentes de salinidad (Koch y Dawes 1991; Ye y Zhao 2003). Esta misma variación en la capacidad de

tolerar incrementos de la salinidad, ha sido también demostrada recientemente entre poblaciones mediterráneas

de C. nodosa, originarias de una laguna hipersalina (44 ups; Mar Menor, Región de Murcia) y de zonas costeras

abiertas cercanas con menor salinidad (37 ups; Fernández-Torquemada y Sánchez-Lizaso 2011). De la misma for-

ma, la tolerancia a condiciones de hipersalinidad de poblaciones mediterráneas de esta especie podría diferir de la

de poblaciones del Atlántico donde el régimen salino es muy diferente al de las aguas mediterráneas; sin embargo

no se dispone de estudios en esta línea en praderas de C. nodosa atlánticas peninsulares y de las Islas Canarias.

Otros factores que pueden condicionar la tolerancia de las especies a la salinidad y que tampoco han sido muy es-

tudiados en este contexto, son la estacionalidad (tanto del ciclo productivo como de las condiciones ambientales)

y la sinergia con otros factores que pueden verse alterados por los vertidos hipersalinos (Fernández-Torquemada y

Sánchez-Lizaso 2011). Adicionalmente, se han descrito diferencias ecotípicas y/o genotípicas entre praderas pro-

fundas y someras de P. oceanica y C. nodosa (Drew 1978b; Procaccini et al. 2001; Olesen et al. 2002; Miglaccio et

al. 2005) que podrían resultar también en importantes variaciones en su capacidad de tolerar el estrés hipersalino.

Respecto al conocimiento básico existente sobre las propiedades particulares de la fisiología, ultraestructura y

morfología, que explican la diferentes capacidades de las angiospermas marinas mediterráneas para tolerar el in-

cremento de la salinidad, éste (sin contar las contribuciones de esta tesis) representa una proporción muy peque-

ña del disponible para el conjunto de angiospermas marinas, expuesto en la Tabla 1 y comentado en el aparta-

do 4 de este capítulo. En este sentido, sólo dos estudios aportan cierta evidencia del efecto del estrés hipersalino

en la fotosíntesis (y respiración) de C. nodosa (Drew 1978a; Pagés et al. 2010). Por su parte, Gacia et al. (2007)

observaron alteraciones en el contenido de carbohidratos no estructurales en rizomas de P. oceanica, la señal iso-

tópica del carbono (δ13C) y la concentración de nutrientes (nitrógeno y fósforo) en sus tejidos, en relación a la

influencia de un vertido de salmuera. Por tanto, la comprensión de los mecanismos que acotan los rangos de to-

lerancia al estrés hipersalino de las angiospermas marinas mediterráneas a los diferentes niveles de organización

22

del ecosistema (molecular, fisiológico, morfológico, poblacional, etc), representa hoy día una importante laguna

de conocimiento hacia la que se han dirigido los objetivos de esta tesis doctoral, y que se detallan a continuación.

6. OBJETIVOS Y ESTRUCTURA DE LA TESIS

La presente Tesis Doctoral ha sido desarrollada a partir de los resultados obtenidos en el mar-

co de los proyectos OSMOGRASS I (Ref. 02YSGTB/2007/1.3), financiado por el Ministerio de Me-

dio Ambiente y Medio Rural y Marino, correspondiente al Programa Nacional de Ciencias y Tecnolo-

gías Medioambientales, y OSMOGRASS II (CTM2009-08413MAR), financiado por el Programa de

Investigación Fundamental no Aplicada del Plan Nacional de I+D+i del Ministerio de Economía y Competitividad.

El objetivo general del proyecto fue profundizar en el conocimiento básico las respuestas ecofisiológicas y vege-

tativas que determinan la capacidad de las angiospermas marinas mediterráneas (Posidonia oceanica y Cymodocea

nodosa) al estrés hipersalino. Se espera además, que este conocimiento contribuya a entender el alcance del im-

pacto de los vertidos hipersalinos (salmueras) de las plantas desalinizadoras en estos valiosos hábitat bentónicos y,

en consecuencia, al establecimiento de criterios científicos y objetivos de control y seguimiento de dicho impacto.

El desarrollo y consecución de dichos objetivos se planteó mediante dos aproximaciones experimentales:

i) simulación in situ del incremento de la salinidad en una pradera natural de P. oceanica (Capítulo 1),

ii) simulación experimental de la exposición de P. oceanica y C. nodosa a un incremento crónico de la salinidad, de-

sarrollada en un sistema de condiciones controladas de laboratorio a escala de mesocosmos (Capítulos 2, 3, 4 y 5).

A continuación se exponen los objetivos específicos de la tesis, y que han dado lugar a los 5 capítulos en que está

estructurada:

23

ESTUDIO DE LAS RESPUESTAS DE LA ANGIOSPERMA MARINA MEDITERRÁNEA POSIDONIA OCEANICA A INCREMENTOS DE

SALINIDAD SIMULADOS IN SITU (CAPÍTULO 1)

Como se ha explicado en el apartado 5.3, este trabajo fue realizado en el contexto de los estudios pioneros que

permitieron establecer los límites de tolerancia de las especies mediterráneas al incremento de la salinidad. En este

caso, se planteó una aproximación experimental complementaria, que corroborara los límites establecidos en los

estudios anteriores, y basada en: 1) la manipulación in situ de la salinidad, ii) análisis de las respuestas de las plantas

en su ambiente natural y iii) exposiciones al estrés hipersalino a escalas temporales intermedias (meses) que per-

mitieran observar la máxima expresión de dichas respuestas (incluyendo una posible recuperación). Para ello, se

empleó agua de mar con dos intensidades diferentes de incremento salino (+1 y +2.5 ups del valor medio ambiente,

37.6 ups), y preparadas a partir de la salmuera producida por una planta desaladora piloto. Las diferentes solucio-

nes hipersalinas fueron conducidas a una pradera de P. oceanica a través de un sistema de tuberías submarinas,

para su distribución en una serie parcelas experimentales (3 m2), cuyo perímetro se delimitó por unas paredes de

poliéster ancladas la fondo marino, y que mantenían la solución experimental simulando el efecto de estratificación

de la capa hipersalina en la parte media-basal de la pradera. A lo largo del periodo experimental, se midieron una

serie de descriptores morfométricos, fisiológicos y poblacionales en plantas control y afectadas por los vertidos,

con el fin de determinar los efectos de la salmuera en función de los diferentes grados de estrés aplicados, y sus

implicaciones para la determinación del umbral de tolerancia de P. oceanica a las condiciones de hipersalinidad.

ESTUDIO DE LOS EFECTOS DEL INCREMENTO DE SALINIDAD EN LA FOTOSÍNTESIS, CRECIMIENTO Y SUPERVIVENCIA DE P. OCEANICA

(CAPÍTULO 2)

A tenor de la experiencia acumulada durante el transcurso del estudio previo desarrollado en praderas

naturales, se descartó la opción de continuar la línea de investigación con experimentos in situ, altamente

costosos en términos de logística, tiempo y economía. Pero además, la necesidad de avanzar en aspectos

ecofisiológicos de las respuestas al estrés hipersalino, obligaba a adoptar aproximaciones experimentales en

las que aquellos factores diferentes del que se pretende manipular (salinidad), estuvieran más controlados que

en el medio natural. En efecto, la experimentación in situ planteaba dos problemas principales: i) la dificultad

de mantener estables los niveles de los tratamientos experimentales (debido, por ejemplo, a la acción del

oleaje y temporales), y ii) la imposibilidad de aislar el efecto del tratamiento de la influencia de otros factores

24

ambientales, cuya variación pudiera enmascarar o confundir las respuestas únicamente debidas a la salinidad.

La opción alternativa considerada más adecuada fue la realización de experimentos de laboratorio bajo con-

diciones controladas, lo que a su vez, planteaba nuevos problemas, ya que i) el mantenimiento de las especies

de angiospermas marinas como P. oceanica, se ha mostrado particularmente problemático en condiciones

de laboratorio, ii) se haría necesario mantener las plantas durante periodos lo suficientemente prolongados

(al menos 1 o 2 meses) que permitan la expresión de las respuestas aclimatativas y, por último, iii) se reque-

riría desarrollar un sistema a escala de mesocosmos, que permitiera un control riguroso de las condiciones

ambientales óptimas de las plantas. Así, el sistema de mesocosmos fue diseñado y desarrollado para tal fin

en colaboración con Emilio Cortés Melendreras, Director del Acuario Marino Municipal de la Universidad y

Ayuntamiento de Murcia, e instalado en los laboratorios del Centro Oceanográfico de Murcia del Instituto

Español de Oceanografía, emplazamiento donde se han llevado a cabo todos los trabajos presentados en esta

tesis. Los resultados del capítulo 2, junto con los presentados en los capítulos 3 y 4, corresponden al primer

experimento realizado en este sistema de mesocosmos experimental, desarrollado durante el periodo estacio-

nal de otoño-inverno de 2008. Los detalles técnicos del sistema de mesocosmos se explican en el Anexo 2.

Concretamente en el capítulo 2, se analizan las respuestas fisiológicas y vegetativas de plantas de P. oceanica

expuestas durante 47 días a distintos niveles de incrementos de salinidad: 37 (control), 39, 41 y 43 ups. Dichos

resultados describen los efectos de los tratamientos hipersalinos en descriptores obtenidos a partir de curvas de

Fotosíntesis-Irradiancia, balance de carbono, técnicas de fluorescencia (PAM), análisis de pigmentos fotosintéti-

cos y de propiedades ópticas foliares (absorbancia de luz PAR por tejido foliar). Además de dichos descriptores

fisiológicos, se analizaron las variaciones de descriptores del tamaño, crecimiento y supervivencia de los haces. Los

resultados de este capítulo corroboran los límites de tolerancia de P. oceanica a nivel fisiológico, y aportan evidencias

relevantes sobre los mecanismos que explican la elevada sensibilidad de esta especie al incremento de la salinidad.

ESTUDIO DE LOS EFECTOS DEL INCREMENTO DE SALINIDAD EN FOTOSÍNTESIS, CRECIMIENTO Y SUPERVIVENCIA DE C. NODOSA

(CAPÍTULO 3)

Como en el caso del capítulo 2, este capítulo pretende caracterizar las repuestas fisiológicas y vege-

tativas de C. nodosa frente a diferentes grados de incremento de la salinidad simulados en el mesocos-

mos experimental, y en particular las respuestas relacionadas con la fotosíntesis, la composición de pig-

25

mentos y ciertas propiedades ópticas de las hojas. Para este experimento se emplearon plantas de praderas

mediterráneas de ambientes costeros abiertos, es decir, adaptadas a las mismas condiciones que las plantas de

P. oceanica empleadas en el cap. 2 (salinidad media estable de aprox. 37 ups). El diseño experimental fue el mis-

mo que el empleado para P. oceanica en el capítulo 2, con lo cual se pretende discutir la presumible mayor to-

lerancia fisiológica de C. nodosa, a través del análisis comparativo de los resultados de ambos experimentos.

ESTUDIO DE LOS EFECTOS DEL INCREMENTO DE SALINIDAD EN LAS RELACIONES HÍDRICAS DE P. OCEANICA Y C. NODOSA

(CAPÍTULO 4)

En este capítulo se estudian las relaciones hídricas foliares de P. oceanica y C. nodosa y los efectos del incremento

de salinidad en las mismas, aspecto clave para entender el comportimiento fisiológico de estas plantas marinas, nunca

antes examinado para especies mediterráneas. Los descriptores que reflejan el comportamiento de las relaciones

hídricas son el potencial hídrico (Ψw), potencial osmótico (Ψπ), y la presión de turgor (Ψp), y fueron obtenidos me-

diante técnicas psicrométricas. Se analizó además, el papel de la concentración de algunos solutos orgánicos (i.e. car-

bohidratos no estructurales y aminoácidos libres) en los procesos de ajuste osmótico en respuesta al estrés hipersalino.

ANÁLISIS DE LA IMPORTANCIA DE LA VARIACIÓN INTER- E INTRA-ESPECÍFICA EN LA DETERMINACIÓN DE LAS TOLERANCIAS

FISIOLÓGICAS DE P. OCEANICA Y C. NODOSA FRENTE AL INCREMENTO DE SALINIDAD (CAPÍTULO 5)

Este capítulo corresponde a un experimento de mesocosmos realizado con ambas especies durante el período es-

tacional de primavera-verano de 2009. En este caso, plantas de P. oceanica y C. nodosa fueron expuestas a dos trata-

mientos salinos (i.e. 37-control; 43 ups-hipersalino) durante un período experimental de 62 días. El objetivo principal

que se persiguió con este estudio fue el de evaluar la existencia de variación intra-específica en la plasticidad fisioló-

gica de ambas especies, y en sus capacidades de tolerar el incremento de la salinidad. Así, para cada especie y condi-

ción salina, se emplearon simultáneamente plantas de praderas situadas en los extremos del gradiente batimétrico de

distribución de ambas especies en la zona y, por tanto, adaptadas a condiciones ambientales muy diferentes (someras

versus profundas). En este experimento se analizaron las respuestas de las plantas a múltiples niveles (fotosíntesis, re-

laciones hídricas, osmolitos orgánicos, etc), se cuantificó el grado de plasticidad de dichas respuestas a nivel de cada

variable, ecotipo y especie, y se discuten sus consecuencias a nivel del crecimiento y supervivencia del organismo.

26

La parte final de la presente Tesis Doctoral corresponde una sección de Discusión General, donde

se aporta una visión global e integrada de las respuestas ecofisiológicas de P. oceanica y C. nodosa al estrés hi-

persalino descritas en esta tesis frente a incrementos de salinidad, sus relaciones con mecanismos de toleran-

cia, variación intra- e inter-específica, y su eficacia para el mantenimiento de la adecuación biológica a nivel de

organismo. Finalmente se presentan las principales Conclusiones de los diferentes estudios realizados, y se ad-

juntan dos anexos: Anexo 1, donde se presentan los datos de los descriptores empleados en los experimen-

tos, pero obtenidos en plantas de las praderas naturales originarias de ambas especies, y que se han empleado

con el propósito de comparar el estado de las plantas en condiciones naturales y de laboratorio; y el Anexo 2,

donde se presenta una descripción técnica detallada del sistema de mesocosmos, su diseño y funcionamiento.

129 ABSTRACT

29 INTRODUCTION

32 MATERIAL AND METHODS

38 RESULTS

50 DISCUSSION

Juan Manuel Ruiz Fernández, Lázaro Marín-Guirao and José Miguel Sandoval Gil. 2009Responses of the Mediterranean seagrass Posidonia oceanica to in situ simulated salinity increase

Botanica Marina 52: 459-470

29

1.1. ABSTRACT

The dominant Mediterranean seagrass Posidonia oceanica inhabits sublittoral environments

with very stable salinity regimes; the species is considered highly sensitive to even moderate in-

creases in salinity caused by hypersaline effluents (brine) from desalination plants. We analyzed

the effect of salinity increase on seagrass vitality and survival by means of an in situ mesocosm ex-

periment. To this end, we used the brine (70-75 psu) produced by a pilot desalination plant, which

was diluted with seawater to obtain brine solutions of 1 psu (high salinity increase, HS) and 2.5 psu

(very high salinity increase, VS) over the mean natural salinity (37.5 ± 0.16 psu) and then inters-

persed a set of experimental units in a nearby P. oceanica meadow. At the end of the experimental

period (3 months), these treatments had produced differential effects on all seagrass descriptors,

i.e., intense and significant in the VS experimental units and more modest (or even negligible) in

the HS ones. Seagrass meadow declined through a significant decrease in shoot density in both

HS (12.4 ± 3.4 %, mean ± SE) and VS (18.5 ± 3.05 %, mean ± SE) in comparison to control expe-

rimental units (< 5%). Surviving shoots had reduced size and leaf growth rate than control plants,

but more generally a reduction in the photosynthetic leaf surface caused by the increase in the

proportion of necrotic leaf area (up to 60% in VS treatments). Non-structural carbohydrate (so-

luble and reserve) concentrations decreased in rhizomes, probably in relation to physiological os-

moregulation processes. All these responses support the hypothesis that the threshold of salinity

tolerance of P. oceanica is very close to the upper limit of its natural salinity range (ca. 38 psu).

1.2. INTRODUCTION

Seawater desalination by reverse osmosis (RO) is nowadays a growing industry considered

as an alternative and efficient solution for alleviating the rising demand for water in many arid

coastal regions worldwide (Morton et al. 1996), but it is not free of significant environmental risks.

Approximately 40-50% of the total volume of seawater used by RO desalination plants results in

a hypersaline (40-70 psu) waste (brine) which is diverted to the adjacent marine ecosystems and

has a high potential for altering benthic marine communities (Chesher 1975; Morton et al. 1996;

Castriota et al. 2001; Einav et al. 2002; Fernández-Torquemada et al. 2005a; Gacia et al. 2007).

1R E S P O N S E S O F T H E M E D I T E R R A N E A N S E A G R A S S P O S I D O N I A O C E A N I C A T O I N S I T U S I M U L A T E D S A L I N I T Y I N C R E A S E

30

Brine discharges may sporadically contain chemical substances (e.g. biocides) and organic matter

(Einav et al. 2002), but high salt concentrations is in all probability the major stressor for benthic

communities.

Seagrass meadows are dominant and highly productive communities in estuarine and ma-

rine coastal habitats of many tropical and temperate areas (Green and Short 2003). They pro-

vide valuable ecological and socio-economic functions and services to the coastal ecosystem,

but increasing human pressures have dramatically reduced their distribution and condition du-

ring recent decades (Phillips and Durako 2000). Increases in nutrient availability, sediment

loading or light attenuation are the factors that have received most attention as direct or indi-

rect causes of reported seagrass declines. Salinity (alone or in combination with other factors)

is another relevant ecological factor in determining the structure, function and distribution of

seagrass communities (McMillan and Moseley 1967; Walker and McComb 1990; Montagne

and Ley 1993; van Katwijk et al. 1999; Kahn and Durako 2006); seagrass meadows are suscep-

tible to changes in salinity regimes induced by modifications of freshwater inflows (McIvor et al.

1994) or by hypersaline effluents from desalination plants (Chesher 1975; Tomasko et al. 2000),

but knowledge of the tolerance of seagrass species to salinity fluctuations is still very limited.

There is a certain amount of quantitative information on the osmoregulatory capacity of se-

agrass species and their tolerances to salinity, mostly concerning species living in coastal environ-

ments with fluctuating salinity regimes (estuaries, coastal lagoons and closed embayments; see

Touchette 2007 for a review). As in many aquatic macrophytes, osmoregulatory adjustments in

seagrasses include, among others, changes in ion concentrations, synthesis of compatible solutes

and changes in cell ultrastructure (Tyerman et al. 1989; Iyer and Barnabas 1993; Adams and Bate

1994a; Pak et al. 1995). These mechanisms enable seagrasses to cope with and acclimatise to

gradual and/or pulsed changes in seawater salinity, but when salinity changes persist or become

more intense, the adverse effects of ionic and osmotic stress on metabolism can compromise the

productivity and survival of seagrass species (Ralph 1998; Hellblom and Björk 1999; Kamermans

et al. 1999; Fernández-Torquemada et al. 2005b; Kahn and Durako 2006; Koch et al. 2007b).

Therefore, knowledge of the salinity thresholds of seagrass species is crucial to understanding and

31

predicting their capacity to withstand the more drastic and chronic changes of salinity regimes

caused by human impacts, such as hypersaline discharges from desalination plants.

The recent increase in the desalination of seawater along the Mediterranean coast of Spain

(Medina San Juan 2001) has given rise to major concern about the ecological consequences of

the impact of brine effluents on the endemic dominant seagrass Posidonia oceanica (L.) Delile

(Sánchez-Lizaso et al. 2008), whose extent and condition have already been altered by other

human activities during recent decades (Ruiz et al. 2001; Ruiz and Romero 2003). P. oceanica

meadows grow under a stable and narrow salinity range (36.5-38 psu in the Western basin and

38-39.5 in the Eastern basin; Klein and Roether 2001) and hence are expected to have a reduced

capacity to respond to and withstand salinity increases caused by brine effluents, in comparison

with estuarine species. To date, only two studies have evaluated this hypothesis, both confirming

the high sensitivity of this seagrass species to even a small increase in salinity over its natural ran-

ge. Both studies obtained consistent results using different experimental approaches and suggest

critical tolerance thresholds of 39 psu (short-term, 15-day laboratory experiments; Fernández-

Torquemada and Sánchez-Lizaso 2005) and 39.3 psu (long-term, field study of a P. oceanica

meadow exposed to the impact of brine discharges over a six-year period; Gacia et al. 2007).

In the present study we performed a field mesocosm experiment to simulate the effects

of an increase in salinity caused by brine effluents on a P. oceanica meadow. This approach is

complementary to those used in previous studies, because it allows: i) in situ manipulation

of salinity, ii) the study of intact, whole plants in their natural environment, and iii) responses

that are assessed at medium time-scales (months). Nonetheless, manipulation of environ-

mental variables in the field is by no means a trivial task and requires considerable effort. In

this study, we simulated hypersaline conditions in situ on P. oceanica plots through a com-

plex experimental system that conducted and delivered the brine produced in a pilot des-

alination plant to an adjacent natural seagrass stand. Several seagrass descriptors were moni-

tored in 3 m2 experimental units exposed to two levels of salinity increase during a 3-month

period and compared with unmanipulated plants maintained under natural salinity conditions.

32

1.3. MATERIAL AND METHODS

1.3.1. STUDY SITE

The experiment was conducted in a locality on the south-eastern coast of Spain (37º 49.427´

N, 0º 44.827´ W; San Pedro del Pinatar, Murcia Region; Fig. 1.1). A pilot 200 m3 s-1 desalination

RO plant was deployed by the Spanish public company ACSEGURA (Murcia, Spain) in the

harbour facilities. Brine produced was used to obtain experimental hypersaline solutions. Experi-

ments were conducted in a dense (695 ± 12.1, mean ± SE shoots m-2) and shallow (10-12 m depth)

Posidonia oceanica meadow located 350 m from the harbour facilities (hereafter, “experimental

area”; Fig. 1.1). Previous surveys determined that this distance was sufficiently far from the influen-

ce of the impact of the harbour on marine environment and seagrass condition (data not shown).

1.3.2. EXPERIMENTAL SET-UP

In May 2001, twelve 3 m2 quadrat plots (experimental units) were deployed in dense se-

agrass patches in the experimental area selected. They were evenly distributed (separated from

each other by 5-10 m) covering a total surface meadow area of approximately 5000 m2. All

units were delimited, positioned and labelled. Three sets of four randomly selected units were

assigned to the following experimental treatments: i) natural (unmanipulated) salinity, ii) high

salinity increase (HS) and iii) very high salinity increase (VS). Salinity increments are relative to

the mean natural salinity (see below). Samples of seagrass and seawater variables in all experi-

mental units were taken between May 2001 and December 2002, covering an annual growth

cycle characteristic of Posidonia oceanica. The whole study period was divided into three suc-

cessive phases: i) pre-treatment, i.e., before the start of experimental salinity treatments (426

days, from May 2001 to June 2002), ii) experimental period, i.e. during experimental brine

addition (ca. 100 days, from July to October 2002), and iii) post-treatment phase, i.e. after

cessation of experimental brine addition (ca. 80 days, from October to December 2002).

33

The two selected levels of salinity increment were 1 psu over the mean natural salinity for the

HS treatment and 2.5 psu over the mean natural salinity for VS treatment. Therefore, considering

a reference salinity value of 37.6 psu for the study area (time series 1996-2001; Spanish Institute

of Oceanography unpublished data), the expected mean salinity for the HS treatment was 38.6

psu, that for the VS treatment being 40.1 psu (Table 1.1). These are just below and above the

upper salinity threshold established for P. oceanica from previous studies (Fernández-Torque-

mada and Sánchez-Lizaso 2005; Gacia et al. 2007). To attain these hypersaline conditions, brine

produced by the pilot plant (70-75 psu) was diluted with seawater; mixing was performed in two

independent 2000 L tanks (placed close to the shoreline) to which seawater was supplied by a

water pump. From each tank, diluted brine was channelled through a 20 cm diameter underwater

pipeline to the experimental area, where it divided into four smaller (6 cm diameter) pipes (Fig.

1.1), each leading into its corresponding HS or VS experimental unit following the experimental

design previously assigned (see above).

Brine plumes from desalination plants form a dense and persistent hypersaline layer over the

sea bottom covering the basal part of photosynthetic tissues of P. oceanica shoots. To simulate

this “layer effect”, all experimental units (including the Control treatment) were surrounded by a

0.25 m high topless cage made of impermeable plastic (polyester) cloth, sealed at its base to the

seabed using small iron pegs pushed into the sediment (Fig. 1.2). Plastic cages were installed in

June 2001 and maintained throughout the study period. The possible interaction of the structure

with the plants and their environment was an issue of major concern in this experiment. Firstly, it

was designed to have an elastic behaviour to minimise physical interactions with water movement

(e.g. erosion-sedimentation). Secondly, care was taken to prevent the encasement of the plants

interfering with fluxes of nutrients and CO2, as cage dimensions allowed most of the leaf biomass

(ca. two thirds of the top canopy) to be allocated out of the cage. Even so, the existence of pos-

sible procedural artefact effects on meadow and shoot descriptors was evaluated by comparison

of caged control units with another four un-caged control units randomly interspersed within the

experimental area. None of the seagrass descriptors analysed showed significant differences bet-

ween caged and un-caged units over the whole study period [see analysis of variance (ANOVA)

test used for each seagrass variable in the ‘Satistical analysis’ section], and therefore we assume

negligible artefact effect in our experiment. For the sake of simplicity, these data are not shown.

34

Care was also taken by the pilot plant operators to avoid or minimise the use of chemical

substances usually employed in RO processes. It is usually assumed that the high dilution ratios

reduce their presence to very low concentrations (Einav et al. 2002) and salinity may be consi-

dered the major factor responsible for the observed effects of brine on seagrasses. Nonetheless

some water quality variables were directly (pH and light) or indirectly (nutrients) measured in

the experiment to assess the possible influence of these substances (see the following sections).

1.3.3. ENVIRONMENTAL VARIABLES

To determine natural salinity (and temperature) conditions, six seawater samples were taken

from just above the seagrass canopy with a frequency of 1 to 3 days each week from February

to September 2002; these samples were always taken just outside the experimental area so as to

avoid possible interferences with brine delivered to the experimental units during the experimen-

tal period.

To assess experimental salinity conditions in HS, VS and Control units, seawater sam-

pling was performed on a total of 25 dates throughout the experimental period, i.e., with

a frequency of 1 to 3 days, depending on weather conditions. To this end, seawater samples

were taken by divers in the nodes of a 1 m2 quadrat subdivided into four sub-quadrats (i.e.

n = 9 samples) placed at the centre of the experimental unit. Samples were always taken at

the height of the basal part of P. oceanica leaves, i.e. 5-10 cm above the sea bed. For each

sampling date, salinity means (and standard errors) are estimated for each experimental unit

(n = 9 replicates) and treatment (n = 4 replicates). In addition, seawater samples were taken

at different heights above the sea bed (0, 5, 10, 20 and 50 cm) to characterize the formation

of the brine layer in the experimental units (i.e. vertical stratification). Within each unit, this

sampling was performed at four points arranged at regular intervals following one of the cen-

tral axes of the unit area. It was repeated on four dates between August and September 2002.

35

Figure 1.1. Posidonia oceanica: location of the study site and the experimental area, indicating the position of the pilot desalination plant and 2000 L tanks employed for brine dilution. Also shown are the routes of underwater pipelines deployed to conduct experimental hypersaline solutions HS and VS to the experimental area. The shaded area represents the distribution of the Posidonia oceanica meadow.

Figure 1.2. Posidonia oceanica: image of the seagrass experimental unit (3 m2) surrounded by the topless plastic cage.

36

Salinity and temperature were measured using a conductivity meter ORION 125Aplus con-

nected to a DuraProbeTMConductivity Cell (Orion Research Inc., Beverly, USA). The accuracy

of salinity measurements was calibrated with a high precision (± 0.001) Beckman RS7B induc-

tion conductivity-salinity meter (Beckman Instruments Inc., Fullerton, USA). Light availability

and pH in seawater samples were also determined to assess possible alterations of water quality

by chemical additives or particulate organic matter present in brine effluent. pH was measu-

red using a CRISON 506 pH/mV meter (Crison Instruments, Barcelona, Spain). Continuous

illuminance measurements (units of lux; 1.0 µmol photons m-2 s-1 ≈ 40.0 lux) were made with

underwater ONSET Stowaway LI logger photometers (Onset Corp., Bourne, USA) placed in

three randomly selected experimental units, each belonging to one of the three experimental

treatments. Mean daily-integrated values (lm m-2 d-1) were estimated for each treatment and

compared by one-way ANOVA. No differences in incident light were found between treatments

(P > 0.05, F= 0.895, d.f. = 2, 282, n = 95; days between June and September 2002), those we

consider this variable is without relevance for our study and light data are not shown here.

1.3.4. SEAGRASS RESPONSE VARIABLES

At the meadow level, two seagrass variables were measured: a) the percentage of meadow

cover and b) the net change in shoot density. To this end, in May 2001, a 2500 cm2 fixed quadrat

was placed at the centre of each experimental unit. Meadow cover was measured using a 2500

cm2 PVC frame divided into 25 cm2 sub-quadrats superimposed on the fixed quadrat; the percen-

tage of meadow cover was calculated from the number of sub-quadrats with seagrass presence.

This descriptor was measured in December 2001 (i.e. pre-treatment phase) and exactly one year

later (December 2002, i.e. post-treatment phase). To estimate net changes in shoot density, the

number of shoots in fixed quadrats was counted at the beginning and at the end of each of the

following time intervals: May 1st - December 15th 2001 in the pre-treatment phase, and June 1st -

August 28th - October 10th - December 13th 2002 in the experimental period and post-treatment

phase. Differences between final and initial counts of each quadrat and time interval were norma-

lised to the initial shoot density and expressed as the percentage of net change in shoot density;

negative values indicate shoot decline due to mortality and/or reduced shoot branching.

37

Shoot size (as biomass), the number of leaves per shoot and leaf growth were measured

in shoots collected on five sampling occasions in the pre-treatment phase (August and Oc-

tober 2001; January, March and June 2002) and on two sampling occasions during the ex-

perimental period (August and October 2002). Collected shoots (n = 8 per experimental

unit) had been previously marked to determine leaf growth rate as new biomass (mg DW

shoot-1 day-1) following the method adapted from Zieman (1974) (see Romero 1989). For

each marked shoot, the sections of leaves below the mark (new growth) were separated,

dried and weighed, the number of leaves counted and shoot size estimated as total shoot leaf

biomass (g DW shoot-1). In August 2002, the length and width of necrosis marks on leaves

were also measured to quantify the proportion of necrotic photosynthetic tissue per shoot.

Non-structural carbohydrates (NCs) and total nitrogen content were determined in

dried (finely ground) leaf and rhizome tissues obtained from shoots collected in the experi-

mental units (i.e. n = 8 replicates per experimental unit, treatment and sampling time). NCs

(soluble and starch) were determined on rhizome fragments (4 to 5 cm from rhizome apex)

using the method described in Alcoverro et al. (2001), based on Yemm and Willis (1954).

Soluble carbohydrates were solubilised from dry and ground tissues by sequential extrac-

tions in 95% (v/v) ethanol at 80ºC for 15 min. Ethanol extracts were evaporated and the re-

sidues dissolved in de-ionised water for sugar analysis by the spectrophotometric resorcinol

assay. Starch was extracted overnight from the ethanol-insoluble residue in 1 N KOH and

determined spectrophotometrically using an anthrone assay. Both analyses were standardi-

sed to sucrose. Total nitrogen content (% DW) was measured in dried plant tissues (young

leaves and rhizomes) to evaluate any effect of brine treatments on the plant N metabolism

and to highlight a possible influence of nutrients present in brine effluents. Total N content

was determined as %N per dry weight unit using a Carlo-Erba CNH elemental autoanalyser.

38

1.3.5. STATISTICAL ANALYSIS

Differences in mean seawater salinity and pH among experimental treatments (four levels:

HS, VS, Control and “Natural”) were analyzed by one-way ANOVA. A two-way ANOVA was

used to assess significant treatment effects (three levels: HS, VS and Control) on meadow des-

criptors (percentage of meadow cover and net change in shoot density), before and after the

experimental period (two levels: December 2001 and December 2002). We used univariate

methods of repeated measures ANOVA (one-factor rmANOVA; Crowder and Hand 1990)

to assess the effects of salinity treatments on the time course of shoot features and physiologi-

cal descriptors in experimental units (i.e. subjects) over the entire period studied. Experimen-

tal treatments (HS, VS and Control) were the between-subject factor and time (i.e. sampling

events) was the repeated-measures (within-subject) factor (six levels for leaf growth rate and

seven levels for the remaining variables). The effect of treatments on leaf necrosis measured

in August 2002 was analysed using one-way ANOVA. Prior to application of factorial ANO-

VA, all data were checked for normality and homoscedasticity and, when necessary, transfor-

med. In the case of rmANOVA, the assumption of sphericity was assessed using Mauchly’s

sphericity test and whenever necessary, corrected degrees of freedom and significance levels

(Huynh-Feldt adjustment) were used. Post-hoc mean comparisons (Student-Newman-Keuls,

SNK; Zar 1984) were performed on the between-subject factor to identify specific treatment

level(s) causing significant effects. All treatment effects were considered significant at P < 0.05.

1.4. RESULTS

1.4.1. NATURAL AND EXPERIMENTAL CONDITIONS

In the experimental area, water temperature followed a typical marked seasonality with a

minimum value in winter (13ºC) and a maximum value in summer (28ºC). Mean natural salinity

did not follow any seasonal pattern and showed very low variation through the entire period

studied with a minimum value of 37.29 and a maximum of 38.05 psu, i.e. total range ampli-

tude of 0.7 psu (Table 1.1 and Fig. 1.3). During the experimental period mean natural salinity

39

(37.59 ± 0.16 psu) did not differ from that observed in the entire study period nor from long-term

time series, and was not significantly different from those values measured in the controls (Table

1.1), suggesting a negligible influence of brine injected in neighbouring HS and VS experimental

units. Mean salinities of the HS (38.4 ± 0.27 psu) and VS (39.22 ± 0.80 psu) treatments were

significantly higher than those of natural and control seawater and were significantly different

from one another (SNK, P < 0.0001; Table 1.1 and Fig. 1.3). These values were lower than tho-

se previously established for experimental salinity treatments (see material and methods section

and Table 1.1) and represented actual salinity increase of 0.6 and 1.6 psu above mean natural

salinity, instead of the expected 1 and 2.5 psu for HS and VS, respectively. The coefficient of

variation of salinity values in HS (range 37.36-37.93 psu) and VS (37.69-41.60 psu) treatments

during the experimental period were, respectively, 2.5 and 5.6 times higher than that of mean

natural salinity (range 37.36-37.93 psu) (Table 1.1). This clearly indicates a highly fluctuating sa-

linity regime in HS and VS treatments in comparison to the more stable salinity in natural and

control experimental units (Fig. 1.3). Extreme observations in the VS treatment (40-41.5 psu)

were caused by a malfunction of the experimental system over 3 days. Salinity remained constant

through the vertical profile in the control experimental units (Fig. 1.4), but increased at 10-20 cm

height over the bottom in HS and VS experimental units, confirming the formation of a hyper-

saline layer. This layer was disrupted on days with rough weather; this disruption is probably the

major source of salinity fluctuations in HS and VS treatments during the experimental period

(Fig. 1.3, Table 1.1). Spatial variability of salinity within the experimental units (i.e. among repli-

cates) may also account for this temporal variability, but probably to a minor extent, based on

its low coefficient of variation (0.62 ± 0.065 % and 0.27 ± 0.11 % for HS and VS treatments).

Mean pH values were slightly lower in the VS treatment (Table 1.1), but no signi-

ficant differences were detected between treatments (one-way ANOVA, P > 0.05).

40

Figure 1.3. Temporal variability in mean salinity measured in natural seawater (crosses) and in control (empty circles) and hypersaline (black circles) treatments HS (a) and VS (b). Values are means ± SE, n = 4.

41

Table 1.1. Mean (SE) seawater salinity established for experimental salinity treatments (expected values) and real values measured during the experimental period in the experimental units (observed values).

Figure 1.4. Vertical profile of mean salinity ± SE (n = 4) in experimental units of each experimental treatment between0 and 50 cm above the sea bottom.

42

1.4.2. SEAGRASS VARIABLES

The experimental treatments HS and VS had a significant effect on the meadow cover and

the net change of shoot density measured in 2500 cm2 fixed quadrats (two-way ANOVA; Table

1.2, Fig. 1.5). For both variables, no significant differences were found between treatments in the

pre-treatment period (SNK, Table 1.2), nor for control between the pre-treatment (December

2001) and post-treatment (December 2002) phases. On the other hand, significant differences

between brine treatments (HS and VS) and controls were evident after the experimental period

in 2002 for both variables, as expressed in the significant interaction term in the two-way ANO-

VA (Fig. 1.5, Table 1.2). Meadow cover was significantly reduced by approximately 40% both

in HS and VS treatments in relation to control treatments. Shoot density decreased by 12-14%

relative to initial shoot density (May 2002) for both treatments at the end of the experimental

period (October 2002) and further decreased (18.5%) in the VS treatment three months af-

ter the experimental treatments ceased (i.e. post-treatment phase, December 2002) (Fig. 1.5).

Temporal variation of selected shoot variables followed the seasonal annual pattern des-

cribed for P. oceanica meadows of the Spanish Mediterranean coast (e.g. see Alcoverro et al.

1995 and Annex 1; Fig. 1.6). Seasonal effects represented the major source of variation for all

selected variables in the entire period studied (“Time” in Table 1.3). In the pre-treatment phase

(May 2001-June 2002), no significant differences were detected between experimental units

previously assigned to the three experimental treatments for any variable and any sampling time

(T1-T5; SNK, P > 0.05). On the other hand, significant effects were observed for the HS and

VS treatments during and after the experimental period (July-October 2002; T6-T7 in Table

1.3) in comparison to control plants (Fig. 1.6). At the end of the experimental period, shoot

size mass was reduced by 17% (not statistically significant) and 66% in HS and VS treatments,

respectively, in comparison to controls. The number of leaves per shoot was initially reduced

only in the VS treatment, but at the end of the experimental period, the mean value of this

variable in HS treatments was also significantly lower than in controls (18 and 25%, respecti-

vely). Leaf growth declined moderately (26%), but significantly at the end of the experimen-

tal period, but only in plants from the VS treatment. During the experimental period (August

43

2002), the percentage of leaf necrosis was 2.3-fold higher in shoots from the HS treatment and

10.3-fold higher in shoots from the VS treatment when compared to control (5.6 ± 1%) (Fig. 1.7).

Concentrations of NCs in P. oceanica rhizomes (Fig. 1.8) followed the characteristic seasonal

pattern reported for this seagrass species, with maximum mean values in late summer and autumn

(Alcoverro et al. 2001; Annex 1). This seasonal variation was more pronounced for starch than

soluble sugars. Experimental treatments caused significant reductions of both soluble and reserve

(starch) carbohydrate concentrations (relative to control) only during the experimental period,

precisely when carbohydrate concentrations were at their highest (August 2002, Fig. 1.8; T6,

post-hoc SNK test, Table 1.4). This reduction was 29-36% for soluble sugars and approximately

25% for starch, with no significant differences between HS and VS treatments. Such differen-

ces disappeared at the end of the experimental period due to the reduction in carbohydrate

concentrations in control plants (Fig. 1.8), probably reflecting the use of this carbon source for

the typical growth of new leaves in autumn (Alcoverro et al. 2001). Nitrogen concentrations in

rhizomes were consistently higher than in leaves (Fig. 1.9), in accordance with the role of the

rhizome as a storage organ in this seagrass species; significant temporal variability (Table 1.4)

reflects seasonal variation in external N availability (Annex 1), but also mobilisation of internal

resources to support leaf growth or retranslocation from leaves (Invers et al. 2002). No signifi-

cant differences in the nitrogen content of seagrass tissues (leaves and rhizomes) were observed

in relation to experimental treatments during the experimental period (Table 1.4 and Fig. 1.9).

44

Figure 1.5. Posidonia oceanica: means ± SE net shoot mortality estimated for each experimental treatment in the different phases of the study period (see materials and methods section). Estimates of net shoot mortality in the pre-treatment phase are referenced to the initial shoot counts measured in May 2001 and those obtained in the experimental period and post-treatment phase are referenced to initial shoot counts measured in June 2002. HS, high salinity increase; VS, very high salinity increase.

Table 1.2. Posidonia oceanica: summary of two-way ANOVA performed to assess effects of experimental treatments (control, HS and VS) on meadow cover and net shoot mortality.

45

Figure 1.6. Posidonia oceanica: variation in (a) shoot size, (b) number of leaves and (c) leaf growth rate (means ± SE) through the entire period studied for each experimental treatment: control (triangles, dotted line), high salinity increase (HS; circles, solid line) and very high salinity increase (VS; squares, dashed line).The horizontal grey bars indicate the duration of the experimental period.

46

Figure 1.7. Posidonia oceanica: proportion (means ± SE) of necrotic leaf area by treatments in August 2002. HS, high salinity increase; VS, very high salinity increase.

Table 1.3. Posidonia oceanica: summary of the repeated measures ANOVA to assess the effect of the experimental treatments (control, HS and VS) on the selected shoot features measured in experimental units at different sampling times (T1-T7) during the entire period studied.

47

Table 1.4. Posidonia oceanica: summary of the repeated measures ANOVA performed to assess the effect of the experimental treatments (control, HS and VS) on non-structural carbohydrates and nitrogen content measured in tissues in experimental units and sampling times (T1-T7).

48

Figure 1.8. Posidonia oceanica: temporal variation in (a) soluble and (b) reserve (starch) non-structural carbohydrates (means ± SE) throughout the entire period studied for each treatment: control (triangles, dotted line), high salinity increase (HS; circles, solid line) and extremely high salinity increase (VS; squares, dashed line).

49

Figure 1.9. Posidonia oceanica: temporal variation in total nitrogen content in (a) leaves and (b) rhizomes (means ± SE) throughout the entire period studied for each experimental treatment: control (triangles, dotted line), high salinity increa-se (HS; circles, solid line) and very high salinity increase (VS; squares, dashed line).The horizontal grey bars indicate the duration of the experimental period.

50

1.5. DISCUSSION

Results obtained in this field experiment provide new evidence to support the hypothe-

sis that the Mediterranean seagrass P. oceanica is highly sensitive to an increase in salinity.

Despite the high temporal variability of mean salinity, measurable differences were obtai-

ned between brine treatments (HS= 38.4 ± 0.27 psu and VS= 39.2 ± 0.80 psu) and control

(natural) conditions. These values were lower than but close to those initially fixed for the

experiment (i.e. expected values in Table 1.1), indicating efficacy of the experimental de-

sign and set-up in simulating in situ salinity increase treatments. Temporal variability of mean

salinity in brine treatments mainly reflected the dilution of the hypersaline layer by hydro-

dynamic forces (e.g. orbital currents), as described for brine plumes discharged by desali-

nation plants in shallow coastal habitats (e.g. Fernández-Torquemada et al. 2005a; Gacia

et al. 2007). Therefore, although it was not possible to maintain stable salinity conditions

in our field experiment, this may well reflect the behaviour of real brine effluents in nature.

Seagrass structure and vitality were significantly affected in plants exposed to brine

treatments over three months between summer and autumn. As explained in the previous sec-

tions, no artefactual effects of experimental structures (i.e. plastic cages) on seagrass variables

were detected, and there were no effects of physico-chemical properties of brine effluents other

than salt (turbidity, nutrients and pH); therefore, we can assume that salinity increase is the

major factor responsible for the reported plant responses. Decrease in shoot density, reduced

shoot size and leaf growth and the increase in leaf necrotic surface area are all well documented

symptoms of hypersaline stress reported from studies performed both in the laboratory (e.g.

Fernández-Torquemada and Sánchez-Lizaso 2005; Koch et al. 2007b) and in the field (e.g.

Tomasko et al. 2000; Gacia et al. 2007). The intensity of such effects is also consistent with the

experimental level of salinity increase, in spite of the temporal variability that we detected. Plants

from HS treatments showed significant but very slight effects in the number of leaves per shoot,

leaf necrosis and shoot density compared with the more intense effects in these and other varia-

bles (shoot size and leaf growth) in plants exposed to VS treatment. Shoot decline was modest,

but enough to cause a deterioration of seagrass structure at the meadow level (i.e. percentage of

51

meadow cover). Decreases in shoot density (12.4-18.5 % in HS and VS treatments, respectively)

were of the same order as those observed in laboratory experiments (Fernández-Torquemada

and Sánchez-Lizaso 2005) at similar salinities (38-41 psu). However, in these laboratory expe-

riments plants were exposed to hypersaline conditions for only 15 days, whereas in our field

experiment shoot decline continued for a further three months after the experimental period

(i.e. a total of six months). In situ field experimentation allows the clonal integrity of the plants

and the sediment-root system to be maintained, which may compensate for or buffer the effects

of salinity stress much better than plant fragments maintained under aquarium conditions.

This high sensitivity of P. oceanica to hypersaline conditions might be considered an unusual

response, as seagrasses are able to compensate for hyperosmotic stress by an array of physiolo-

gical, biochemical and ultra-structural adjustments working over time scales from hours to weeks

(see Touchette 2007 for a review), as is already known for algae (e.g. Lobban and Harrison 1997)

and terrestrial vascular plants (e.g. Khan and Weber 2006). As demonstrated for some seagrass

species (e.g. Tyerman 1989), plants accumulate ions in the cell to restore initial changes in turgor

pressure induced by salinity increase. However, high ion concentrations (especially Na+ and Cl-)

can disrupt electrochemical membrane potentials and inhibit the activity of enzymes, and hence

they must later be replaced by K+ and organic osmolytes, such as free amino acids, organic

acids and soluble carbohydrates (Tyerman 1989; Lobban and Harrison 1997; Touchette 2007).

Most of these compatible solutes also have osmoprotectant properties on macromolecules and

membranes, reducing the inhibitory effects of ions on enzymes (Kirst 1989). Some ultrastructu-

ral changes (e.g. ultrastructure and numbers of chloroplast and mitochondria, invaginations of

plasmalema membranes, etc.; Jagels and Barnabas 1989; Iyer and Barnabas 1993) are thought to

be seagrass responses to offset the high costs of these osmorregulatory mechanisms. There is

some evidence of the existence of osmoregulatory mechanisms in Posidonia australis Hooker f.

(Tyerman 1982; Tyerman et al. 1984), but it has never been studied in the Mediterranean species

P. oceanica. In this species concentrations of proline, the most common osmolite used by plants

(including some seagrass species) are extremely low (Pirc and Wollenweber 1988), and carbo-

hydrates (or other organic osmolytes) may play a major role in osmorregulatory adjustments of

this species with high capacity for carbon storage (Touchette and Burkholder 2000). In fact, the

52

reduction in both soluble and reserve carbohydrates in the rhizomes of P. oceanica shoots ex-

posed to hypersaline treatments suggests their mobilisation to supply osmoregulatory demands

from epidermal leaf cells. Soluble carbohydrates increased in leaves of Thalassia testudinum

Banks ex König exposed to high salinities (Koch et al. 2007b), but decreased in Ruppia maritima

L. leaves, although in this later case, this is probably due to their conversion to more soluble forms

that would better facilitate osmotic adjustment in this species (e.g. proline; Murphy et al. 2003).

The modest effects of the HS treatment on plant vitality suggest that osmorregulatory me-

chanisms may still be effective at this level of salinity increase (0.81 psu over the mean natural

salinity), but not at the higher salinities of the VS treatment (1.63 psu over the mean natural

salinity). In accordance with the well-known annual pattern of variation described for P. oceani-

ca (Alcoverro et al. 2001), carbohydrates concentrations were at their annual maximum during

the experimental period. Nonetheless, despite this high availability of internal carbon resour-

ces, no further decrease in carbohydrates concentrations was observed in plants exposed to VS

treatment. This fact, together with the more intense effects of this experimental treatment on

seagrass variables (i.e. shoot size, growth rate and survival), suggests that the osmoregulatory

capacity of plants was overwhelmed or inhibited at this higher level of salinity stress. As hyper-

salinity stress persists and/or increases, plants become more overtaxed by the high energy cost

required for the maintenance of ionic balance (Touchette 2007); furthermore, osmotic and ionic

stresses can lead to the denaturation of membranes and organelles (Iyer and Barnabas 1993)

and the inhibition of photosynthesis and respiration (Ralph 1999; Fernández-Torquemada et

al. 2005b) in some seagrass species. In algae, hyperosmotic stress causes inhibition of enzymes

involved in carbon metabolism and protein synthesis (Kirst 1989; Lobban and Harrison 1997).

None of these physiological responses has yet been studied in P. oceanica (nor in many other

seagrass species), but they could account for the adverse effects of salinity increase on structure,

fitness and survival of P. oceanica shoots reported in this and previous studies (Fernández-Tor-

quemada and Sánchez-Lizaso 2005; Gacia et al. 2007). Other indirect mechanisms might also

operate to accelerate shoot mortality during the experimental period. Thus, for example, the

high proportion of necrotic leaf area recorded in August 2002 for shoots from the VS treatment

(ca. 60%, Fig. 1.7) results in a severe reduction of the effective photosynthetic surface and hence

53

in the flux of oxygen to below-ground tissues. Tissue anoxia can impair the mobilisation of car-

bohydrate reserves, allow the invasion of reduced phytotoxins from sediments, preventing respi-

ratory demands from being met, which has been shown to cause plant collapse and shoot death

(Zimmerman and Alberte 1996; Pedersen et al. 2004). Nonetheless, all these physiological me-

chanisms have not as yet been studied in the context of hyperosmotic stress in P. oceanica (nor

in many other seagrass species), and hence the above interpretations must be taken with caution

and corroborated by more specific research. Furthermore, not all shoot decline is explained by

hyperosmotic stress, as shoot density further decreased once salinity had returned to natural

salinity values. This suggests that plants stressed under hyperosmotic conditions had a reduced

capacity to reacclimatise to hypo-osmotic conditions, although this must largely depend on the

duration of the hyperosmotic stress (see Fernández-Torquemada and Sánchez-Lizaso 2005).

Results obtained in this study indicate that the salinity threshold for P. oceanica is within the

narrow range of salinity used in this experiment, which is consistent with those established in a

laboratory experiment at constant salinity (39 psu; Fernández-Torquemada and Sánchez-Lizaso

2005) and in a field study under more variable salinity (39.3 psu; Gacia et al. 2007). These values

are very close to the mean salinity of the VS treatment (39.22 psu), although symptoms of sali-

nity stress were also observed at the lower salinities of the HS treatment (38.4 psu). Care must

be taken when generalising from these mean values, as salinity was not constant. Nonetheless,

the bulk of evidence from this and previous studies reinforces the hypothesis that P. oceanica

is highly sensitive to even small salinity increments over its natural range (37.0-38.05 psu in this

study, Table 1.1). Higher salinity thresholds (50 to 65 psu) have been demonstrated for a number

of temperate and tropical seagrass species, including some Posidonia spp (Tyerman et al. 1984;

Lirman and Cropper 2003, Fernández-Torquemada et al. 2005b; Koch et al. 2007b), but these

cope with the wider and fluctuating salinity ranges characteristic of their natural habitats, i.e. 20-

60 psu in estuaries and hypersaline embayments (see Touchette 2007 for a review). Therefore, it

is reasonable to find that the upper salinity threshold for P. oceanica is very close to the upper li-

mit of the narrow and stable salinity range (36-38 psu) characteristic of the oceanic waters where

this species has evolved in the Mediterranean Sea. This result has relevant practical implications,

as brine effluents might have to be strongly diluted with seawater to avoid deleterious effects on

54

P. oceanica meadows. Moreover, it is evident that a better understanding is needed of the osmorre-

gulatory capacity of this (and other) Mediterranean seagrass species to resist hypersaline conditions.

55

Posidonia oceanica meadow. Experimental site

56

Detail of the Posidonia oceanica meadow

57

259 ABSTRACT

60 INTRODUCTION

62 MATERIAL AND METHODS

70 RESULTS

76 DISCUSSION

58

Lázaro Marín-Guirao, Jose Miguel Sandoval-Gil, Juan Manuel Ruiz, Jose Luis Sánchez-Lizaso. 2011Photosynthesis, growth and survival of the Mediterranean seagrass Posidonia oceanica in response to simulated salinity

increases in a laboratory mesocosm system

Estuarine, Coastal and Shelf Science 92: 286-296

59

2.1. ABSTRACT

This study aims to examine the effect of increased salinity on the photosynthetic activi-

ty of the Mediterranean seagrass Posidonia oceanica in a laboratory mesocosm system. To do

this, large rhizome fragments were transplanted in a mesocosm laboratory system and main-

tained at 37 (ambient salinity, control treatment), 39, 41 and 43 (hypersaline treatments) for 47

days. Pigment content, light absorption, photosynthetic characteristics (derived from P vs. E

curves and fluorescence parameters), and shoot size, growth rates and net shoot change were

determined at the end of the experimental period. Both net and gross photosynthetic rates

of plants under hypersaline conditions were significantly reduced, with rates some 25-33% and

13-20% lower than in control plants. The pigment content (Chla, Chlb, Chlb:a molar ratio, to-

tal carotenoids and carotenoids:Chla ratio), leaf absorptance and maximum quantum yield of

PSII (Fv/Fm) of control plants showed little or no changes under hypersaline conditions, which

suggests that alterations to the capacity of the photosynthetic apparatus to capture and pro-

cess light were not responsible for the reduced photosynthetic rates. In contrast, dark respi-

ration rates increased substantially, with mean values up to 98% higher than in control leaves.

These results suggest that the respiratory demands of the osmoregulatory process are likely

to be responsible for the observed decrease in photosynthetic rates, although alterations to

photosynthetic carbon assimilation and reduction could also be involved. As a consequence, leaf

carbon balance was considerably impaired and leaf growth rates decreased as salinity increased

above the ambient (control) salinity. No significant differences were found in the percenta-

ge of net shoot change, but mean values were clearly negative at salinity levels of 41 and 43.

Results presented here indicate that photosynthesis of P. oceanica is highly sensitive to hyper-

saline stress and that it likely account for the decline in leaf growth and shoot survival repor-

ted in this and previous studies in response to even small increments of the ambient salinity.

2PHOTOSYNTHESIS, GROWTH AND SURVIVAL OF THE MEDITERRANEAN SEAGRASS POSIDONIA OCEANICA IN RESPONSE TO SIMULATED SALINITY INCREASES IN A LABORATORY MESOCOSM SYSTEM

60

2.2. INTRODUCTION

The reverse osmosis industry uses seawater to obtain freshwater for human consumption.

It is an expanding industry in some countries as a result of the shortage of freshwater resources

(Morton et al. 1996). The main impact of this activity on the marine coastal environment is re-

lated to the effects of increased salinity caused by hypersaline discharges (i.e. brine) on benthic

organisms and communities (Einav et al. 2002; Sadhwani et al. 2005; Lattemann and Höpner

2008). As brine and seawater have different densities, a hypersaline layer is formed, expanding

over the sea bed, and this can have a negative effect on benthic communities (Fernández-Tor-

quemada et al. 2005a, 2009). This means that we must widen our scientific knowledge of the

mechanisms used by benthic organisms to acclimate or tolerate hypersaline conditions so that

we can assess and predict the consequences of brine impacts on marine coastal ecosystems.

Salinity is a critical environmental factor that determines the abundance and distribution

of seagrass communities (Montague and Ley 1993; Adams and Bate 1994b), which are rele-

vant components of coastal and estuarine ecosystems worldwide (Green and Short 2003). The

endemic Mediterranean seagrass Posidonia oceanica forms extensive, continuous meadows

at depths of between 0.5 and 40 m. These meadows are widely recognised has having great

ecological and socio-economic importance for the functioning of many coastal ecosystems

(Boudouresque et al. 2009). Since the year 2000, the desalination industry along the Medi-

terranean coast of Spain has undergone considerable development, and brine discharges now

flow through near-shore marine environments inhabited by P. oceanica meadows (Palomar and

Losada 2010). P. oceanica is considered a stenohaline seagrass species inhabiting infralittoral

environments of open coasts and is not usually present in estuaries and coastal lagoons (Boudo-

uresque et al. 2009; but see Pergent et al. 2002). This distribution pattern has led researchers

to hypothesise that P. oceanica may be highly sensitive to even moderate salinity increases and

hence prone to be negatively affected by brine discharges. In accordance with this hypothesis,

some recent studies have reported the reduced capacity of P. oceanica to withstand salinity

increases associated with brine effluents (Fernández-Torquemada and Sánchez-Lizaso 2005;

Gacia et al. 2007; Sánchez-Lizaso et al. 2008; Ruiz et al. 2009). Using various experimental

61

approaches, all of these studies showed a decrease in leaf growth rates and biomass, an increase

in necrosis marks on photosynthetically active leaf tissues and a decrease in plant survival rates

as salinity increased above a mean value of 39.1 (Fernández-Torquemada and Sánchez-Lizaso

2005; Gacia et al. 2007), or even lower (38.4; Sánchez-Lizaso et al. 2008; Ruiz et al. 2009).

Such threshold mean values are very close to the upper limit of the natural salinity range (36.5-

38.2) typically measured in the localities of the Mediterranean coast of Spain where aforemen-

tioned studies were performed, confirming the high sensitivity of these seagrass populations to

even small increments in ambient salinity. However, all the previously cited studies based their

conclusions on plant growth and survival rates, without providing any evidence of the physio-

logical mechanisms responsible for the sensitivity of P. oceanica to hypersaline conditions.

Photosynthesis is the most important physiological process governing plant productivity, and

has often been shown to be reduced under hypersaline conditions both in marine macroalgae (Kirst

1989) and in seagrasses (Touchette 2007). Hypersaline stress can alter photosynthesis and respi-

ration rates in seagrasses depending upon the species tested, the range of saline conditions consi-

dered, the duration of the exposure and the acclimation period, amongst other factors (Biebl and

McRoy 1971; Kerr and Strother 1985; Dawes et al. 1989; Ralph 1998; Murphy et al. 2003; Fernández-

Torquemada et al. 2005b; Koch et al. 2007a). However, unlike terrestrial plants (e.g. Huchzermeyer

and Koyro 2005), indirect and direct evidence of the mechanisms involved in salt-stress photosyn-

thesis alterations of seagrass leaves is very scarce and limited to very few seagrass species. These

mechanisms include reductions in pigment content, changes in the number and ultrastructure of

chloroplasts, impaired photosystem function, over-reduction of the photosynthetic transport chain

and inhibited activity of carbon assimilation and reduction enzymes (Jagels 1983; Beer et al. 1980a;

Ralph 1998, 1999; Iyer and Barnabas 1993; and see Touchette 2007 and references therein). Since

an impaired plant carbon budget caused by hypersaline stress through photosynthetic alterations

can result in long-term effects on seagrass abundance and survival rates, further studies should

be carried out on a higher number of seagrass species using different experimental conditions.

Our study represents the first to assess the impact of increased salinity on the photosynthetic

performance (and respiration) of P. oceanica. We hypothesise that the photosynthetic capacity of

62

P. oceanica is sensitive to hypersaline stress and that it can compromise plant vitality and survival

of this seagrass species. To this end, vegetative fragments (i.e. large rhizome-connected shoots)

of P. oceanica were exposed to chronic salinity treatments of 37 (i.e. ambient salinity) 39, 41 and

43 simulated in a laboratory mesocosm system for 47 days. As mentioned above, this salinity

range includes the upper threshold of salinity tolerance previously established for this seagrass

species (38.5-39.5). Photosynthetic responses (based on both oxygen evolution and chlorophyll

fluorescence techniques), pigment content and leaf absorptance, together with leaf growth rates

and morphology and shoot survival rates, were determined at the end of the experiment period.

2.3. MATERIAL AND METHODS

2.3.1. FIELD PLANT COLLECTION AND MESOCOSM SYSTEM

In October 2008, a number of large P. oceanica fragments with intact rhizomes and roots

were collected by divers in a shallow (6 m deep), dense meadow (shoot density 908 ± 50 shoots

m-2, meadow cover 32.1 ± 4.1%) off the south-western coast of the Murcia Region (37º 34´ N,

1º 12´ W; Isla Plana, Murcia, Spain). In this meadow, salinity was almost constant throughout the

year (37-38 psu, Practical Salinity Units; Annex 1) with no extreme fluctuations observed (except

for some very short and sporadic pulses of low salinity caused by rainfall). The plants were kept in

black plastic bags to prevent over-exposure to light, stored in large coolers (200 L) with seawater

and transported to the laboratory of the Spanish Oceanography Centre in Murcia (Spain). The

plants were immediately (within 3 h) transplanted into the aquaria of a mesocosm system for

acclimation (of 1 week) prior to the experiment (see below). The transplantation units consisted

of a large, densely branched rhizome fragment bearing 40-60 connected shoots attached to the

bottom of a plastic net cage (base 22 x 40 cm, height 10 cm) filled with coarse sediment which

had been pre-sieved to remove any animals and large particles of organic matter (Fig. 2.1). Lar-

ge rhizome fragments of P. oceanica were used instead of small pieces since initial experiences

showed that the survival and growth rates of the smaller ones rapidly declined in aquarium condi-

tions after the first 2 weeks.

63

The mesocosm system consisted of four 1500 L glass aquaria subdivided into three 500 L

sections (hereafter ‘sub-aquarium’) each one with their own illumination system. Each of the four

independent large aquaria was supplied with its own systems of seawater circulation and filtration,

with precise control of temperature, irradiance, salinity, pH and nutrients (see Annex 2 section for

a detailed description). Each sub-aquaria was independent respect to some factors (e.g. irradian-

ce) but not for others (e.g. seawater). Therefore, the three sub-aquaria of the large aquaria are

under the same experimental conditions (salinity, temperature, light, water quality), and hence

each large aquaria represented a single, different treatment level, and plants in the transplantation

units corresponded to a experimental population in which we take individual shoots at random

for measurements. To face with the problem of non-independence both among replicates (wi-

thin treatments) and among treatments, was also one of our major concerns in this experiment

due to the particular design of the mesocosm system. But in our case, the use of this design was

unavoidable for several reasons: (a) the use of smaller aquaria or tanks and plant fragments was

not appropriate for the time scale and species required in this study, (b) replication of the large

aquaria has obvious economical and logistical constraints, and (c) other alternatives such as field

experiments can be even more complex because the manipulation of salinity in situ is particularly

complicated (e.g. Chapter 1). But attending to previous works (e.g. Cayabyab and Enríquez

2007), the use of an individual shoot (the whole shoot or part of a tissue) as sampling unit justified

for this kind of physiological studies if caution is taken in preventing or minimizing dependen-

ce among replicates and treatments. In this sense and following recommendations in Oksanen

(2001) and Quinn and Keough (2002), we also identified and avoided all possible factors that

could potentially contribute to introduce dependence within and among treatments due to the

characteristics and behaviour of our particular biological system or due to uncontrolled external

factors that can confound the effect of the experimental factor. To this end the following criteria

and actions were considered: i) within the ramets, we avoided the influence of internal gradients

of resources allocation (nutrients, photosynthetic products, etc.) characteristic of this clonal plant

by using only individual shoots (vertical shoots) of 2-3 years old, located away from the apical

parts of the ramet which are known to act as internal sinks, ii) the selection of samples for the

different descriptors (depending on sample size, n) was always a random process, iii) ramets

were enough large to avoid the sampling of neighbour shoots, iv) there is no reason to suspect

64

the existence of some kind of spatial gradient caused by some external or internal factor or bet-

ween sub-aquaria of the same large aquaria (this aspect was carefully controlled, see below, v)

treatment levels were randomly assigned to each aquarium and also there is no reason to suspect

the existence of some kind of uncontrolled or unknown factor between aquaria (or in some part

of the lab) that can confound the effect of salinity and ,vi) we did not find significant differences

between aquaria for some plant variables (e.g. some descriptors derived from P vs. E curves,

Fv/Fm, photosynthetic pigments; ANOVA, P > 0.05; unpublished data) during the acclimation

period (see below). In addition, to avoid possible influences of spatial environmental micro-gra-

dients, the relative position of the transplantation units within the aquaria were regularly changed

(approximately twice a week). The functioning of the mesocosm system (as well as the conti-

nuous or periodical measurements explained in the Annex 2, i.e. temperature, irradiance, salinity,

pH and nutrients) allow us to ensure that there are not differences from one aquaria to another

except for salinity, which was the manipulated experimental factor. Therefore, we can logically

and reasonably attribute differences of the variables measured among tanks to differences in sa-

linity rather than to confounded influences derivate from possible non-independence problems

It is widely known that keeping large, slow-growing seagrass species such as P. oceani-

ca in aquarium conditions is particularly difficult. However, results obtained in previous trials

have indicated that this system is able to maintain healthy plants with 100% survival rates for

more than two months, long enough to achieve the objectives of this experiment. Also in this

way and to address questions such as the possible stress caused by artificial conditions that

could confound the effect of experimental treatments, a set of reference field values corres-

ponding to seasonal campaigns are shown in the section of Annex 1. In the comparison, it can

be appreciate that values of physiological descriptors obtained from plants maintained under

mesocosm conditions were similar to that obtained from plants growing in the same season

and in the same natural meadow from where experimental plants where collected, hence corro-

borating the optimum physiological status of the plants at the end of the experimental period.

65

2.3.2. EXPERIMENTAL DESIGN AND SET-UP

Three transplantation units were placed in each sub-aquarium i.e. a total of nine transplanta-

tion units per aquaria (Fig. 2.1). For a week prior to administering the experimental treatments,

the plants were acclimated to the mean prevailing environmental conditions at the plant collec-

tion site during the season in which the experiment was performed, i.e. a temperature of 21ºC,

salinity of 37, and a saturating irradiance of ca. 300 µmol quanta m-2 s-1 measured on the leaf tips

on a 12/12 h light:dark cycle (i.e. 12.96 mol quanta m-2 day-1). Environmental data had been ob-

tained using underwater continuous CT and PAR irradiance recorders (Compact-CT and MDS-

MkV/L, respectively; Alec Electronics, Japan; see Annexe 1). After the acclimation period, one

aquarium was maintained with ambient conditions (control, 37) while salinity was increased in the

other three aquaria to produce the following experimental hypersaline conditions: 39, 41 and 43,

respectively. This salinity range was selected based on the increased salinity threshold levels of

P. oceanica established in previous studies (Fernández-Torquemada and Sánchez-Lizaso 2005;

Gacia et al. 2007; Ruiz et al. 2009). Each experimental treatment was randomly assigned to one

of the four 1500 L aquaria.

Figure 2.1. Posidonia oceanica plants in the mesocosm system.

66

In order to achieve these salinity levels, high quality artificial marine salt (Seachem Reef

Salt™) was dissolved in 2000 L auxiliary tanks before the solution was transferred to the reservoir

tank of the aquaria. This hypersaline solution was mixed with the aquarium seawater at a slow rate

until the desired salinity level was reached. Adjusting the salinity levels took about 4 h, with these

levels then maintained for a total experimental period of 47 days - enough to observe early phy-

siological responses to hypersaline stress before the occurrence of more severe lethal effects. As

explained above, the environmental parameters were monitored daily, with the tanks, aquaria and

filters cleaned every day in order to prevent the appearance of epiphytes and macroalgal blooms.

Additionally, the transplantation units were periodically repositioned within the sub-aquaria to

avoid possible gradient effects. The selected response variables were measured at the end of the

experiment period, using the individual shoot as the sampling unit (replicates) for all measure-

ments. All shoots of a sample were randomly collected from the experimental population formed

by the nine transplantation units contained in each large aquarium (i.e. treatment level). For each

variable, a strategy was followed that allowed a homogenous distribution of the sampling effort

throughout the aquarium (see below). Shoots less than 2-3 years old were excluded from sam-

pling in order to avoid or minimise possible dependence effects caused by the influence of in-

ternal resources allocation gradients characteristic of these clonal plants (e.g. Marbá et al. 2002;

Olivé et al. 2009). For similar reasons, the collection of neighbouring shoots was also avoided.

2.3.3. CHLOROPHYLL FLUORESCENCE

The chlorophyll a fluorescence emission of dark-adapted leaves was measured with a di-

ving-PAM portable fluorometer (Walz, Germany) in order to calculate the maximum quantum

yield of PSII (Fv/Fm), a measurement of the photochemical efficiency of PSII (Krause and Weis

1991; Larkum et al. 2006a). All measurements were performed on plants adapted to darkness

overnight (i.e. before switching on the illumination system) to ensure full oxidation of reaction

centres and primary electron acceptors. For Fv/Fm calculation, the minimum (Fo) and maxi-

mum (Fm) fluorescence were measured by exposing photosystems to a saturating pulse (0.8

s) of white light using dark-adapted leaves that had been pre-illuminated with a weak red mo-

dulated light. In order to measure fluorescence, each individual leaf was held in the DCL-8

67

leaf clip holder so that a constant distance between the leaf and the fibre optic probe could be

maintained. Prior to the experiment, the fluorescence parameters of the P. oceanica leaves were

measured, confirming the typical pattern of variation as a function of leaf age already descri-

bed for other seagrass species (Thalassia testudinum, Durako and Kunzelman 2002; Enríquez

et al. 2002). In order to prevent this source of variation from masking possible effects of the

treatment on Fv/Fm, fluorescence measurements were taken at regular intervals of 4 cm from the

leaf base to the apex in all leaves on a shoot. The maximum value obtained was selected as the

Fv/Fmvalue of the individual shoot. The same measurement was taken in six randomly-selected

shoots per sub-aquarium (i.e. n = 18 replicates per treatment). Since fluorescence-based me-

thods allow measurements without altering the state of the plants in the aquaria, these measure-

ments were taken once a week to assess any changes in Fv/Fm throughout the experiment period.

2.3.4. RESPIRATION AND PHOTOSYNTHESIS RATES

The photosynthesis-irradiance curves (P vs. E) of P. oceanica leaves were determined fo-

llowing methods proposed by Walker (1985) and Cayabyab and Enríquez (2007). Photosynthesis

rates were measured polarographically using a DW3 chamber with a Clark type O2 electrode

system (Hansatech, UK) connected to a controlled temperature circulating bath (P-Selecta,

Spain). The incubation chamber was filled with filtered seawater and maintained at the same

temperature as the plants in the aquaria, i.e. 21ºC. Four replicated leaf segments with a surface

area of approximately 2 cm2 were taken from the middle section of the youngest fully-developed

leaf and used to determine photosynthesis rates for each test treatment. These leaves have been

shown to have the maximum photosynthetic performance (Alcoverro et al. 1998) as well as the

highest Fv/Fm values previously measured in P. oceanica shoots. Each leaf segment was obtained

from four individual shoot randomly selected from the experimental shoot population of each

aquaria. Leaf segments were first incubated in darkness for 15 min to determine the initial dark

respiration rate (initial-Rd), before being exposed to increasing intensities of irradiance (E) of 14,

37, 66, 84, 143, 250, 654, 1010, 1496, 2271 µmol quanta m-2 s-1. After the final light treatment, leaf

segments were again exposed to darkness to determine the final dark respiration rate (final-Rd).

Leaf segments were illuminated using a tungsten-halogen light source (LS2, Hansatech, UK) and

68

light intensities within the chamber calibrated using the fibre quantum sensor of a diving-PAM

fluorometer. During the incubation period, 5 mM NaHCO3 was added to the seawater in the

chamber to prevent carbon limitation, with N2 also bubbled into the water to maintain oxygen

concentrations within a saturation range of 20-80%. Oxygen readings were continuously re-

corded on a computer through a transducer unit (Oxigraph, Hansatech, UK) connected to the

electrode. Increases in oxygen were measured for each incubation interval as µmol O2 cm-2 h-1

and plotted against their respective E values to construct the P vs. E curve, illustrating the ty-

pical saturation kinetics of the photosynthetic response. The maximum net photosynthesis rate

(net-Pmax) was determined by averaging the maximum values above the saturating irradiance

(Ek), with gross photosynthesis (gross-Pmax) then calculated as the sum of net-Pmax and final-Rd.

Photosynthetic efficiency (α, µmol O2 cm-2 h-1/µmol quanta m-2 s-1) was calculated as the slo-

pe of the regression line fitted to the initial lineal part of the P vs. E curve. The compensation

irradiance (Ec) was taken as the intercept on the X-axis and Ek as the ratio Pmax/α. The P:Rd

ratio was used as a proxy of the daily leaf metabolic carbon balance. P was derived by multi-

plying gross-Pmax by the number of light hours (i.e.12 h), with Rd taken as total respiration over

a 24 h period considering final-Rd for the light hours and initial-Rd for the dark period (12 h).

2.3.5. LEAF ABSORPTION AND PIGMENT CONTENT

Leaf light absorption and pigment content were determined as described by Cayabyab and

Enríquez (2007) on three 2 cm2 leaf segments obtained from three individual shoots randomly

collected from each sub-aquarium (i.e. n = 9 replicates per treatment aquarium). The absor-

bance (D) of intact leaf segments was read spectrophotometrically (Thermo Evolution 300,

Fisher Scientific, Spain) in the PAR range (380-750 nm) with a 1-nm resolution using the opal

glass technique (Shibata 1959). Leaf absorption was expressed as absorptance (A), which is the

fraction of incident light absorbed (Kirk 1994) and was calculated from absorbance readings

using the equation A =1-10-D. Absorptance was calculated using: (1) the average light absorp-

tion for the range 400 nm and 700 nm; and (2) light absorption at 680 nm, i.e. the Chla peak.

69

Leaf absorption by non-pigmented structures was corrected using bleached leaves as

a reference in the spectrophotometer and the spectra obtained were corrected for resi-

dual scattering by subtracting the absorbance at 725 nm. After this analysis, pigments were

extracted from leaf segments using 80% acetone mixed with a MgCO3 solution to pre-

vent the acidification of the extract (Dennison 1990). The extracts were stored in the dark

for 24 h and absorbance was then read spectrophotometrically at 470, 646 and 663 nm

using 1 ml quartz-glass cuvettes. The concentrations of chlorophyll a and b and total carote-

noids were calculated using the equations proposed by Lichtenthaler and Wellburn (1983).

2.3.6. LEAF MORPHOLOGY, LEAF GROWTH RATE AND SHOOT SURVIVAL

At the beginning of the experimental period, all of the shoots in each transplantation unit were

counted, and six randomly-selected shoots per sub-aquarium (n = 18 replicates per treatment

aquarium) were marked for leaf growth measurements using the Zieman (1974) method adap-

ted for P. oceanica by Romero (1989). At the end of the experimental period, all of the marked

shoots were collected, the number of leaves per shoot was counted and the length and width of

each leaf measured to calculate shoot size on a per area basis (cm2 shoot-1). Newly-formed leaf

segments (i.e. those below the mark) were separated from the rest and measured to estimate

shoot leaf growth rate (cm2 shoot-1 day-1). Leaf growth rates were also calculated for the youngest

new leaves that had appeared since the experimental period had begun (i.e. youngest leaves wi-

thout marks). The surface area of necrosis marks on the leaves was also measured and expressed

as the proportion of necrotic photosynthetic tissue per shoot. Shoot counts were repeated at the

end of the experimental period in order to estimate shoot survival rates. Differences between ini-

tial and final shoot numbers of each transplanting unit were normalised to initial shoot numbers

and expressed as the percentage of net change in shoot number (n = 9); negative values indica-

ted a net decline in the initial shoot number due to mortality rates higher than shoot recruitment

(i.e. shoot division or rhizome branching) and/or reduced shoot recruitment (Ruiz et al. 2009).

70

2.3.7 STATISTICAL ANALYSIS

One-way ANOVA was used to assess the significant effects of the treatment (a fixed

factor with four salinity levels: 37, 39, 41 and 43) on response variables. The effect of sa-

linity treatments on Fv/Fm time trends was analysed using two-way ANOVA, with sali-

nity and time (8 sampling times) as fixed factors in the model. Before carrying out the

ANOVA test, the data were checked for the assumptions of normality and homoscedas-

ticity, and transformed when necessary. Post-hoc mean comparisons (Student-Newman-

Keuls, SNK; Zar 1984) were performed to identify specific treatment level(s) causing sig-

nificant effects. Treatment effects were considered statistically significant at P < 0.05.

2.4. RESULTS

The mesocosm system designed for this experiment was able to maintain high water

quality conditions throughout the experimental period. Seawater pH values of the four

aquaria showed daily variations between 8.02 and 8.18, with maximum values during

the light period and minimum in the dark period as a result of the photosynthetic acti-

vity of the seagrass. This daily pattern of pH variation was equal in all aquaria and was

within the natural range of this parameter measured in shallow P. oceanica canopies (In-

vers et al. 1997). Dissolved nutrient (nitrate and phosphate) concentrations measu-

red throughout the experimental period were always within the levels typically repor-

ted for Mediterranean oligotrophic waters inhabited by P. oceanica (Margalef 1985).

Fv/Fm values for each experimental treatment throughout the experimental period

are shown in Fig. 2.2. The salinity treatments did not have any significant effect on mean

Fv/Fm values (Table 2.1), which ranged from 0.781 ± 0.0012 to 0.803 ± 0.0004 for all sam-

pling times and treatments. Differences in time trends were only significant in the last

week (interaction term in Table 2.1 and Fig. 2.2), but this was due to a very small (less than

2%) increase in Fv/Fm values in the 41 and 43 salinity treatments above the control values.

71

Table 2.1. Summary of the ANOVA test carried out to assess significant effects of salinity treatments on time trends of Fv/Fm throughout the experimental period. df = degrees of freedom; MS = Mean Squares; n.s. = not significant; **P< 0.01. ***P <0.001.

Figure 2.2. Maximum mean quantum yield (Fv/Fm) of Posidonia oceanica leaves exposed to the different experimental treatments throughout the experimental period:37 (control, black circles), 39 (white circles), 41 (black triangles) and 43 (white triangles). Zero on the X-axis (time, days) marks the start of the experimentaltreatments and the negative section shows the acclimation period.Values are means ± SE, n = 18.

72

The salinity treatments had significant effects on photosynthetic parameters derived from

P vs. E curves (Figs. 2.3 and 2.4, Table 2.2). Maximum net and gross photosynthetic rates (net-

Pmax and gross-Pmax) were significantly and consistently lower than the mean control values for

all salinity treatments, although the differences were greater in the case of the net-Pmax (25-33%)

than for the gross-Pmax (13-20%). The dark respiration rates (both initial and final-Rd) were the

most affected parameter, with substantial increases in plants subjected to hypersaline treatments

(50-86% for initial-Rd and 76-98% for final-Rd) in comparison to those subjected to the control

treatment. As such, the P:Rd ratio was 4.55 ± 0.47 for control plants and a significant 49-56% lower

in plants which underwent hypersaline treatments. No significant changes were observed in α of

plants exposed to hypersaline treatments when compared to control plants, although a slight re-

duction (15%) in this parameter was observed in the most severe hypersaline treatment (43). For

the 39 and 41 hypersaline treatments, Ek and Ec irradiances significantly decreased and increased,

respectively, as initial-Rd increased, while α remained constant. However, some departure from

this pattern was observed for Ec and Ek at 43 due to the mild (not significant) decrease in initial-

Rd and α observed in this treatment. Light absorption spectra of P. oceanica leaves obtained in

each experimental treatment are shown in Fig. 2.5. Leaf light absorption (both PAR averaged and

at 680 nm) measured in control leaves was 1.6-4.6% lower in hypersaline treatments, although

this was only significant for the 680 nm absorptance (Table 2.3). There were no significant di-

fferences in pigment content (Chla, Chlb, Chlb:a molar ratio and total carotenoids) between

the different treatments (Table 2.3). Despite this lack of statistical significance, it must be noted

that the lowest mean values of the Chlb:a molar ratio were found in the most severe hypersaline

treatment of 43. The carotenoid:Chla ratio increased significantly by 6% in treatments ranging

from 37 to 41, but showed similar mean values as control plants in the 43 treatment (Table 2.3).

73

Figure 2.3. Oxygen evolution rate as a function of irradiance measured inPosidonia oceanica leaves after 47 days of exposure to each experimental treatment: 37 (control, black circles), 39 (white circles), 41(black triangles) and 43 (white triangles). Values measured in the dark(0 μmol quanta m-2 s-1) are the initial-Rd and those measured in light conditions are net photosynthetic rates. Values are means ± SE, n = 4.

Table 2.2. Mean values ± SE of P-E curve parameters of P. oceanica at the end of the experiment period, and summary of the one-way ANOVA carried out to test significant differences between salinity treatments. df = degrees of freedom;MS = mean squares; F = Fisher statistic;P = probability; *P < 0.05. **P < 0.01.***P < 0.001; n.s.= not significant. Groups of homogeneous means obtained using the post-hoc SNK test are indicated with the symbol <

74

Figure 2.4. Variation of photosynthetic parameters derived from P vs E curves for the different salinity treatments. Net-Pmax = net maximum photosynthe-tic rate; gross-Pmax = gross maximum photosynthetic rate; initial-Rd = initial dark respiration rate; final-Rd = final dark respiration rates; α = photosyntheticefficiency; Ek = saturation irradiance; Ec = compensation irradiance. Values are means ± SE, n = 4.

75

Figure 2.5. Light absorption spectra of P. oceanica leaves exposed to salinity treatments. Vertical grey bars are error bars of the mean (n = 9).

Table 2.3. Mean values (± SE) of pigment concentrations and leaf absorptance of Posidonia oceanica leaves obtained for each experimental treatment at the end of theexperimental period, and summary of the one-way ANOVA carried out to test significant differences between treatments. df = degrees of freedom; MS = mean squares; F = Fisher statistic; P = probabi-lity (0.05). n.s. = not significant. Groups of homogeneous means obtained using the post-hoc SNK test are indicated with different letters.

76

The vegetative responses of P. oceanica plants exposed to experimental increases in salinity

are shown in Fig. 2.6 and Table 2.4. Neither the shoot size nor the number of leaves per shoot

was affected by the salinity treatments. The leaf growth rate of whole shoots subjected to the hy-

persaline treatments was consistently 17% less than the mean values measured in control plants,

but the ANOVA test was unable to detect this effect due to the high variability of this response

variable between individual shoots under control conditions. However, growth rates of new leaf

tissue that had formed during the experimental period (i.e. unmarked leaves) showed a signifi-

cant and consistent negative relationship with treatment salinity (Pearson correlation coefficient

R =-0.962, P < 0.05), and was a statistically significant 34% lower for plants subjected to the 43

treatment than those in the control aquarium. Mean values of the surface area of the necrotic

leaf tissue were less than or equal to 10% and always observed at the apical senescent part of the

leaves, indicating normal values for this variable obtained in natural meadows at similar depths

and in the same season (e.g. Ruiz et al. 2009). The percentage of net shoot change (Fig. 2.5)

was very close to zero (from 0.007 ± 0.7 to -0.36 ± 1.29%) for the 37 (control) treatment and

39 treatment and clearly more negative (-3.9 ± 2.2 to -5.5 ± 2.1%) for the 41 and 43 treatments.

Nonetheless, such differences were very small and variability among replicates was high enough

to render the ANOVA test unable to detect significant treatment effects for this variable.

2.5. DISCUSSION

Plants in the transplantation units kept in the mesocosm system under control conditions (i.e.

37) had mean leaf growth rates and photosynthetic performance (Pmax) values within the ranges

found in previous studies for P. oceanica meadows in the same season (autumn) and at the same

depth (Alcoverro et al. 1998; Ruiz and Romero 2001, 2003). Fv/Fm values also fell within those of

healthy plants of other seagrass species (0.7-0.8 e.g. Ralph 1999; Durako and Kunzelman 2002;

Ralph et al. 2002; Cayabyab and Enríquez 2007). Mean values of these plant variables obtained

in the control treatment were also very close to those previously measured in plants from the

same collection site during the same season (Annex 1), indicating that the experimental manipu-

lation caused few or negligible stress on plants.

77

Figure 2.6. Mean (SE, n = 18) shoot size, number of leaves per shoot, leaf growth rates of the whole shoot and of thenewly-formed leaves, leaf necrotic area percentage and the net shoot change of Posidonia oceanica measured at the end of the experimental period.

78

Table 2.4. Summary of the one-wayANOVA carried out to assess differences in shoot variables between salinity treatments measured at the end of the experimental period. n.s. = not significant. df =degrees of freedom; MS = mean squa-res; F = Fisher statistic; P = probability. 1Log (x+1) transformed data; 2 arc sin (√(x+1)) transformed data.

79

Moreover, the survival rates (ca. 100%) of the P. oceanica in the transplantation units in the con-

trol aquaria throughout the 6 week experimentation period are certainly worth highlighting. This

is quite a notable success, considering that this species has been particularly problematic when

used for laboratory experimentation due to its supposed low plasticity and strict ecological requi-

rements. Significant shoot mortality (15-30% of the initial shoot population) has been commonly

reported for fragments of P. oceanica and other seagrass species kept in control conditions in labo-

ratories for experimental periods shorter than that of this study (e.g. Fernández-Torquemada and

Sánchez-Lizaso 2005; Cayabyab and Enríquez 2007). What is interesting about these results is

that the mesocosm system used in this study maintains an optimal physiological status and vitality

of plants under experimentation, as well as minimising further stress caused by experimental ma-

nipulation that could confound the main effect of experimental treatments (i.e. salinity increase).

This study has provided experimental evidence that the photosynthetic capacity of P. oce-

anica is highly sensitive to the increases in salinity simulated in our mesocosm system. In effect,

photosynthetic rates (both net- and gross-Pmax) were significantly and consistently reduced at

salinities immediately higher than that of the control treatment (37), the later being similar to the

mean ambient salinity measured in the field at the plant collection site in the same season (37.2-

37.3). Partial inhibition of photosynthetic O2 production or carbon assimilation has consistently

been reported for temperate and tropical seagrass species exposed to experimental hypersaline

conditions (Ogata and Matsui 1964; Biebl and McRoy 1971; Beer et al. 1980a; Kerr and Strother

1985; Fernández-Torquemada et al. 2005b; Koch et al. 2007b). Such a reduction in photosyn-

thetic rates can be explained by alterations in the structure and function of the photosynthetic

apparatus caused by hypersaline stress, such as those reported previously for some seagrass spe-

cies: changes in ultrastructure and number of chloroplasts (Jagels and Barnabas 1989; Iyer and

Barnabas 1993), declines in chlorophyll content (Ralph 1998; but see Van Katwijk et al. 1999) and

in photochemical efficiency of the PSII (Ralph 1998, 1999; Murphy et al. 2003; Koch et al. 2007b).

However, in our experiment, changes in light absorptance, chlorophyll content and Fv/Fm due

to increased salinity were minor or did not occur at all, indicating little or no alterations to the

capacity of photosynthetic structures to capture and process light. Only a slight decrease in the

photosynthetic efficiency (α) was observed in the most severe hypersaline condition (43), and

80

this was probably related with a reduction in the absorption cross section of the photosynthetic

units (Falkowski and Raven 2007) as indicated by the lowest Chlb:a ratios found in this treatment.

In any case, our results indicate that the hypersaline conditions tested in this study (i.e. up to 116%

seawater) caused very little stress to the photosynthetic apparatus compared to that reported in

previous studies on other seagrass species, probably due to the more severe hypersaline condi-

tions tested in these studies (e.g. up to 250% seawater in Ralph 1998, 1999). Our results are more

consistent with studies using terrestrial plants which indicate that the photosynthetic apparatus

is not significantly damaged until very low water deficits are reached due to very high salinity

levels or longer periods of exposure to stress (Berkowitz 2000; Huchzermeyer and Koyro 2005).

In contrast, dark respiration rates for P. oceanica leaves were highly responsive to hypersaline

stress. The significant increases in this variable observed in all hypersaline treatments is more likely

to account for the reduction of net-Pmax reported in this study. Similar responses have been repor-

ted for other seagrass species exposed to hypersaline stress (Biebl and McRoy 1971; Drew 1978a;

Kerr and Strother 1985), probably reflecting the increased respiratory demand in order to cope with

osmoregulatory adjustments (Jagels 1983; Kirst 1989; Kahn and Durako 2006; Touchette 2007) or

the operation of metabolic pathways with higher respiratory costs (e.g. C-2 or C-4-like metabolism;

Azcón-Bieto and Talón 2000). Nonetheless, as reported for terrestrial plants under water and salt

stress (e.g. Lambers et al. 2006a,b), this response has been seen to be not as consistent with that

found in photosynthetic rates in seagrasses (Touchette 2007). In effect, in some seagrass species

the respiration rates decreased as salinity increased, or were unaffected (Ogata and Takada 1968;

Fernández-Torquemada et al. 2005b). Further to the stimulation of respiration rates, other salt-

induced effects on plant metabolism could be responsible for the reduced photosynthetic capacity

of P. oceanica leaves under hypersaline stress as suggested by the decrease in gross-Pmax. Thus,

for instance, in salt-stressed plants, experimental evidence suggests that the inhibition of electron

transport rate, photophosphorylation and key stromal enzymes is involved in photosynthetic car-

bon assimilation and reduction (e.g. Kramer and Boyer 1995; Farias de Aragão et al. 2005; Huch-

zermeyer and Koyro 2005; but see Berkowitz 2000). Changes in the solute environment around

enzymes (particularly the ionic environment) under hyperosmotic stress have been suggested to be

the major cause of the inhibited photosynthetic metabolism in terrestrial and aquatic plants (Kirst

81

1989; Kramer and Boyer 1995). Some, albeit very limited, evidence supports the existence of

such inhibitory mechanisms in seagrasses. For example, Drew (1978a) reported a marked reduc-

tion in 14C accumulation in the seagrass Cymodocea nodosa in response to salinity increase, and

indicated that both enhanced respiratory demands and the interference of hyperosmotic stress

with the photosynthetic mechanisms were involved. Also, Beer et al. (1980a) found a consistent

and marked reduction in RuBisCo activity in Halophila stipulacea and Halodule uninervis as NCIa

concentration increased in in vitro assays. An inhibition of the photosynthetic carbon metabolism

could explain other photosynthetic responses reported in this study. Thus, the decrease in Ek ob-

served in hypersaline treatments means that less light is required to saturate photosynthesis and

compensate for impaired leaf carbon balance. Nonetheless, lower Ek values can also mean that

photosystems receive a higher amount of excitation energy capable of causing photochemical

damage (Athar and Ashraf 2005). Considering that Fv/Fm values of P. oceanica leaves were una-

ffected by hypersalinity, the observed increase of the carotenoids: Chla ratio as salinity increased

suggests that the photoprotective dissipation process can operate effectively to prevent accu-

mulated photoinhibitory damage to photosystems (e.g. the xanthophyll cycle; Demmig-Adams

1990; Demmig-Adams and Adams 1996; Athar and Ashraf 2005; Huchzermeyer and Koyro

2005). Evidence of the existence of these mechanisms in seagrasses has been found previously

(e.g. Ralph et al. 2002), but since our results do not represent robust evidence of the existence of

such mechanisms in P. oceanica under hypersaline stress, such an interpretation should be addres-

sed in this and in other seagrass species through further and more specific experimental work.

Despite evidence of possible compensatory responses (i.e. decrease in Ek), the daily leaf

carbon balance (as indicated by the P:Rd ratio) was significantly impaired under hypersaline con-

ditions and, as a consequence, the leaf growth rate decreased as salinity increased. In contrast

to previous studies of P. oceanica under similar hypersaline conditions (Fernández-Torquemada

and Sánchez-Lizaso 2005; Gacia et al. 2007; Ruiz et al. 2009), it is worth noting that these phy-

siological alterations did not result in significant lethal effects at the organism level, as reflected

by the lack of any impact on leaf necrosis and the small net shoot decrease (less than 6%) re-

corded in plants subjected to the hypersaline treatments. The discrepancy between these results

and mortality responses obtained in the aforementioned studies probably reflects variability in

82

experimental conditions rather than any inconsistency in seagrass response to increased salinity.

The physiological changes reported in this study seem to represent sub-lethal effects occurring

at early stages of salinity stress. Based on the impact of altered photosynthetic (and respiration)

metabolism on leaf carbon balance and growth observed after 45 days, it should be expected

that if hypersaline stress persists for longer periods, plants would become more exhausted and

lethal effects more significant. Accordingly, significant shoot decline (12-14%) has been obser-

ved in experimental P. oceanica plots 100 days after exposure to increased mean salinities (38.4-

39.2; Ruiz et al. 2009-Chapter 1). Nonetheless, some authors has pointed out the possibility

of long-term acclimation to increased salinities based on the presence of this seagrass species

in some hypersaline lagoons of the Mediterranean Sea (e.g. Pergent et al. 2002; Tomasello et

al. 2009). Tomasello et al. (2009) provided evidence of the genetic isolation of a P. oceanica

meadow confined in a hypersaline lagoon of Sicily and suggest a possible selection of particular

genotypes adapted to persistent stressfull hypersaline conditions. It should be emphasised that

the physiological responses reported in this study must be limited to the particular experimental

conditions used (e.g. seagrass populations growing under almost constant salinity, chronic expo-

sure to hypersaline conditions, plants collected in autumn, etc.) and additional research is nee-

ded in order to extrapolate them to the more variable and complex circumstances (e.g. pulsed

and discontinuous salinity regimes, synergism with other abiotic factors, seasonal variability, etc)

typically found in seagrass meadows exposed to natural or anthropogenic salinity fluctuations.

83

Posidonia oceanica meadow.Isla Plana, Mazarron (Murcia Region)

84

Posidonia oceanica in the mesocosm system

85

387 ABSTRACT

87 INTRODUCTION

90 MATERIAL AND METHODS

99 RESULTS

107 DISCUSSION

86

Jose Miguel Sandoval-Gil, Lázaro Marín-Guirao, Juan Manuel Ruiz.The effect of salinity increase on the photosynthesis, growth and survival of the Mediterranean seagrass Cymodocea nodosaEstuarine, Coastal and Shelf Science , In Press

87

3THE EFFECT OF SALINITY INCREASE ON THE PHOTOSYNTHESIS, GROWTH AND SURVIVAL OF THE MEDITERRANEAN SEAGRASS CYMODOCEA NODOSA

3.1. ABSTRACT

TherearemajorconcernsintheMediterraneanSeaovertheeffectsofhypersalineeffluents

from seawater desalination plants on seagrass communities. However, knowledge concerning

the specificphysiological capacitiesof seagrasses to tolerateor resist salinity increases is still

limited. In this study, changes in the photosynthetic characteristics, pigment content, leaf light

absorption, growth and survival of the seagrass Cymodocea nodosa were examined across a

range of simulated hypersaline conditions. To this end, large plant fragments were maintained

under salinities of 37 (control ambient salinity), 39, 41 and 43 (practical salinity scale) in a labo-

ratory mesocosm system for 47 days. At the end of the experimental period, net photosynthesis

exhibitedamodest,butsignificant,decline(12–17%)inalltestedhypersalineconditions(39–43).

Atintermediatesalinitylevels(39–41),thedeclineinphotosyntheticrateswasmainlyaccounted

forbysubstantialincreasesinrespiratorylosses(approximately98%ofthecontrol),thenegative

effectsofwhichonleafcarbonbalancewereoffsetbyanimprovedcapacityandefficiencyof

leaves to absorb light, mainly through changes in accessory pigments, but also in optical proper-

tiesrelatedtoleafanatomy.Conversely,inhibitionofgrossphotosynthesis(by19.6%compared

to the control mean) in the most severe hypersaline conditions (43) reduced net photosynthesis.

In this treatment, the respiration rate was limited in order to facilitate a positive carbon balance

(similar to that of the control plants) and shoot survival, although vitality would probably be

reduced if such metabolic alterations persisted. These results are consistent with the ecology

of Mediterranean C. nodosa populations, which are considered to have high morphological and

physiological plasticity and a capacity to grow in a wide variety of coastal environments with

varying salinity levels. The results from this study support the premise that C. nodosa has a hig-

her tolerance to hypersaline conditions than the highly-sensitive Posidonia oceanica, the other

dominant Mediterranean seagrass, which is limited to marine environments with stable salinities.

3.2. INTRODUCTION

Seagrass meadows represent the dominant habitat in marine infralittoral environments of the

MediterraneanSea(Procaccinietal.2003).Theecologicalandsocio-economicalrelevanceof

88

these seagrass communities has been widely documented and recognised worldwide, as has their

high vulnerability to a variety of anthropogenic disturbances concentrated in the coastal zone

(Kenworthyetal.2006;Shortetal.2007).IntheMediterraneanSea,hypersalineplumes(i.e.,

brines) from seawater desalination plants are recent human impacts that potentially threaten the

conservationofseagrasshabitats(Sánchez-Lizasoetal.2008;Boudouresqueetal.2009;Ruizetal.

2009;PalomarandLosada2010).Ineffect,increasesinseawatersalinityhavebeenshowntoalter

seagrasscommunitiesinthiscoastalzoneandothers(Chesher1975;Tomaskoetal.2000;Fernán-

dez-Torquemadaetal.2005a;Gaciaetal.2007;Ruizetal.2009).Consequently,anunderstanding

of the mechanisms seagrass species employ to tolerate salinity increases is crucial for forecasting

andmonitoringtheconsequencesofbrineimpactsonthesevulnerableandvaluableecosystems.

However,despitethefactthatsalinityisamajorecologicalfactorinfluencingseagrassdistribu-

tionandstructureworldwide(Chesher1975;MontagueandLey1993;Castriotaetal.2001;Einav

etal.2002;Fernández-Torquemadaetal.2005b;Gaciaetal.2007),salinitytolerancerangeshave

onlybeenestablishedforafewseagrassspecies.Furthermore,thestructuralandphysiologicalme-

chanisms by which seagrasses cope with natural or anthropogenically-induced changes in salinity

regimesarestillpoorlyunderstood(Drew1978a;Tyerman1989;forareview,seeTouchette2007).

Theacquisitionofparticularadaptationsinordertomaintainosmoticequilibrium(e.g.osmo-

regulation) and key physiological functions (e.g. photosynthesis) is one of the basic properties

enablingseagrassestosuccessfullyevolveinmarineenvironments(Arber1920;KuoandDen

Hartog2000).Thephysiologicalcapacityofseagrassestotolerateincreasesinsalinityisspecies

specificandcloselyrelatedtothesalinitycharacteristicsoftheenvironmentsinwhichtheygrow.

Species limited to coastal environments with constant salinity levels are considered to be less tole-

rant to changes in this parameter than more euryhaline species, which are able to grow in a broader

rangeofcoastalhabitatswithwidelydifferentsalinitylevels(Tyerman1989;KuoandDenHartog

2000;Kochetal.2007b;Touchette2007).Thesetwoextremesarerepresentedbythedistinct

ecologies of the two most common Mediterranean seagrasses, Posidonia oceanica (L.) Delile

and Cymodocea nodosa (Ucria) Ascherson, and their differential tolerances to hypersaline stress.

89

The distribution of the larger, slow-growing P. oceanica is usually limited to open coastal

waterswithvirtuallyconstantsalinities (e.g.36.5–38on thesoutheasterncoastofSpain:Ruiz

et al. 2009; but seeMeinesz et al. 2009 andTomasello et al. 2009).Conversely, the sma-

ller, fast-growing C. nodosa is commonly found in a wider range of marine environments with

varying salinity levels (Drew 1978b;Procaccini et al. 2003;Boudouresqueet al. 2009).Den-

se and highly productive C. nodosa meadows are common in open coastal waters with cons-

tant salinities, estuaries with fluctuating salinities and temperatures, and confined hypo- and

hypersaline water bodies along the Mediterranean coast. Accordingly, C. nodosa has greater

morphological and functional plasticity than P. oceanica and has been recognised as being a

more eurybiontic species, better adapted than P. oceanica to cope with changes in environ-

mentalconditions(Drew1978b;TerradosandRos1991;PérezandRomero1994;Cebriánetal.

2000;MarbàandDuarte2001;Olesenetal.2002;Cancemietal.2002;Agostinietal.2003).

Thus, based on previous knowledge, it is expected that C. nodosa meadows are more tole-

rant to natural or anthropogenically-induced salinity increases than those of P. oceanica. Results

obtained from recent studies are in agreement with this general hypothesis. Plant growth, bio-

mass and mortality in P. oceanica have been shown to be highly sensitive to very small salinity

increases(39)abovemeanambientvalues(Fernández-TorquemadaandSánchez-Lizaso2005;

Gaciaetal.2007;Ruizetal.2009),while thesesameplantvariables remainedunaffected in

C. nodosa atmuchhigher salinity thresholds (greater than41) (Fernández-Torquemadaand

Sánchez-Lizaso2006;Pagèsetal.2010).Thismarkedinter-specificdifferenceinresistinghy-

persaline stress is likely to be determined by the physiological adaptive mechanisms involved

in the capacity of each species to tolerate or avoid osmotic and ionic stress (e.g. Kramer and

Boyer1995;Versluesetal.2006).Thenatureofsuchmechanismsremainsunclearinseagras-

sesingeneral(Touchette2007)andhasbeenvirtuallyignoredinthespecificcaseofMedite-

rranean seagrasses. Among these mechanisms, photosynthesis is one of the most important,

beinggenerallyaffectedbyhypersalineconditions(Touchette2007),althoughinformationon

this topic is scarce and only available for a few seagrass species. The limited evidence available

indicates that hypersalinity can reduce photosynthetic performance in seagrasses (and hen-

ce their growth and survival rates) by altering the structure of their photosynthetic apparatus

90

(IyerandBarnabas1993),itscapacitytoharvestlight(McMillanandMoseley1967;Ralph1998)

anditsPSIIphotochemicalefficiency(Ralph1998,1999;Kochetal.2007b),and/orbyaltering

photosyntheticcarbonmetabolism(e.g.Beeretal.1980a).Obtainingfurtherinformationregar-

ding the effects of hypersalinity on the photosynthetic physiology of Mediterranean seagrass

species is thereforecrucial tounderstanding their specific tolerances to increased salinity, and

also to establishing scientific and technical criteria for preventing and controlling human im-

pactsinvolvingchangesinseawatersalinity(e.g.desalinationplants;PalomarandLosada2010).

In this study,weexaminedfor thefirst time: (a) thebehaviourof thephotosyntheticphy-

siology of Mediterranean C. nodosa populations in response to sudden and chronic salinity in-

creases, simulated ina laboratorymesocosmsystem;and(b) relationshipwith leafcarbonba-

lance and growth and shoot survival. To this end, pigment content (chlorophyll a, chlorophyll

b and total carotenoids), leaf optical properties,maximumphotochemical efficiency (Fv/Fm),

photosynthesis-irradiance curves (i.e. photosynthetic parameters), leaf growth and shoot featu-

res and survival were measured for C. nodosa plants under ambient salinity (i.e. 37) and com-

pared with measurements performed on plants simultaneous exposed for 45 days to various

degrees of chronic salinity increase. This study formed part of a broader experiment aimed at

analysing the physiological responses of Mediterranean seagrass species to salinity increa-

ses, which simultaneously involved both P. oceanica and C. nodosa in order to provide reliable

comparisons between these species.Only data regarding C. nodosa photosynthesis (and res-

piration) are presented here, which are discussed in relation to previously-published data ob-

tained during the same experiment for P. oceanica (Marín-Guirao et al. 2011; Chapter 2).

3.3. MATERIAL AND METHODS

3.3.1. FIELD PLANT COLLECTION AND MESOCOSM SYSTEM

InOctober2008,scubadiverscollectedlargeC. nodosa fragments (shoots connected to basal

rhizomeswithintactrootsystems)fromadense,shallowmeadow(atadepthof5–6m)colonising

infralittoralsedimentsonthesoutheasterncoastofSpain(IslaPlana,MurciaRegion;37º34´20.86´´

91

N,1º12´28.16´´W).Thislocalitywascharacterisedbyhighlyoligotrophicwaterswithconstantsali-

nity(37–38psu-PracticalSalinityScale)andseasonalfluctuationsindownwardirradiance(5.3–19.8

molquantaday-1)andtemperature(14.6–24.8ºC)(seeAnnex1),whicharetypicalconditionsfor

shallowinfralittoralhabitatsoftheSpanishMediterraneancoast(Margalef1985;Hofrichter2004).

The plants were transported in large coolers to the laboratory, arriving within 4 hours of co-

llection.Heretheywereintroducedintotheaquariaofthemesocosmsystemforacclimatisation

(seebelow).Fiveplantfragmentswereplacedinaplasticbasket(22x40cmbaseand10cmin

height)filledwithcoarsesediments,whichhadbeenpreviouslywashedtoremoveanimalsand

largeorganicparticles.Thisrepresentedanexperimentalunit(EU;Fig.3.1).Allplantfragments

included the apical meristem and were carefully selected to have comparable basal rhizomes,

withsimilarlengths(35–45cm)andnumbersofshoots(5–10),inordertostandardisetheexperi-

ment.TheoverallnumberofshootsperEUwasbetween40and50,withatotalof24EUsbeing

prepared.SixEUswererandomlydispersedthroughoutthecross-sectionofeachlargeaquarium

ofthemesocosmsystem(seebelow),togetherwithEUscontainingP. oceanica fragments, which

weresimultaneouslyexaminedinthesameexperiment(Marín-Guiraoetal.2011;Chapter2).

Themesocosm (Fig. 3.1) consistedof four large (1500L), independent aquaria,

each with an autonomous circuit of circulating seawater and independent control of

waterflow,temperature,irradiance,pHandnutrients(formoredetails,seeAnnex2).

Asexplainedhereand in theChapter2 (sectionofMaterialandmethods), theprecisecon-

trol of environmental parameters within the mesocosm system was one of our major con-

cerns.Theaimwas to: (a)maintain spatialhomogeneityof such factorswithinandbetween

theaquaria, (b) tosimulateanaverageenvironmentalbackground representativeof thatex-

periencedby theplants innaturalconditions,and(c) toavoidartificialconditions thatcould

cause additional stress for the plants. Previous and current experiments have demonstrated

that this mesocosm system can successfully maintain healthy plants of C. nodosa and P. oce-

anica for periods of up to 4months, with almost 100% survival and performances similar to

those observed under natural conditions. This is noteworthy, considering that performing such

experiments under laboratory conditions is not feasible for most seagrass species, especially

92

the larger ones. Investigations involving such species are usually undertaken in small aqua-

ria on plant fragments of restricted size, which means the plants can only be kept healthy for

periods of about 2 or 3weeks (e.g. Fernández-Torquemada and Sánchez-Lizaso 2005;Ca-

yabyabandEnríquez2007;Pagèsetal.2010).Thisclearly limitsthetemporalscaleofexpe-

rimentalmanipulationsandplantresponses(Oksanen2001).Inordertoassesspossibleaddi-

tionalstresscausedbytheartificialconditions,measurementsfromtheaquariawerecompared

with those obtained simultaneously in the natural meadow (see section 3.3.7 and Table 3.6).

Figure 3.1. General view of the mesocosms system (up) and C. nodosa in the experimental units (down).

93

3.3.2. EXPERIMENTAL DESIGN

For plant acclimatisation, the EUs were maintained in the aquarium for 1 week prior to

the start of the experimental treatments, under environmental conditions similar to tho-

se experienced by the natural population: a temperature of 21 ± 0.1ºC, a salinity of 37

± 0.1 psu and a saturating irradiance of ca. 300 ± 30 μmol quanta m-2 s-1. A 12/12 hour

photoperiod was selected, in order to provide a daily light exposure of 12.9 mol quan-

ta m-2 day-1, which was close to that obtained by averaging daily integrated light cur-

ves recorded in the field at the top of themeadow leaf canopy (Marín-Guirao et al. 2011).

Followingtheacclimatisationperiod,salinitylevels(psu)wereadjustedtoobtaintheexpe-

rimentaltreatmentselectedforeachaquarium,whileallotherparameters(light,temperature,

pH,nutrientsandwaterflow)werekeptconstant.Tothisend,oneaquariumwasmaintainedat

ambientsalinity(i.e.37;thecontroltreatment),whilesalinitywas increasedintheotherthree

aquariumstoobtainhypersalinetreatmentsof39,41and43.Salinitytreatmentswererandomly

assigned to each large aquarium.These salinity levelswere achievedby addinghighquality

marinesalt(Seachem®)followingtheprotocoldescribedbyMarín-Guiraoetal.2011(Chap-

ter2).Theseexperimentalsalinitylevelsencompassedthemeasuredrangeexperiencedinthe

Mediterranean by C. nodosaundernaturalconditions(36.5–38;Annex1)andwhenexposedto

brinedischarges(Fernández-Torquemadaetal.2005a;Fernández-TorquemadaandSánchez-

Lizaso2006).Theexperimentalconditionsweremaintainedforaperiodof47days.Weconsi-

deredboththeintensityanddurationoftheexperimenttobesufficienttoallowspecificplant

stress responses,butnotsogreatas to induceseveremetabolic impairmentand/ordamage.

Allplantresponsevariablesweremeasuredattheendoftheexperimentalperiod.Foreach

variable, except number of shoots (see below for the variable types), the average of two or

three measurements performed on leaves from different shoots provided the value used for

eachEU,whichwe considered the replicatedunit in this experiment.WithinEUs, each leaf

came fromadifferent shoot, collected fromadifferentplant fragment.Onlyhealthy shoots

(i.e.withoutwoundsorherbivoredamage)ofasimilarage(3–5yearsold)wereselected.We

94

avoided collection of the youngest (i.e. those closest to the apical shoot) and neighbouring

shoots within the same plant fragment, to circumvent any effects of spatial dependency

due to internal resource gradients between sinks (i.e. apical, pioneer ramets) and source in-

dividuals (Marbá et al. 2002;Olivé et al. 2009). For each shoot,measurements and analy-

ses were always performed on mature photosynthetic tissues of leaf rank number 1 or 2.

In such an experimental design, differences in plant variables between treatment levels may

besubject toa riskofconfounding, sinceallEUs fromaparticular treatment levelwerepla-

ced in the same largeaquarium (QuinnandKeough2002).This approachwasunavoidable

for several reasonsalreadyexplained in theMaterial andMethods sectionof theChapter2.

Giventhesecircumstances,andbasedonourknowledgeofthespecificbiologicalandexperi-

mentalsystems,wefollowedtherecommendationsofOksanen(2001)andQuinnandKeough

(2002), in order to identify, control and reduce the probability that local factors other than

the treatmentswould confound theeffectsof experimental treatmentsbetween theaquaria.

Inthissense,asexplainedabove,theefficientcontrolofenvironmentalparameterswithinthe

mesocosm systemprovidedus confidence that factorsother than salinitydidnotdifferbet-

weentheaquaria.Furthermore,someplantvariables(Fv/Fm,Pvs.Ecurves,pigmentcontent

andleafgrowth)measuredduringtheacclimatisationperiodexhibitednosignificantdifferen-

cesbetween aquaria (ANOVA,P >0.05).Thus,we are confident that differencesbetween

treatments at the end of the experimental period were due to divergence in time trajectories

imposed by the treatments from the onset of the experiment and not to pre-existing differences.

3.3.3.. CHLOROPHYLL FLUORESCENCE

Measurements of chlorophyll afluorescenceemissionswereperformedusingadiving-PAM

portablefluorometer(Walz,Germany)ondark-adaptedleavesinordertocalculatethemaximum

quantumyieldofPSII(Fv/Fm),whichrepresentsameasureofthephotochemicalefficiencyofthe

PSIIwithallreactioncentresopen(Schreiber2004;Larkumetal.2006a)(seeMaterialandMe-

thods,section2.3.3.,Chapter2,formoredetails).Priortotheexperiment,fluorescenceparame-

ters measured along C. nodosaleavesconfirmedthetypicalvariationpatternasafunctionofleaf

95

age, as previously described for another seagrass species, Thalassia testudinum (Durako and Kun-

zelman2002;Enríquezetal.2002).Sincethissourceofvariationcouldmaskpossibletreatment

effects,fluorescencemeasurementswereperformedatregular1cmintervals,fromleafbaseto

apex,forallleavespresentonashoot(2–3leavespershoot).Fromthissetofmeasurements,the

maximumFv/Fm value was selected to represent the whole shoot. These measurements were

performedonthreerandomly-selectedshootsfromeachEUoneachsamplingoccasion(n=

6replicatespertreatment).Sincethediving-PAMfluorometerallowed in vivo measurements

withoutalteringthestateoftheplants(Beeretal.2001),thesemeasurementswereperformed

once aweek to assess variations inmeanFv/Fm values throughout the experimental period.

3.3.4. RESPIRATION AND PHOTOSYNTHETIC RATES

Photosynthesis-irradiance (P vs. E) curves for C. nodosa leaves were determined, fo-

llowingWalker (1987) andCayabyab and Enríquez (2007), for two shoots from threeof

the sixEUs, randomly selected fromeach aquarium (n = 3 replicatesper treatment).Res-

piration and photosynthetic rates were measured polarographically, using a DW3 cham-

ber with a Clark-type O2 electrode system (Hansatech, UK). The incubation chamber

was filled with filtered seawater and maintained at the same temperature as the aquaria

(21ºC). Leaf segments of approximately 0.6 cm2 from themiddle portion of the firstma-

ture leaf were used for photosynthesis measurements on each shoot. This leaf portion re-

presented the maximum Fv/Fm values, as previously measured on C. nodosa shoots.

TheprotocolemployedforthePvs.Ecurves(andthevariablesobtainedfromthem)are

describedindetailintheChapter2(Materialandmethodssection).Variablesexaminedwere

dark respiration (initial-Rd, μmolO2 cm-2 h-1), the maximum rate of net photosynthesis (net-Pmax,

μmolO2 cm-2 h-1 ),saturatingirradiance(Ek, μmolquantam-2 s-1

), gross photosynthesis (gross-

Pmax, μmolO2 cm-2 h-1),photosyntheticefficiency(α, μmolO2 cm-2 h-1/μmolquantam-2 s-1) and

compensationirradiance(Ec, μmolquantam-2 s-1).Afterthefinallightexposure,leafsegments

werere-exposedtodarkness,inordertodeterminethefinaldarkrespirationrate(final-Rd). The

P:Rd ratio was used as a proxy for the daily leaf carbon balance, P being calculated by multiplying

96

gross-Pmax bythenumberoflighthours(i.e.12),withRdbeingthetotalrespirationovera24-hour

period,usingfinal-Rdforthelightperiod(12hours)andinitial-Rdforthedarkperiod(12hours).

3.3.5. PIGMENT CONTENT AND LEAF OPTICS

PigmentcontentmeasurementswereperformedontwoshootsfromeachEU(n=6repli-

cates per treatment). The leaf portions examined were selected using the same criteria adopted

forthePvs.Ecurves(i.e.maximumFv/Fmvalues).Thepigmentswereextractedfrom0.8cm2

leafsegmentsusing80%acetone(seetheprotocoldescribedinChapter2,MaterialandMe-

thods). The Chla and Chlb concentrations, as well as the total carotenoid concentration, were

expressedaspigmentdensity(i.e.pigmentperabsorptioncross-section;Enríquezetal.1992).

Prior to pigment analysis, the optical properties of intact C. nodosa leaves were measured spec-

trophotometrically,onthesameleafsegments,usingtheopalglasstechnique(seeChapter2).

For each leaf segment, absorbance (D)wasmeasured in the PAR range (380–750 nm)

at a resolution of 1 nm. Leaf absorption was expressed as absorptance (A), which is the frac-

tionof incident lightabsorbed(Kirk1994),calculatedusingtheequationA=1-10-D. The ab-

sorbance value at the 680nmChla peak was used, since it is a good descriptor of average

PARabsorption(Enríquezetal.1992;Olesenetal.2002).Specificabsorptioncoefficientswere

usedasdescriptorsoftheefficiencyofpigmentsand leafmassatabsorbing light inseagras-

ses(Enríquezetal.2002;CayabyabandEnríquez2007).Thechlorophylla-specificandmass-

specificabsorptioncoefficientswereestimatedfromtheexponentintheexponentialfunction

[exp=-ln(1-A)],withvaluesnormalisedtopigmentcontent(a*;m2 mg-1 Chla) and leaf mass

(aw*;cm2 mg-1leafDW),respectively.Variationsintheabsorptionefficiencyofseagrassleaves

are a function of pigment content and also of leafmorphology and/or the packaging of its

mass (Enríquez 2005). In order to determine the contributionof leaf anatomyon variations

in leafopticalproperties, the leafmassperarea index(LMA;gDWcm-2) was estimated by

measuring the area and weight of each leaf segment employed for pigment determinations.

97

3.3.6. LEAF MORPHOLOGY, GROWTH RATE AND SHOOT SURVIVAL

Atthebeginningoftheexperimentalperiod,allshootsineachEUwerecountedandthree

randomly-selected shoots were marked for leaf growth determinations (n = 6 replicates per

treatment)followingthemethodsdescribedbyZieman(1974)andPérezandRomero(1994).

Themarkedshootswerecollectedattheendoftheexperimentalperiod.Foreachmarkedshoot,

the number of leaves was counted and the length and width of each leaf was measured, in order

to calculate shoot size on a per-area basis (cm2 shoot-1). Newly-formed leaf segments (those be-

low the mark) were separated from the rest and measured, in order to estimate shoot leaf growth

rate from leaf elongation (cm2 shoot-1 day-1). The surface area of necrosis marks on the leaves was

also measured and expressed as a proportion of necrotic photosynthetic tissue per shoot. Shoot

counts were repeated at the end of the experimental period to estimate shoot survival. Differen-

cesbetweeninitialandfinalshootnumbersforeachEUwerenormalisedtoinitialshootnumbers

and expressed as a percentage of net change. Negative values indicated a net decline from the

initial shoot number, resulting from higher mortality rates than shoot recruitment rates (shoot divi-

sionorrhizomebranching)and/orreducedshootrecruitmentrates(Ruizetal.2009;Chapter1).

3.3.7. FIELD REFERENCE

Mean values of some plant variables obtained in the laboratory were compared with

reference values measured in the field during the same season and in the same natu-

ral meadows from which the plants were collected for this experiment. These field ob-

servations formed part of a seasonal sampling of environmental and plant variables

performed in these meadows from October 2008 to July 2009 (see Annex 1). Data co-

rresponding to the autumn-winter 2008 campaign are presented in Table 3.6. This com-

parison was considered to be necessary in order to address issues, such as the possible stress

caused by the artificial conditions, which could confound the effects of the experimental

treatments on the physiological and vegetative responses of the studied seagrass species.

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3.3.8. STATISTICAL ANALYSIS

A one-way ANOVA was used to assess significant treatment effects (a fixed factor

with four salinity levels: 37 (control), 39, 41 and 43) on the response variables at the endof

theexperimentalperiod.TheeffectofsalinitytreatmentsonFv/Fm time trends was analysed

usingtwo-wayANOVA,withsalinityandtime(8samplingoccasions)as thefixedfactors in

the model. A non-parametric Kruskal-Wallis test was used when assumptions of normality

and homoscedasticity were not satisfied. Post-hoc mean comparisons (Student-Newman-

Keuls, SNK; Zar 1984) were performed to identify which specific treatment level(s) produ-

cedsignificanteffects.Treatmenteffectswereconsideredstatistically significantatP<0.05.

3.4. RESULTS

The mean maximum quantum yield of PSII (Fv/Fm) for C. nodosa leaves varied bet-

ween 0.773 ± 0.002 and 0.790 ± 0.001. Salinity had a significant effect on this varia-

ble, although this was transitory since it was only detected during sampling episodes 6

and 7 (i.e. 25 and 32 days from the onset of the experimental treatments; Fig. 3.2 and

Table 3.1). These mean values fell within the reference ranges measured in the natu-

ral meadow (Table 3.6) and those obtained from healthy plants of other species (Ral-

ph 1999;Durako andKunzelman 2002; Ralph et al. 2002;Cayabyab andEnríquez 2007).

99

Figure 3.2. Evolution of the mean maximum quantum yield (Fv/Fm) of C. nodosa leaves in each experimental salinity treatment (psu) throughout the experimental period: Control (37, solid circles), 39 (empty circles), 41 (solid triangles) and 43 (empty triangles). Zero at the X-axis (time, days) represents the onset of experimental treatments and the negative part corresponds to the acclimation period. Values are mean ± SE, n = 6.

Table 3.1. Summary of the ANOVA test performed to assess significant effects of salinity treatments on the course of mean Fv/Fm values in C. nodosa leaves throughout the experimental period. df = degrees of freedom; MS = mean squares; p = probability level; ns = not significant; * P < 0.01.

100

ThePvs.Ecurvesobtainedfromthefourtreatmentsattheendoftheexperimentalexposu-

reareshowninFig.3.3.Thesalinitytreatmentshadsignificanteffectsonthephotosyntheticpa-

rametersderivedfromthePvs.Ecurves(Fig.3.4andTable3.2).Themeannet-Pmax of plants

from the control treatment was 1.37 ±0.002μmolO2 cm-2 h-1,whereasthisvaluewassignificantly

decreased,by12–17%,inthehypersalinetreatments.Themeangross-Pmax demonstrated similar

valuesbetweenthe37to41salinitytreatments,butwassignificantlydecreased,by19.6%,inthe

mostseveresalinity(43).Darkrespirationratesvariedsignificantlybetweenthetreatments(Fig.

3.4andTable3.2).Meaninitial-Rdwas16.9%and41.5%higherinthe39and41treatmentcondi-

tions,respectively,thaninplantsfromthecontrol(-0.154±0.004μmolO2 cm-2 h-1).Meanfinal-

Rd exhibited a similar pattern of variation with salinity, but with higher differences between the

treatments:themeanvaluesobtainedfrom39and41were75–85%higherthanthosemeasured

inthecontrol(0.171±0.001μmolO2 cm-2 h-1).Valuesofinitial-andfinal-Rd were reduced by

11%and17%,respectively,inplantsexposedtothemostseveresalinitytreatment(43)inrelation

to the control. Relative to themean control values,meanphotosynthetic efficiency (α) was

significantlyincreasedinthe39and41salinitytreatments(by15–18.5%),whilethesaturationirra-

diance(Ek)decreasedbyapproximately28%.Compensationirradiances(Ec)weresignificantly

different between all the treatments, although the largest effect was observed in plants from the

43salinitytreatment,inwhichthisparameterwasreducedby23%relativetothemeancontrol

value.TheP:Rdratioindicatedahighly-positivedailycarbonbalanceinthecontrolplants(4.008

±0.09).Thisratiowassignificantlylowerinthe39and41salinities(31%and33%,respectively),

but similar to that detected in leaves from plants exposed to the most saline treatment (43).

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Figure 3.3. Oxygen evolution rates (mean ± SE, n = 3) as a function of irradiance obtained for C. nodosa leaves after 47 days of exposure to each experimental salinity treatment: Control (37, solid circles), 39 (empty circles), 41 (solid triangles) and 43 (empty triangles). Values measured in the dark (0 μmol quanta m-2 s-1) correspond to initial Rd values and those measured under illuminated conditions are net photosynthetic rates.

Table 3.2. Summary of one-way ANOVA performed to test significant differences between P vs. E curve parameters of C. nodosa at the end of the experimental period for each salinity treatment. df = degrees of freedom; MS = mean squares; F = Fisher statistic; P = probability level; *P < 0.01, **P < 0.001; ns = not significant. Groups of homogeneous means obtained by the post-hoc SNK test are indicated by different letters.

102

Figure 3.4. Variations in photosynthetic parameters (mean ± SE, n = 3) of C. nodosa leaves derived from P vs. E curves with salinity treatments. Net-Pmax = net maximum photosynthe-tic rate; gross-Pmax = gross maximum photosynthetic rate; initial-Rd = initial dark respiration rate; final-Rd = final dark respiration rate; α = photosynthetic efficiency; Ek = saturation irradiance; Ec= compensation irradiance.

Table 3.3. Mean values (SE) of pigment concentrations in C. nodosa leaves obtained for each experimental treatment at the end of the experimental period, and summary of the one-way ANOVA applied to test significant differences between treatments. df = degrees of freedom (3,20); MS = mean squares; F = Fisher statistic; P = probability level; *P < 0.01, **P < 0.001. Groups of homogeneous means obtained by the post-hoc SNK test are indicated by different letters.

103

Pigment contents (Chla, Chlb and total carotenoids) in C. nodosa leaves were also affected by

the salinity treatments (Table 3.3). Mean Chla concentration decreased as salinity increased, es-

peciallyinthemostseverehypersalinetreatment(43),wheremeanvaluesweresignificantlylower

(18.2%)thanthecontrolmean.TheconcentrationofChlb and the Chlb:a ratio both increased

significantlyinthe39and41salinitytreatments(by6–9%and5–7%,respectively),comparedto

the mean values of the control and 43 treatments. Total carotenoid concentrations were increased

in the tissues from all treatments, although the largest differences were in the leaves from the 39

and41treatments,whichhadmeanvaluessignificantlyhigher(by10–17%)thanthecontrol.The

carotenoid:Chlaratioincreasedsignificantly(by9–15%)inallhypersalinetreatments(Table3.3).

The leaf optics results from C. nodosa leaves obtained from each experimental treatment

areshowninFig3.5andTable3.4.Thesalinitytreatmentshadsignificanteffectsonleafab-

sorptance(A),specificabsorptioncoefficients(a*andaw*)andthe leafmassperarea index

(LMA).Awas0.845± 0.002 in plants from the control treatment,whichwas slightly lower

than that obtained by Silva and Santos (2003) from anAtlantic C. nodosa meadow. Similar

Avalueswere obtained from themost severe hypersaline condition (43), butAwas signifi-

cantlyhigher(0.87–0.88) inplantsexposedto intermediatesalinity increases(i.e.39and41).

Themeanvalueofthespecificabsorptioncoefficientsshowedaconstantlinearincreasebet-

weenthe37and41treatments(R=0.999,P<0.05),butnofurtherincreaseinthisvariablewas

observedinsalinity43whenthiscoefficientwasstandardisedtopigmentcontent(a*)oreven

lower values when it was relativised to leaf mass (aw*).TheLMAof the leaf segments em-

ployedforpigmentandabsorptancemeasurementsdecreasedsignificantly inthehypersaline

treatments, indicating a lower degree of biomass packing in leaves under hypersaline conditions.

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Figure 3.5. Variations in optical properties (mean ± SE, n = 6) of C. nodosa leaves with salinity treatment: light absorption spectra and leaf absorptance (upper panels), and pigment-specific absorption coefficients (a*, aw*) and leaf mass per area (LMA) or biomass packing (lower panels).

Table 3.4.Summary of the one-way ANOVA performed to test significant differences between leaf optical descriptors of C. nodosa at the end of the experimental period at each salinity treatment. df = degrees of freedom; MS = means squares; F = Fisher statistic;p = probability level; *P < 0.01, **P < 0.001. Groups of homogeneous means obtained by the post-hoc SNK test are indicated by different letters.

105

Shootsizeandleafgrowthrateswerenotsignificantlyaffectedbythesalinitytreatments,

although the lowest mean values were observed in shoots from the 41 and 43 salinity treatments

(Table3.5).Thefrequencydistributionofthenumberofleavespershootineachexperimental

treatment(Fig.3.6)exhibitedsignificantdifferencesbetweentreatments(χ2=78.6,P<0.0001),

mainlyduetoanincreaseinthepercentageofshootswithonlyoneleaf(thefirstandyoungest

leaf)inthemostseverehypersalinetreatment(36%ofshootsinthe43treatmentcomparedwith

10–23%intheremainder).Significantdifferenceswerealsoidentifiedintheleafnecroticarea

percentage,althoughtherewasnoapparentrelationshipwithsalinity.Whilesomewhathigher

mean values were observed in plants from the most severe salinity treatments (41 and 43), leaf

necrosis was always found in the apical parts of the oldest leaves, indicating leaf senescence. The

net change in initial shoot numbers was negligible for all experimental treatments (Table 3.5).

Mean values of photosynthetic parameters (Pmax, α and Rd), pigment concentra-

tions and leaf growth rates obtained from the control treatment at the end of the ex-

perimental period were all within those ranges observed in the reference C. nodo-

sa meadow during the season in which the experiment was performed (Table 3.6).

Figure 3.6. Percentage of C. nodosa shoots with 1, 2 or 3 leaves after 47 days of exposure to each experimental treatment.

106

Table 3.5. Mean values (SE) of shoot variables of C. nodosa and summary of the one-way ANOVA and non-parametric Kruskal-Wallis H test performed to assess differences between salinity treatments measured at the end of the experimental period. ns = not significant; MS = mean squares; F = Fisher statistic; P = probability level; *P < 0.05; H = Kruskal-Wallis statistic. Groups of homogeneous means obtained by the post-hoc SNK test are indicated by different letters.

Table 3.6. Reference values of some C. nodosa variables selected for this study measured in the field. SE = standard error.

107

3.5. DISCUSSION

The physiological and vegetative behaviour of those plants maintained at ambient salinity

(37; control treatment) in themesocosm system for47dayswas remarkably similar to thatdi-

rectly observed in their natural meadow during the same season. This indicated the reliability

of the experimental system to reproduce optimum environmental and physiological conditions

for C. nodosa in the laboratory.Shootsurvival in thiscontrol treatmentwas100%,emphasising

the successof themesocosm system inmaintainingplant vitality for experimentalperiods 3–5

times longer than those employed in previous laboratory experiments involving other Medite-

rranean C. nodosa populations (Fernández-Torquemada and Sánchez-Lizaso 2006; Pagés

etal. 2010).More importantly, these results indicated that theartificial conditionselicited little

or negligible stress on the observed plant behaviour, which was, therefore, primarily a respon-

se to the experimental treatments (salinity increases) and not to other confounding effects.

The photosynthetic physiology of the C. nodosa plants used in this study was signifi-

cantly altered following exposure to chronic salinity increases, above the average level at

which they are adapted to grow under natural conditions (i.e. infralittoral Mediterranean ha-

bitats with a constant salinity of 37). Photosynthesis inhibition is a phenomenon generally

observed in this (Drew 1978a), and other (Ogata and Matsui 1964; Biebl and McRoy 1971;

Kerr andStrother 1985;Fernández-Torquemadaet al. 2005b;KahnandDurako2006;Koch

et al. 2007b), seagrass species when exposed to hypersaline stress. Accordingly, net pho-

tosynthesis was partially and consistently inhibited in all hypersaline treatments in the pre-

sent study, although the results obtained for other physiological variables suggested that

the nature of the inhibition differed depending on the severity of the hypersaline stress.

At intermediate salinity increases (39 and 41), gross photosynthesis remained close to control

values and the reduction in net photosynthesis was accounted for by the enhancement of dark

respiration rates observed only in these treatments. An increase in leaf respiration is a common

physiologicalresponseofplantstohypersalinestress,reflectinganenhancedmetabolicdemand

inordertocopewithosmoregulatoryadjustmentsand/orothermetabolicalterationsinducedby

increasedsalinity(Yeo1983;ZhuandMeinzer1999;Munns2002;ParidaandDas2005).

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Anothercleareffectinducedbyhypersalinestresswasanimprovedcapacityandefficiency

of C. nodosaleavestocaptureandprocesslight.Thisconclusionwasbasedonthesignificantly

highervaluesforleafabsorptance,pigmentsandleafmasslightabsorptionefficiency(specific

absorptioncoefficients) andphotosyntheticefficiency (α), anda significantly lower saturation

irradiance (Ek), in plants exposed to the 39 and 41 treatments, compared with control plants.

This enhancement of light absorption was consistent with the increase in accessory pigments

in these treatments (Chlb, carotenoids, Chlb:aandcarotenoids:Chla) and could also be related

to the lower LMA mean values measured in C. nodosa leaves, which are indicative of a less-

packed (more-expanded) photosynthetic leaf biomass. These changes in leaf anatomy have been

shown to modify the effects of complex phenomena characteristics of multicellular tissues on the

optical propertiesof seagrass leaves and their light absorptionefficiency (e.g.packageeffect

andmultiplescatter;Enríquezetal. 1992;Enríquez2005). It isnoteworthy thatmostof these

responses resemble acclimatisation responses demonstrated by aquatic plants (Kirk 1994; Fa-

lkowskyandRaven2007),includingsomeseagrassspecies(RuizandRomero2001;Olesenetal.

2002;CayabyabandEnríquez2007;Ralphetal.2007),tolowlightconditions.Consequently,

the reported photosynthetic responses could be interpreted as a compensatory mechanism dri-

ven by C. nodosa leaves to reduce light requirements and to alleviate thenegativeeffectsof

respiratorylossesontheleafcarbonbalance(e.g.DennisonandAlberte1985;RuizandRomero

2001).No suchphotoacclimatory responseswere reported forP. oceanica when it was simul-

taneouslyexposedtothesameexperimentalconditions(Marín-Guiraoetal.2011;Chapter2).

Contrary to the enhanced respiration detected in intermediate salinity treatments (39 and 41),

respirationratessignificantlydecreased,by11–17%,inthemostseverehypersalinecondition(43),

compared with the control mean values. The complex behaviour exhibited by this variable as sa-

linityincreasedwasconsistentwithsimilarresponsespreviouslydescribedforseagrasses(Ogata

andMatsui1964;OgataandTakada1968;BieblandMcRoy1971;Drew1978a;KerrandStrother

1985;Fernández-Torquemadaetal.2005b).Therefore,inthiscase,inhibitionofgrossphotosyn-

thesis was the cause of the lowered net photosynthesis in the most extreme saline treatment (43).

Suchphotosyntheticinhibitionmaybeaconsequenceofimpairmentsand/orinjuriescausedby

salts at different levels of the photosynthetic process (chloroplast ultrastructure, pigment content,

109

photochemicalefficiency,photophosphorylationactivity,electrontransportrateandenzymes

involved in carbon assimilation and reduction), as has been demonstrated in terrestrial plants

andmacroalgaeexposedtohypersalinestress(Kirst1989;KramerandBoyer1995;Atharand

Ashraf2005;HuchzermeyerandKoyro2005).Itwasnotpossibletodiscerntheinhibitoryme-

chanism involved on the basis of our results, and direct or indirect evidence is rather scarce for C.

nodosa(Drew1978a),asisthecaseformostseagrassspecies(forareview,seeTouchette2007).

Beeretal. (1980a)providedsomeexperimentalevidencethat theactivityof thecarbon-

fixingenzymeRuBisCopresentintheepidermisofHalodule uninervis was gradually inhibited

by increasing NaCl concentrations in in vitro assays. Inhibition of photosynthesis induced by

hypersaline stress has also been associated with conformational changes of the photosynthetic

apparatus (McMillan andMoseley 1967; Jagels andBarnabas 1989; Iyer andBarnabas 1993;

Ralph1998,1999).Decreasesinpigmentcontentand/orphotochemicalefficiencyhavebeen

reported in some seagrass species in relation to hypersaline stress, as a symptom of damage to

photosyntheticstructures(e.g.Ralph1998,1999;Kammermansetal.1999;Murphyetal.2003;

KahnandDurako2006;Kochetal.2007b;Pagèsetal.2010),althoughsuchsymptomswere

only apparent under hypersaline conditions far more extreme than those studied here. Consis-

tently, negative effects of hypersaline stress on the structure and function of the photosynthetic

apparatusoflandplantsusuallyoccurinextremeconditions(HuchzemeyerandKoyro2005).

The decrease in chlorophyll density (Chla and Chlb) reported in salinity treatment 43 could

alsobe interpretedinthiscontext,althoughitcouldalternativelybeaconsequenceofame-

tabolic down-regulation of light harvesting, in order to adjust light capture to compensate for

reduced carbon assimilation (Demmig-Adams and Adams 1996). In accordance with the latter

possibility, Fv/Fm values provided no evidence that hypersaline treatments caused the accu-

mulated damage to the photosystems and antennae systems, even in the most severe hyper-

salinetreatment.Whatever themechanism involved, thepartial inhibitionofgrossphotosyn-

thesis reported in the most severe hypersaline treatment should contribute in the impairment

oftheleafcarbonbalance.AsclearlyindicatedbythehighleafP:Rratioestimatedfromthis

treatment (similar to the control), this adverse situation was primarily counterbalanced by limi-

110

ting the dark respiration rate to a level below that of the control mean. This particular respiration

rate behaviour could be interpreted as a disruption of cellular respiration or, alternatively, as

a control mechanism to cope with the demands of an altered metabolism, to enable survival

under extremely stressful conditions. A similar control of respiratory activity has been typica-

llydescribed in terrestrialplants (KramerandBoyer 1995;Lambersetal.2006b)andmarine

macrophytes (Ogata andTakada 1968;Kirst 1989) followingexposure tohypersaline condi-

tions and has been associated with a general decline in carbon metabolism and the starvation

of respiratory substrates, or its diversion to osmoregulatory and osmoprotectant functions.

In addition to these possible acclimatisation mechanisms, our results suggest that C. nodosa

maydevelopadditionalmechanismsinordertohelpitcopewithhypersalinestress.Forinstan-

ce,the increasedtotalcarotenoidcontent(andthecarotenoid:Chla ratio) observed in all the

hypersaline treatments could be related to the activation of general protective mechanisms by

the photosynthetic structures against photo-damage and oxidative stress induced by hypersaline

stress(Demmig-AdamsandAdams1996;Ralphetal.2002;AtharandAshraf2005;Paridaand

Das2005).ThisevidencewasalsofoundinthedominantMediterraneanseagrassP. oceanica

underthesameexperimentalconditions(Marín-Guiraoetal.2011;Chapter2).Inthatseagrass

species, leaf light absorption,photosyntheticefficiency,photosynthetic rates (net andgross)

andleafcarbonbalance(withP:R45–56%lowerthancontrolmeans)wereallmorenegatively

affected by the same hypersaline treatments than C. nodosa, even for the lowest salinity increase

(39).Theseseverephysiologicalalterationsclearlyinterferedwiththemetabolicrequirements

for growth and survival in P. oceanica shoots. Comparatively, the metabolic carbon balance of

C. nodosaleaveswasmuchlessaffectedwithinthesamesalinityrange(31–33%,andonlyinthe

intermediate salinity treatments), with no apparent disruption of leaf growth and shoot survi-

val. Thus, under the same experimental conditions, it is clear that P. oceanica is physiologically

more sensitive to hypersaline stress than C. nodosa, and that the plasticity of the photosynthe-

ticphysiologydescribedinthisstudylikelyrepresentsanadaptationmechanism.Evidenceof

such differential adaptation mechanisms was also found during the experiment documented

here, relating to the role of other key physiological aspects in the responses of these seagrass

speciestohypersalinestress,suchasthewaterrelations(Sandoval-Giletal.2012;Chapter4).

111

Previous short-term laboratory experiments established that C. nodosa is able to survive and

sustaingrowthinsalinitiesofupto41–44(Fernández-TorquemadaandSánchez-Lizaso2006;

Pagèsetal.2010).Ourresultsareconsistentwiththeseearlierstudies,butalsoprovidephysiolo-

gical evidence that these salinity levels must be close to the plant’s physiological tolerance thres-

hold. In effect, as explained before, the partial inhibition of gross photosynthesis and the redu-

ced respiratory activity reported from 43 salinity treatment enables plants to survive in this severe

stress condition, but presumably with reduced vitality, since some of the internal resources re-

quiredforgrowthandbiomassmaintenancemustbereallocatedtocopewithstressmetabolism

(Lichtenthaler 1996). The leaf loss reported for shoots from this treatment is consistent with such

asituation,andothersdescribedforseagrassesinrelationtootherstressors(e.g.lightlimitation;

Ralphetal.2007).Obviously,thistolerancethresholdisonlyvalidforC. nodosa populations

growing in open coastal waters of the Spanish Mediterranean Sea with a mean constant salinity

of37–38,andcannotbeextrapolated tootherhabitats, regionsor situationswithadifferent

salinity regime. There are, for instance, highly productive C. nodosa populations in hypersaline

lagoonsintheMediterraneanSea(e.g.upto47intheMarMenorlagoon;TerradosandRos

1991)forwhichahighertolerancethresholdisexpected(Fernández-TorquemadaandSánchez-

Lizaso2011),asaresultoflong-termgenotypicselection(e.g.Tomaselloetal.2009)andthe

possible existence of interactive effects between salinity and other key environmental factors that

alsovarybetweenhabitattypes(e.g.vanKatwijketal.1999;Kochetal.2007b).However,this

issue deserves further research on C. nodosa,combininglaboratoryexperimentsandfieldwork.

In conclusion, our study provides experimental evidence that Mediterranean popula-

tions of C. nodosa adapted to marine infralittoral environments with stable salinity are phy-

siologically more tolerant than P. oceanica to sudden and chronic increases in external sa-

linity.Thisfindinghas severalgeneralecological implications.Firstly, it is consistentwith the

distinct biologies and ecologies exhibited by these two seagrass species, and thus explains

their abundance and distribution throughout the various marine habitats off the Medite-

rranean coast. Secondly, it allows us to predict that natural populations of C. nodosa adap-

ted to Mediterranean open coastal environments will be more resistant to anthropic indu-

ced salinity increases (e.g. brine discharges from desalination plants) than neighbouring

112

meadows of the more sensitive P. oceanica. Nonetheless, care must be taken when extrapo-

lating conclusions from controlled laboratory experiments performed on short to medium ti-

mescales to more complex environmental situations in the nature over extended periods.

113

Shallow Cymodocea nodosa meadow (Murcia Region)

114

Cymodocea nodosa in the mesocosm system

115

4117 ABSTRACT

117 INTRODUCTION

120 MATERIAL AND METHODS

126 RESULTS

132 DISCUSSION

116

Jose Miguel Sandoval-Gil, Lázaro Marín-Guirao, Juan Manuel Ruiz. 2012Tolerance of Mediterranean seagrasses (Posidonia oceanica and Cymodocea nodosa) to hypersaline stress: water relations and

osmolyte concentrations

Marine Biology 159: 1129-1141

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4TOLERANCE OF MEDITERRANEAN SEAGRASSES (POSIDONIA OCEANICA AND CYMODOCEA NODOSA) TO HYPERSALINE STRESS: WATER RELATIONS AND OSMOLYTE CONCENTRATIONS

4.1. ABSTRACT

The present study examines for the first time the effects of increased salinity on water relations

and osmolyte (carbohydrates and amino acids) concentrations in two Mediterranean seagrass

species, Posidonia oceanica and Cymodocea nodosa, which are adapted to growth in environ-

ments with contrasting salinity and have a known differential sensitivity to alterations in ambient

salinity. The specific aim was to obtain insights into their respective capacities to cope with na-

tural or anthropogenically induced (e.g. desalination plants) hypersaline stress and its ecological

implications. To this end, large plant fragments of both seagrass species were maintained for 47

days in a laboratory mesocosm system under ambient salinity (37 psu; control) and three chronic

hypersaline conditions (39, 41 and 43 psu). Analyses of leaf tissue osmolality indicated that both

species followed a dehydration avoidance strategy, decreasing their leaf water potential (Ψw) as

the external salinity increased, but using different physiological mechanisms: whereas P. oceanica

leaves exhibited a reduction in osmotic potential (Ψπ), C. nodosa leaves maintained osmotic sta-

bility through a decrease in turgor pressure (Ψp) probably mediated through cell hardening pro-

cesses. Accordingly, the concentrations of soluble sugars and some amino acids (mainly Pro and

Gly) suggested the activation of osmoregulatory processes in P. oceanica , but not in C. nodosa

leaves. Osmotic adjustments probably interfered with leaf growth and shoot survival of P. oce-

anica under hypersaline stress, whereas C. nodosa showed a more efficient physiological capacity

to maintain plant performance under the same experimental conditions. These results are con-

sistent with the more euryhaline ecological behaviour of C. nodosa and contribute to unders-

tanding the high vulnerability shown by P. oceanica to even mild increments in seawater salinity.

4.2. INTRODUCTION

Seagrasses are clonal marine plants, forming one of the most ecologically relevant habitats

of temperate and tropical coastal marine environments (Green and Short 2003). Among other

primary factors (light, temperature and nutrient availability), salinity has been shown to be a cri-

tical factor influencing seagrass structure, productivity and distribution (McMillan and Moseley

1967; Zieman 1974; Walker and McComb 1990; Vermaat et al. 2000; Fernández-Torquemada

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et al. 2005a). The capacity to osmoregulate is a basic property of seagrasses that has allowed

them to evolve in and colonise marine environments (Arber 1920), but research in this area has

been scarce. As in terrestrial and aquatic plants (Hsiao 1973; Bisson and Kirst 1995; Verslues et al.

2006), seagrasses have developed different physiological strategies to enable growth under the

wide range of salinity regimes characteristic of the different coastal habitats in which they have

evolved (Tyerman 1989; Kuo and den Hartog 2000; Short et al. 2007; Touchette 2007). During

recent decades, human activities have had a profound impact on the environmental characteris-

tics of such coastal habitats, including changes in salinity due to water regulation infrastructures

(e.g. Boudouresque et al. 2009). The understanding of the tolerance of seagrasses to changes

in external salinity is still very poor, so our capacity to forecast the impact of salinity changes on

seagrass habitats is very limited. The analysis of plant water relations under varying conditions of

osmotic stress has traditionally been used as a basic tool for understanding the specific strategies

that each species employs to tolerate changes in external salinity (e.g. Kramer and Boyer 1995;

Ashraf and Athar 2009; Kahn and Weber 2006; Verslues et al. 2006), but very few studies have

focused on seagrasses (Tyerman 1982, 1989; Tyerman et al. 1984; Murphy et al. 2003; Koch et

al. 2007b; Touchette 2007), and no studies have been done on Mediterranean seagrass species.

Seagrass tissues have low water and osmotic potentials (Ψw and Ψπ, respectively) relati-

ve to seawater, which allows them to maintain water influxes and turgescence (Tyerman 1989;

Touchette 2007). As salinity increases, the Ψw gradient between the tissue and the external

medium is modified, decreasing water uptake and the capacity to maintain cell turgor. Under

such circumstances, the main mechanisms elicited by marine macrophytes (seagrasses and ma-

croalgae) to restore water status is the accumulation of additional solutes, a process known as

osmotic adjustment or osmoregulation. In the short term (minutes to hours and days), osmotic

adjustment can be easily achieved through cytosolic ion accumulation, mostly of Na+ and Cl-

(Flowers et al. 1977; Bisson and Kirst 1979; Tyerman 1982; Kirst 1989; Munns 2002; Touchette

2007). However, accumulation of these ions can exert adverse effects on metabolic processes

and they must, in the long term (days to weeks), be replaced by organic osmotically-active

metabolites termed osmolytes or compatible solutes (Wyn Jones and Gorham 1983; Kirst 1989;

Hasegawa el al. 2000; Marcum 2006). The type of compatible solutes produced depends on

119

species and other factors, and their functions are not restricted to only osmotic adjustment, but

also include protection of biochemical structures against ionic stress (Kramer and Boyer 1995).

Soluble carbohydrates, some soluble nitrogen-containing compounds (mainly amino acids such

as proline), and organic acids are among the most common types of osmolytes that have been

identified in seagrasses (Brock 1981; Pulich 1986; Tyerman 1989; Adams and Bate 1994a; Mur-

phy et al. 2003; Touchette 2007). Some authors have suggested that the high levels of proline

and soluble sugars measured in Mediterranean Cymodocea nodosa populations could account

for the osmotic stability of this seagrass species in a wide variety of saline environments (Pelle-

grini and Riouall 1973; Augier et al. 1976; Drew 1978a; Pirc and Wollenweber 1988; Pirc 1989).

However, to date, no direct or indirect evidence exists regarding the role of these metabolites

in osmoregulatory functions of Mediterranean seagrass species. Plants can also regulate Ψw

gradients under hypersaline conditions by passive solute accumulation resulting from loss of

water (reduced Ψp) and by cell wall hardening processes (Kramer and Boyer 1995; Verslues

et al. 2006). Cell wall hardening allow to species to develop rigid cell walls (i.e. high elastic

modulus, ε), which in turn allows plants to lose turgor without a substantial change in cell volu-

me and hence with minimal interference on cell metabolism and growth (Verslues et al. 2006).

Evidence of all these strategies has been reported for algae (Bisson and Kirst 1995) and some

seagrass species (Tyerman 1989), but its operation in Mediterranean seagrasses is unknown.

In the Mediterranean Sea, Posidonia oceanica and Cymodocea nodosa are the most abundant

seagrass species, forming extensive meadows at depths of up to 40 metres (Procaccini et al. 2003).

Hypersaline effluents delivered from desalination plants (or brines) actually represent a new po-

tential threat to seagrass communities (Morton et al. 1996; Boudouresque et al. 2009; Palomar and

Losada 2010). P. oceanica L. (Delile) is a large, slow-growing species that colonises open coastal

habitats with a very narrow range of salinity (e.g. from 36.5 to 38 psu on the Spanish Mediterra-

nean south-eastern coast; Ruiz et al. 2009), whereas the smaller C. nodosa (Ucria) Ascherson is

also present in estuarine systems with highly variable salinities and confined hypersaline lagoons

(e.g. Terrados and Ros 1991; Pérez and Romero 1994). This differential ecological behaviour is

consistent with the physiological and morphological plasticity described for both seagrass species

(Drew 1978b; Pirc and Wollenweber 1988; Pirc 1989; Olesen et al. 2002; Pérez and Romero 1994;

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Marbà et al. 2002), from which it is assumed that C. nodosa must be a more eurybiontic species

with a wider range of tolerance to key environmental factors than P. oceanica. Accordingly, recent

experimental evidence supports the hypothesis that the tolerance threshold of C. nodosa to hy-

persalinity is higher than that of P. oceanica (Fernández-Torquemada and Sánchez-Lizaso 2005,

2006; Gacia et al. 2007; Ruiz et al. 2009; Marín-Guirao et al. 2011), but the knowledge of the

overall mechanisms by which these species cope with hypersaline stress is still rather incomplete.

In the present study we investigate, for the first time, the physiological strategies utilised by

the Mediterranean seagrass species P. oceanica and C. nodosa to maintain osmotic equilibrium

under chronic hypersaline conditions similar to those imposed by brine effluents delivered from

desalination plants. Based on their respective biological and ecological attributes, C. nodosa was

expected to show greater efficiency in maintaining osmotic equilibrium than P. oceanica. This

basic knowledge is crucial to understanding the ecology of Mediterranean seagrass assembla-

ges, but also for forecasting the effects of anthropogenically induced salinity increments. To this

end, large, intact fragments of both species were maintained under different levels of chronic

salinity increment, simulated in a laboratory mesocosm system during 47 days. At the end of the

experimental period, we specifically determined the effects of hypersaline treatments on: i) water

relations in leaf tissues, ii) the concentrations of free amino acids (in leaves) and carbohydrates

(both soluble and reserves in leaves and rhizomes) and iii) leaf growth and shoot survival rates.

4.3. MATERIAL AND METHODS

4.3.1. FIELD PLANT COLLECTION AND EXPERIMENTAL DESIGN

Large P. oceanica and C. nodosa fragments (i.e. shoots connected to a basal rhizome with

the root system intact) were collected on October 2008 by scuba-divers in dense, shallow mea-

dows (6 meters depth) from a location off the south-eastern coast of Spain (37º 34´20.86´´N,

1º 12´28.16´´O; Isla plana, Murcia Region). In this location, meadows of both species were

separated by only a few tens of meters and hence exposed to identical environmental condi-

tions. Such conditions are typical of marine infralittoral environments near the Spanish Medi-

121

terranean coast (Margalef 1985), with a very narrow range of mean seawater salinity throughout

the year (37.1 to 37.6 psu-Practical Salinity Scale) and large seasonal fluctuations of light (5.3 to

19.8 mol quanta day-1) and temperature (14.6 to 24.8ºC) (Annex 1). Plants were collected by

hand by divers from randomly separated sites within the meadow of each species in the study

area in order to obtain an experimental plant pool with representative local genotypic variabi-

lity. Collected plants were transported to the laboratory in large coolers within 4 hours of their

collection, and then introduced into the aquaria of the mesocosms system for acclimation (see

below). To this end, three fragments of a single species were mounted in a plastic basket (22 x

40 cm base and 10 cm height) filled with coarse sediments previously washed to remove animals

and large organic particles, thus comprising a “transplantation unit”, which we considered the

experimental unit (EU). All plant fragments included the apical meristem were carefully selected

to be of similar size (35-45 cm) with similar shoot numbers (15-25 shoots) in order to standar-

dize the experiment. The total number of shoots per EU was 40 to 50 shoots for P. oceanica

and 50 to 60 for C. nodosa. A total of 24 EUs were prepared for each seagrass species. Six

EUs of each species were placed randomly in each large aquarium of the mesocosm system.

The mesocosm system basically consisted of four independent, large (1500L) aquaria, each one

with its own system of seawater circulation and filtration and precise control of temperature, irra-

diance, salinity, pH and nutrients, as extensively described in the Annex 2 and Methods sections of

Chapters 2 and 3. As explained in in previous Chapters, each large aquaria is divided in three 500L

sub-aquaria in which 2 EU of each species is placed; each sub-aquarium has an individual control

of some factors (e.g. illumination), but not for others (e.g. seawater circulating system) and hence

a sub-aquarium cannot be considered as an independent units. Actions taken to avoid possible de-

pendence effects associated to this kind of experimental designs have been explained in previous

Chapters. We did not find significant differences between aquaria for some plant variables (water

and osmotic potentials and total carbohydrate content in leaves; ANOVA, P > 0.05; unpublis-

hed data) during the acclimation period, and therefore we can assume that differences between

treatments at the end of the experimental period were due to divergence in time trajectories since

the onset of experimental treatments and not to pre-existing differences. Finally, measurements

obtained in the aquaria at the end of the experimental period were compared with those obtained

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simultaneously in the natural meadow in order to assess possible additional stress introduced

by the artificial system that could potentially mask plant responses to hypersaline treatments.

For plant acclimation, EUs were maintained in the aquarium for one week prior to the onset

of experimental treatments, under the same mean environmental conditions experienced by the

natural populations during the experimental period: a temperature of 21 ± 0.1ºC, salinity of 37 ±

0.1 psu and a saturating irradiance of ca. 300 ± 30 µmol quanta m-2 s-1. A 12/12 hr photoperiod

was selected to obtain a daily light exposure of 12.96 mol quanta m-2 day-1. After the acclimation

period, salinity levels were adjusted to obtain the selected experimental treatments, while all

other parameters (light, temperature, pH and water flow) were kept constant. One aquarium

was maintained at ambient salinity (i.e. 37, control treatment), while salinity was increased in the

other three aquariums following the protocol described in Marín-Guirao et al. 2011 (Chapter 2)

to obtain the following hypersaline treatments: 39, 41 and 43 psu, respectively. The experimental

salinity levels were selected in accordance with the upper thresholds of salinity tolerance assu-

med for these seagrass species (e.g. Fernández-Torquemada and Sánchez-Lizaso 2006; Ruiz

et al. 2009) and also in accordance with the maximum and minimum mean values measured in

hypersaline plumes created by brine effluents from desalination plants (37-42 psu; Fernández-

Torquemada et al. 2005a; Gacia et al. 2007). The experimental conditions were maintained for a

period of 47 days, which we considered to be long enough to induce specific plant stress respon-

ses, but not so long as to induce more severe metabolic impairment and damage. All plant res-

ponse variables were measured at the end of the experimental period. For each variable (except

for shoot number) and species, the average of two or three leaves (depending on the variable

type) was used as the value for each EU so that the number of replicates was the same for each

treatment (aquaria i.e. n = 6). Within the EU, each leaf belonged to a different shoot collected

from a different plant fragment. Only healthy shoots (i.e. without wounds or herbivore damage)

of a similar age (3-5 years old) were selected. We avoided collection of the youngest shoots (i.e.

those closest to the apical shoot, which are considered a sink site in the clonal structure of the

seagrass; Olivé et al. 2009) and neighbouring shoots within the same plant fragment, to avoid

spatial dependence effects associated with the influence of internal resource gradients from

these clonal plants (Marbá et al. 2002; Olivé et al. 2009). Within each shoot, measurements and

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analyses were always performed in mature photosynthetic tissues of leaf rank 2-3 for P. oceanica and

rank 1-2 for C. nodosa.

4.3.2. LEAF WATER RELATIONS

For determination of water relations, measurements of leaf-tissue osmolality (mmol kg-1 FW)

were performed on 2 shoots per EU (i.e. a total of 12 measurements per species and treatment)

using a Wescor Vapor Pressure Osmometer 5520 (Logan, Utah). For each shoot, osmolality

was measured both in fresh and frozen (at -20ºC for 2 h) blotted leaf segments to obtain water

(Ψw) and osmotic (Ψπ) potentials respectively, according to Tyerman (1982) and Boyer (1995).

Tyerman et al. (1984) demonstrated the existence of an osmotic pressure gradient along seagrass

leaves, ranging from minimum pressure values at the base (sheath) toward maximum values

in the blade. In previous trials, we also observed the existence of such pressure gradients in P.

oceanica and C. nodosa leaves, which allowed us to establish the optimal distance from the leaf

base at which osmolality measurements stabilised to a maximum along the leaf blade (unpublis-

hed data). This distance was used as the criteria to cut leaf segments for osmolality measure-

ments, and was 20-25 cm for P. oceanica and 3-5 cm for C. nodosa. Leaf segments needed to

cover the bottom of the osmometer sample holder were as follows: a 6.5 mm diameter leaf-disc

for P. oceanica and two leaf segments of 5 mm length for C. nodosa. For osmolality measure-

ments on fresh tissues from both species, a time delay of 10 min was used to equilibrate the os-

mometer sample-chamber, following the protocol described by Tyerman (1982) and Murphy et

al. (2003). Instantaneous measurements were done on frozen leaf-tissues, since the equilibration

of the sample-chamber was immediate. In order to avoid water evaporation of leaf tissues during

handling, leaf segments were cut fully submerged in treatment seawater and handling time was

minimised (Tyerman 1982; Wullschleger and Oosterhuis 1986; Murphy et al. 2003). Osmolality

measurements were expressed in megapascals (MPa), using the van`t Hoff relation (Tyerman

1982; Nobel 2009), and the leaf-tissue turgor pressure (Ψp) of each leaf segment was then calcula-

ted as the absolute difference between Ψw and Ψπ measured in that segment (Kramer and Boyer

1995). The external medium osmolality for each experimental treatment was also determined by

measurements of seawater in 6.5 mm sample discs, following the standard protocol (Wescor Inc.).

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4.3.3.CARBOHYDRATES AND AMINO ACIDS

Four shoots per EU were sampled for analytical determinations. The concentrations of non-

structural carbohydrates (soluble sugars and starch) were measured in two shoots, and free amino

acids (FAA) in the other two shoots (i.e. a total of 12 measurements per species and treatment).

Carbohydrates were analysed in mature leaf tissues and rhizome (the apical 1 cm) tissues of each

shoot, following the method described in Invers et al. (2004) and based on Yemm and Willis

(1954). In the case of the leaves, the basal third (10-15 cm) of photosynthetically developed tissue

was used for analysis, since this is where these metabolites are at the highest concentrations (e.g.

Pirc 1985). Soluble carbohydrates were solubilised from dried (finely ground) tissues by sequential

extractions with 95% (v/v) ethanol at 80ºC for 15 min. Ethanol extracts were evaporated in a ther-

mostated vacuum centrifuge (Univapo 100H, Unijet II) and dissolved in deionised water for analy-

sis. Starch was extracted overnight from the ethanol-insoluble residue in 1 N KOH, and determi-

ned spectrophotometrically using an anthrone assay. Both analyses were standardised to sucrose.

For analysis of free amino acids (FAA) concentrations, frozen (-70ºC, 24 h) leaf tissues were

ground in 0.05 N HCl and centrifuged for 5 min at 10.000 rpm. The supernatant was filtered in

a microfuge using low-binding regenerated cellulose Millipore ultra-free filters, to exclude pepti-

des with molecular weights higher than 10.000 Da. FAA concentration were measured in a 1200

Infinity Series HPLC (Agilent Technologies, US©) coupled to a MSD TOF mass spectrometer

(Agilent Technologies, US©) with an ESI (Electrospray Ionisation) source, following the me-

thod described by Zoppa et al. (2006). The amino acid analyzer was calibrated with amino acid

standards (Sigma-Aldrich©). Initial analyses showed that the amino acid proline was not detec-

ted in leaf tissues of P. oceanica, or was only detectable at concentrations very close to analytical

detectable limits, as reported by other authors in previously published studies (e.g. Augier et al.

1976; Pirc and Wollenweber 1988). Since this amino acid has been shown to have a crucial role

in osmoregulation, both in land plants (Stewart and Lee 1974) and in seagrasses (Brock 1981;

Tyerman 1989), we analysed this amino acid separately using an acidic ninhidrin reagent colo-

rimetric assay (Bates et al. 1973). Leaf tissues were frozen (-80 ºC, 24 h) in sulphosalicylic acid

(3%) prior to homogenization and centrifugation. An aliquot of the supernatant was incubated

at 100 ºC for 1 h in a solution of acetic acid, distilled water and ortho-phosphoric acid. Finally,

125

toluene was used to extract the organic phase with the chromophore and readings were taken

immediately at a wavelength of 520 nm. Proline concentrations were determined from a stan-

dard curve. All amino acid concentrations were expressed on a fresh weigh basis (µmol g-1 FW).

4.3.4. LEAF GROWTH AND SHOOT SURVIVAL

Results of these plant variables obtained in this experiment have been already presented in

Chapters 2 and 3 for P. oceanica and C. nodosa respectively.

4.3.5. FIELD REFERENCE

Mean values of all plant variables obtained in the laboratory were compared with re-

ference values measured in the field in the same season and in the same natural mea-

dows from which plants of both species were collected for transplanting in the aquaria.

This field sampling formed part of a seasonal sampling of environmental and plant varia-

bles performed in these meadows from October 2008 to July 2009 (see Annex 1). Data

corresponding to the autumn/winter 2008 campaigns are presented in Table 4.1. This

comparison was considered to be necessary to address questions such as the possi-

ble stress caused by artificial conditions that could confound the effect of experimental

treatments on the physiological and vegetative responses of the studied seagrass species.

4.3.6. STATISTICAL ANALYSIS

A one-way analysis of variance (ANOVA) was used to test the effect of experimental

treatments (a fixed factor with 4 salinity levels: 37-control, 39, 41 and 43 psu) on each plant

response variable for each seagrass species. Prior to analysis, data were checked for norma-

lity and homocedasticity, and transformed when necessary. When significant differences

were found between treatments, a post-hoc mean comparisons test (Student-Newman-Keuls

test, SNK; Zar 1984) was performed to identify mean homogeneous groups. Treatment

effects were considered statistically significant at P < 0.05. A non-parametric Kruskal-Wallis

test (Quinn and Keough 2002) was applied when ANOVA assumptions were not met.

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4.4. RESULTS

As the salinity levels were increased, the water potential of the external medium (Ψsw) pro-

gressively decreased from -2.74 MPa in ambient seawater (control, 37 psu) to -3.24 MPa in the

43 psu treatment (Fig. 4.1). Leaf tissues from both seagrass species always maintained mean Ψw

values and osmotic (Ψπ) potentials below Ψsw values in all experimental treatments, although

these were clearly more negative in C. nodosa (Ψw = -3.69 to -4.52 MPa; Ψπ = -4.81 to -5.18

MPa) (Fig. 4.1b) than in P. oceanica (Ψw = -3.04 to -3.74 MPa; Ψπ = -3.61 to -4.14 MPa) (Fig.

4.1a). Under ambient seawater conditions (i.e. control treatment), mean values of Ψw, Ψπ and

Ψp for C. nodosa leaves were, respectively, 21.4%, 43.6% and 160% higher than corresponding

values for P. oceanica. The mean values of Ψw and Ψπ of both species in the control treatment

were in the range obtained for these variables in the field (Table 4.1), indicating that the re-

ported differences between the two species were also found under natural conditions. Under

hypersaline treatment conditions (39-43 psu), such differences between species persisted, but

were quantitatively reduced to 7-22% for Ψw, 21-25% for Ψπ and 40-70% for Ψp, due to the

significant effects of salinity increases on these variables in both seagrass species. The mean

leaf Ψw of P. oceanica leaves exposed to hypersaline treatments was significantly reduced (21-

23%) compared to control mean values, although differences between hypersaline treatments

were not statistically significant. In C. nodosa leaves this variable was also significantly reduced

Table 4.1. Reference field values of water relations variables (Ψw, water potential; Ψπ, osmotic potential; Ψp, turgor pressure; MPa), carbohydrate (% DW) and proline (μmol g-1 FW) con-tents, and shoot growth (cm2 shoot-1 day-1).

127

in hypersaline treated versus control plants and showed a strong and significant linear corre-

lation with external salinity (R2 = 0.993, P < 0.01). The osmotic potential (Ψπ) of P. oceanica

leaves showed similar mean values between hypersaline treatments (-3.9 to -4.1 MPa), although

these were significantly lower (10-15%) than control mean values. In contrast, mean Ψπ values

for C. nodosa leaves were quite similar (-5.18 to -4.81 MPa), and were not significantly diffe-

rent between any experimental treatments (Fig. 4.1b). With respect to turgor pressure (Ψp),

mean values obtained for P. oceanica leaves were similar in ambient seawater and at a salinity

39 psu (0.57-0.64 MPa), but significantly decreased by 35% and 44% during the 41 and 43 psu

treatments, respectively. C. nodosa leaves experienced a more significant decrease in Ψp du-

ring the hypersaline treatments (ca. 40-61%) relative to control mean values (1.49 ± 0.2 MPa).

Figure 4.1. Leaf tissue water relations (MPa): water potential (Ψw), osmotic potential (Ψπ), and turgor pressure (Ψp) measured in leaf segments of a) Posidonia oceanica and b) Cymodocea nodosa plants exposed to experimental salinity treatments. The lines inside the bars represent the seawater osmolality of the treatments expressed in pressure units. Significant differences (P < 0.05) between treatments were tested by one-way ANOVA and the different letters represent the different groups of homogeneous means obtained in the post-hoc SNK test. In the case of Ψw and Ψπ of P. oceanica, differences between treatments were described by the same letters. Values are reported as the mean (n = 6) and standard error.

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The mean carbohydrate concentrations in leaves and rhizomes of both seagrass species in the

control treatment were within (or similar to) the ranges of those variables measured in their natural,

respective meadows (Table 4.1; Fig 4.2). Soluble sugar concentrations in P. oceanica leaves (Fig.

4.2a) displayed a close, positive, linear correlation with seawater salinity (R2 = 0.96, P < 0.05) and

reached mean values which were up to 34% higher than control mean values (37 psu) in the most

severe hypersaline treatment at 43 psu. However, the ANOVA test was unable to detect the sig-

nificance of this treatment effect, due to the low power of the test caused by the high variability of

replicates obtained for this variable for all treatment groups. Mean values for the starch content

in P. oceanica leaves were similar in all experimental treatments. Mean soluble sugar concentra-

tions in C. nodosa (Fig. 4.2b) leaves were significantly increased (23-41%) in 39 and 41 psu treated

leaves relative to the control leaves, but no significant differences were detected between control

leaves and leaves exposed to 43 psu in this species. Significant differences were also observed in

the starch content of C. nodosa leaves, but this pattern of variation did not display a clear corre-

lation with experimental salinity levels. No significant salinity effects were observed for both so-

luble sugars and starch in the rhizome tissues of P. oceanica and C. nodosa (Fig. 4.2c, Fig. 4.2d).

With respect to the free amino acid (FAA) composition of leaf tissues, qualitative and quan-

titative differences were apparent between both seagrass species (Table 4.2). In P. oceanica,

asparagine (Asn) was the most abundant (80% of the total FAAs, out of the 8 amino acids de-

tected by this analysis) followed by lysine/glutamine (Lys/Gln) and aspartic acid (Asp) (5-7% of

the total FAA); the sum of the individual concentrations of the rest of the amino acids (proline-

Pro, glycine-Gly, serine-Ser, glutamine-Glu, tryptophan-Trp) represented less than 2% of the

total FAAs. In C. nodosa leaves, 8 amino acids were also detected, but in this case Gly and Pro

accounted for 97% of the total FAAs (62 and 35%, respectively). Concentrations of both the to-

tal FAAs and most of the individual FAAs, was significantly affected by the experimental salinity

treatments, although the response pattern varied depending on the type of amino acid and the

species. In P. oceanica, all amino acids detected, except Ser and Lys/Gln, increased in concen-

tration with salinity. For hypersaline treated leaves, Pro content steadily increased with salinity

to values that were 85% higher than control mean values, while Gly concentrations were 30-72%

higher. Other amino acids, such as Asn and Asp, showed similar increases, but only for the most

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severe hypersaline treated leaves (i.e. 43 psu). Interestingly, Trp concentrations increased by

200% in the 41 and 43 psu treated leaves in relation to control leaves. The total FAA content in

P. oceanica leaves was similar between 37 and 41 psu, but significantly increased by 62% at 43

psu, mainly due to the an increase in Asn. In C. nodosa, Pro and Gly concentrations at control

salinity were, respectively, 87.6 and 30-fold higher than in P. oceanica. Under natural conditions

(i.e. Table 4.1), the mean Pro content in C. nodosa leaves was ca. 50-fold higher than in P. oce-

anica. The concentration of Pro in C. nodosa leaves was reduced by 51% in 39-psu-treated leaves

and by 21% in 41-psu-treated leaves, with respect to mean control values, and Gly concentra-

tions were 31-61% lower following all hypersaline treatments. The remainder of the detectable

amino acids showed a similar significant decline with increasing salinity in this seagrass species.

While the leaf growth rate of P. oceanica significantly declined with increasing salinity, no

significant treatment effect was found for this variable in shoots of C. nodosa. The mean values

of C. nodosa leaf growth rates obtained in both the control and hypersaline treatments were

similar to the upper part of the range of this variable measured in the C. nodosa meadow under

natural conditions (Table 4.1). In P. oceanica, some mortality occurred in EUs at 41 and 43 psu,

as indicated by the slightly (not significant) negative percentages of the net shoot change. Net

shoot changes for C. nodosa EUs were almost zero (0% to +1.1%) for all experimental treatments.

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Figure 4.2. Content (as percentage of dry weight) of starch (black bars) and soluble carbohydrates (open bars) in leaf and rhizome tissues of a, c) Posidonia oceanica and b, d) Cymodocea nodosa plants obtained in the experimental salinity treatments. Differences between treatments (one-way ANOVA, P < 0.05) are indicated by different letters according to the post hoc SNK test. Data are presented as the mean (n = 6) and standard error.

131

Table 4.2. Free amino acid concentrations (µmol g -1 FW) in P. oceanica and C. nodosa leaf tissues after salinity treatment. Values represent the mean (n = 6) and standard error. A summary of the one-way ANOVA applied to assess differences between treatments is indicated (F statistic and significance level: *** P < 0.001, ** P < 0.01, *P < 0.05; ns = not significant). Groups of homogeneous mean values obtained in the post hoc analysis (SNK) are indicated by different letters. Cases in which a non-parametric Kruskall-Wallis test was used (a) F correspond to the H statistic.

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4.5. DISCUSSION

As reported for other seagrass species (e.g. Tyerman 1989), both seagrasses yielded more

negative leaf water potentials (Ψw) than that of seawater in each experimental treatment,

allowing plant tissues to maintain a favourable water balance (i.e. net water influx; Kramer and

Boyer 1995), but the strategy used to achieve such adjustments differed among species and

within treatments in the same species. In P. oceanica leaves exposed to 39 psu, this reduction

in Ψw was achieved by a decrease in osmotic potential (Ψπ), which suggest the activation of

osmoregulatory processes in this treatment (Kramer and Boyer 1995; Azcón-Bieto and Talón

2000; Taiz and Zieger 2003; Verslues et al. 2006); as the severity of the treatment increase (i.e.

Figure 4.3. Mean and standard error of leaf growth (n = 6) and the net change in initial shoot numbers (n = 6) measured in experimental treatments: a, c) P. oceanica and b, d) C. nodosa. Values presented for P. oceanica are those published by Marín-Guirao et al. (2011) for the same experiment. No significant differences between salinity treatments were found for both variables in C. nodosa (one-way ANOVA, P > 0.05).

133

41 and 43 psu), no further decrease in Ψπ was observed, but some loss of turgor pressure (Ψp)

occurred in this species suggesting possible involvement of additional mechanisms mediating

reduction of leaf Ψw (e.g. through passive solute accumulation by dehydration phenomena;

Kramer and Boyer 1995; Verslues et al. 2006). In contrast, C. nodosa did not show evidence of

osmotic adjustments since leaf Ψπ was similar in this species following all treatments. Instead,

in this seagrass species the adjustment in Ψw was achieved by a drastic, significant decrease of

Ψp (up to 61% relative to control mean). Since this remarkable turgor loss was not accompa-

nied by changes in Ψπ (as expected if passive solute accumulation took place), we suggest the

participation of cell wall hardening processes: more rigid cell walls allow cells to harbour higher

turgor pressures as well as lose turgor without significant changes in volume (i.e. without passive

solute accumulation; Kramer and Boyer 1995; Verslues et al. 2006; Touchette et al. 2007). This

interpretation is in agreement with the consistently more negative Ψw and Ψπ values measu-

red in C. nodosa in this study (in relation to P. oceanica), which involves more positive hydric

gradients relative to external water potentials. Plants with these highly negative Ψw and Ψπ

values can develop cell hardening to avoid structural damage caused by high turgor pressures

and excessive water influx (e.g. see Bisson and Kirst 1995; Azcón-Bieto and Talón 2000). This

strategy has been previously described in aquatic plants (Bisson and Bartholomew 1984; Bisson

and Kirst 1995) and in some seagrass species with rigid cell walls, based on more direct experi-

mental determinations of their elastic modulus (Tyerman 1989). On contrary, Pagès et al. (2010)

proposed an elastic cell wall for C. nodosa to cope with increased salinity, although these authors

did not provide any evidence to support this contention. The differences in Ψπ and Ψp bet-

ween both seagrass species reported in this study persisted in their respective natural meadows

throughout an annual growth cycle (Annex 1), supporting the idea that such differences could

reflect distinct, species-specific adaptive strategies to maintain plant water balance as the exter-

nal salinity increases. Obviously, more specific research is required to confirm this hypothesis.

In general terms, and not following a straight forward relationship, the concentrations of

soluble carbohydrates and most of the free amino acids measured in P. oceanica leaves were

higher in hypersaline treatments. These results are consistent with the more negative Ψπ values

of P. oceanica leaves reported for these treatments, and hence with the hypothesis of active

134

osmotic adjustment in this seagrass species. Similar evidence has been obtained for other se-

agrass species (Pulich 1986; Tyerman 1989; Murphy et al. 2003; Ye and Zhao 2003; Koch et

al. 2007b), strongly suggesting a role for these organic compounds as compatible solutes with

osmoregulatory functions in seagrasses. The steady increase in free proline content with salinity

observed in P. oceanica leaves in this study was coherent with the widely recognised role of this

amino acid as an osmoticum in many terrestrial plants (Flowers et al. 1977), mangroves (Parida

and Das 2005), algae (Kirst 1989) and also seagrass species (Stewart and Lee 1974; Brock 1981;

Pulich 1986; Van Diggelen et al. 1987; Tyerman 1989; Berns 2003; Murphy et al. 2003; Ye and

Zhao 2003). Other amino acids have been reported to function as osmotic agents in other

seagrass species (e.g. alanine in Halophila engelmanni; Pulich 1986). No previous evidence for

such role exists for Gly in seagrasses (but see Pulich 1986), which also increased in P. oceanica

leaves following hypersaline treatments. However, it is well known that this amino acid can act as

a precursor for the synthesis of other osmoticum derivates such as betaines (Rhodes and Hanson

1993; Mc Neil et al. 1999), and as a water-stress metabolite (i.e. from enhanced photorespiratory

activity; Wingler et al. 2000) in land plants. Pulich (1986) reported alterations in the nitrogen

and amino-acid metabolism of some seagrass species under exposure to hypersaline conditions,

which could be associated with the production of specific osmolytes such as proline and alanine.

The higher content of asparagine in P. oceanica leaves in the 43 psu treatment (representing ca.

89% of the total FAA content in leaves) suggests similar salt-induced responses in this seagrass

species. The accumulation of these metabolites in leaf tissues, as discussed previously, could also

be a response to metabolic alterations other than osmoregulatory adjustments, especially in the

more severe hypersaline conditions. Thus, for instance, salt-induced accumulation of free amino

acids may be a consequence of protein degradation (and/or inhibition of protein synthesis),

which in turn may be related to stress-imposed injury (Levitt 1980; Souza et al. 2004; Parida

and Das 2005). The accumulation of the amino acid tryptophan in P. oceanica leaves in the 41

and 43 psu treatment (about 200% of the control) was particularly interesting in this context

since tryptophan biosynthesis is activated in various terrestrial species by different environmental

stresses, including salt stress (Zhao et al. 1998; Schimd and Amrhein 1999; Ehlting et al. 2007).

135

In contrast to P. oceanica, trends in Ψπ and both carbohydrate and amino acid content in

leaf tissues did not support a role for osmotic adjustments in C. nodosa under the experimen-

tal conditions used in this study. These results contrast with the high concentrations of certain

compatible solutes in C. nodosa leaves (e.g. Pro and inositols) that are typically found in Me-

diterranean populations of this seagrass species under natural conditions (Pellegrini and Riouall

1973; Augier et al. 1976; Drew 1978a; Pirc and Wollenweber 1988; Pirc 1989) and also in this study

(e.g. proline and glycine). These high levels of compatible solutes could reflect other adaptive

advantages of C. nodosa to cope with hypersalinity such as to play a key role in the osmopro-

tection of macromolecules and membrane stabilisation when ions are concentrated within cells

and tissues (Touchette 2007) and/or, as mentioned previously, to account for the more negative

Ψπ (and higher turgor pressures) observed for C. nodosa under ambient salinity in this study.

In summary, although both seagrass species follow a dehydration avoidance strategy (sensu

Verslues et al. 2006 and Kramer and Boyer 1995) within the studied salinity range (i.e. 37-43

psu), this study provides, for the first time evidence of the physiological mechanisms adopted

by Mediterranean seagrass populations to maintain osmotic equilibrium as the external salini-

ty increase. The efficiency of the strategy adopted not only depends on the ability to main-

tain turgor, but also on the metabolic costs and associated interference with normal metabolic

demands and growth requirements (Flowers et al. 1977; Greenway and Munns 1980). In the

case of P. oceanica, the species seems to accumulate osmolytes as the primary mechanism to

maintain positive turgor, but this process has a high metabolic cost that can potentially inter-

fere with leaf growth and plant survival. Marín-Guirao et al. (2011) (Chapter 2) demonstrated

significant inhibition of photosynthetic rates and enhancement of the respiratory demands of

P. oceanica under the same experimental conditions tested in this study. In such adverse cir-

cumstances, the accumulation of compatible solutes such as soluble carbohydrates and ami-

no acids in leaf tissues can only be explained by salt-induced metabolic impairments or their

translocation (Campos et al. 1999; Souza et al. 2004), which in turn suggest that considerable

amounts of internal resources required for plant maintenance and growth are diverted to sustain

respiration and osmoregulation demands (Munns 2002). The decline of leaf growth and shoot

survival rates of P. oceanica leaves as salinity increase described for the same experiment by

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Marín-Guirao et al. (2011) (Chapter 2) is consistent with this hypothesis. At least in this study,

C. nodosa did not show evidence of osmorregulatory adjustment, but it copes with hypersaline

stress by losing turgor pressure. This seems to be related to its capacity to naturally develop

high turgor pressures and very negative Ψπ under normal salinity conditions (maybe mediated

through cell wall hardening processes). This strategy could represent a more efficient adaptive

advantage of C. nodosa to cope with sudden salinity increments (or to growth in hypersaline

lagoons, e.g. Terrados and Ros 1991) which is consistent with the lack of apparent or signifi-

cant effects of salinity increments on the growth and survival of this seagrass species showed in

this study. This interpretation is also consistent with the high tolerance of plant performance to

salinity increments shown by C. nodosa over broader salinity ranges (Fernández-Torquemada

and Sánchez-Lizaso 2006; Pagés et al. 2010), and supports the initial hypothesis that the more

euryhaline C. nodosa is potentially better adapted to tolerate salinity increments than P. oceanica.

Nonetheless, much care must be taken when extrapolating these results obtained in a sin-

gle experiment performed in our laboratory mesocosm system to the more complex environ-

mental conditions in the field or to other time scales. The high similitude of seagrass status

between laboratory and field conditions (Table 4.1) indicates that our mesocosm system effi-

ciently reproduced natural conditions and hence that no additional stress interfered with plant

responses to targeted experimental manipulations. Nonetheless, despite this notable achieve-

ment, more research is needed to determine the extent to which the reported responses re-

flect general species-specific behaviours, or are limited to the specific conditions of our ex-

periment. Based on the analysis of water relations and osmolyte concentrations, more recent

experiments performed by authors support that both seagrass species have different capacities

to cope with hypersaline stress but the strategy can differ depending on the season, the du-

ration of the stress or the interaction with other key factors (e.g. light). Other factors such as

the type of salinity regime (e.g. chronic vs. fluctuating increments), recovery capacities, relia-

ble determinations of cell wall elasticity parameters, the role of ions or the effect of genoty-

pic variation are other relevant aspects that must be considered in future research in this field.

137

Posidonia oceanica meaodw. Sampling site. Isla Plana, Mazarrón (Murcia Region)

138

Cymodocea nodosa shoots. Shallow meadow (Murcia Region)

1

5141 ABSTRACT

141 INTRODUCTION

144 MATERIAL AND METHODS

153 RESULTS

166 DISCUSSION

2

Jose Miguel Sandoval-Gil, Juan Manuel Ruiz Fernández, Lázaro Marín-Guirao, Jaime Bernardeau-Esteller, Jose Luis Sánchez-LizasoAnalysis of the ecophysiological plasticity of plants from shallow and deep meadows of the Mediterranean seagrasses

(Posidonia oceanica and Cymodocea nodosa) in response to experimental simulation of chronic hypersaline stress

Submitted to Marine Environmental Research

141

5ECOPHYSIOLOGICAL PLASTICITY OF PLANTS FROM SHALLOW AND DEEP MEADOWS OF THE MEDITERRANEAN SEAGRASSES (P. OCEANICA AND C. NODOSA) IN RESPONSE TO EXPERIMENTAL SIMULATION OF CHRONIC HYPERSALINE STRESS

5.1. ABSTRACT

The Mediterranean seagrass species Posidonia oceanica and Cymodocea nodosa each show

a different marine environmental distribution closely related to their distinct salinity tolerance

ranges. In this work, we examined the potential distinct interspecific ecophysiological plastici-

ties that both species can exhibit in response to hypersaline stress, but we also tested the po-

tential implication of ecotypic intraspecific divergences in the development of such plasticities.

To this end, plants from shallow (5–7 m) and deep (18–20 m) meadows of both species were

exposed to a treatment of chronic salinity increase, in a long-term experiment (i.e. 62 days)

developed in a highly controlled mesocosm system. Hypersaline stress caused notable plastic

physiological alterations in P. oceanica and C. nodosa, with appreciable inter- and intraspecific

differences. Although both species were similarly able to osmoregulate by means of organic

solute accumulation, the hypersaline condition induced differential photosynthetic dysfunctions,

respiratory alterations and changes in leaf pigment optics. The metabolic cost as a consequen-

ce of the physiological plasticity integration seemed to compromise the performance of P.

oceanica at the vegetative level but not in the case of C. nodosa. These results confirm that

both the inter- and intraspecific divergences, due to the distinct eco-biological attributes found

and the ecotypic differentiation within species, respectively, play a key role in the responses

which both Mediterranean seagrasses could develop under hypersaline stress conditions, and

that these were consistent with their distinct ecological strategies and salinity tolerance ranges.

5.2. INTRODUCTION

Seagrasses conform to an ecological group of submersed marine plants that developed va-

rious morphological and physiological properties to overcome seawater saline conditions (Larkum

and den Hartog 1989; Tyerman 1989; Kuo and den Hartog 2000; Kuo and den Hartog 2007).

The relatively free ionic and water exchange between leaf blades and the surrounding medium,

and the lower leaf water potential in respect of land plants or aquatic macrophytes, suggest

important seagrass properties determining their successful adaptation to the marine environ-

ment (Tyerman 1989; Touchette 2007). Salinity is a relevant ecological factor determining the

142

distribution and abundance of seagrass species in the different coastal environments worldwide,

revealing differential tolerances to withstanding changes in this factor (McMillan and Moseley

1967; Walker and McComb 1990). In addition, changes in salinity (and in particular salinity incre-

ments) can overcome the tolerance limits of seagrass species and may cause severe disturbance

effects on these and other benthic communities (Morton et al. 1996; Einav et al. 2002; Fernández-

Torquemada et al. 2005a). Seagrasses are highly vulnerable to man-induced changes in environ-

mental conditions, including salinity, and so they are considered biological sentinels of ecosys-

tem change (Ralph et al. 2006; Boudouresque et al. 2009). Moreover, seagrass habitats provide

valuable functions and services to coastal ecosystems and hence knowledge of their tolerance to

changes in environmental conditions is today an issue of major concern in forecasting the conse-

quences of human impacts on coastal ecosystems (Orth et al. 2006; Boudouresque et al. 2009).

In the Mediterranean Sea, the discharge of hypersaline wastes (i.e. brines) delivered from

desalination plants causes a significant salinity increase over large areas of the sea bottom co-

lonized by seagrass beds, and is considered an important threat to these valuable biological

communities (Morton et al. 1996; Fernández-Torquemada et al. 2005; Palomar and Losada,

2010), which dominate infralittoral bottoms up to depths of 40 metres (Procaccini et al. 2003).

In effect, hypersaline stress caused by salinity increases can lead to variable plastic ecophy-

siological alterations of seagrasses (see Touchette 2007 for a review), which can reflect accli-

matative purposes but also, and more generally, non-adaptive (or maladaptive) physiological

responses resulting in salinity stress effects on plant fitness and survival (Lichtenthaler 1996;

Dudley 2004; Ghalambor et al. 2007). Within these ecophysiological alterations, changes in

leaf-water relations, dysfunctions of the photosynthetic machinery, enhancement of respiratory

activities, as well as leaf pigment alterations, are all important responses reported in seagrasses

subject to hypersaline conditions (Ogata and Takada 1968; Beer et al. 1980a; Pulich 1986; Ralph

1998, 1999; Tyerman 1989; Murphy et al. 2003; Kahn and Durako 2006; Koch et al. 2007b).

The Mediterranean seagrass species P. oceanica and C. nodosa notably differ in biological

attributes and ecological strategies (Drew 1978b; van Tyenderen 1991; Cancemi et al. 2002; Ole-

sen et al. 2002), which has been observed to match with differential specific plasticity capacities

143

and tolerances between plant species (Dudley 2004; Valladares et al. 2007; Nicotra et al. 2010).

In effect, P. oceanica L. (Delile) is a large slow-growing and stenohaline species generally adapted

to narrow salinity ranges throughout the Mediterranean Sea (e.g. 36.5–38 psu in oceanic waters

of the southeast of Spain; Ruiz et al. 2009a) and usually not present in hypersaline coastal waters

(but see Pergent et al. 2002 and Tomasello et al. 2009); on the other hand, the smaller C. nodosa

(Ucria) Ascherson is considered a pioneer and more euryhaline species able to form dense and

productive meadows in oceanic, estuarine and hypersaline coastal waters (Terrados and Ros 1991;

Pérez and Romero 1994; Olesen et al. 2002). Such interspecific differences are consistent with

the differential tolerance to hypersalinity shown by both seagrass species in recent studies, mainly

in terms of plant growth and survival (Fernández-Torquemada and Sánchez-Lizaso 2005, 2006;

Gacia et al. 2007; Ruiz et al. 2009; Pagés et al. 2010; Fernández-Torquemada and Sánchez-Liza-

so 2011) but also at the ecophysiological level (Ruiz et al. 2009; Marín-Guirao et al. 2011; Sando-

val-Gil et al. 2012; Sandoval-Gil et al. in press; Chapters 1-4). The bulk of evidence indicates that

C. nodosa is more tolerant than P. oceanica to salinity increments, probably due to some inherent

ecophysiological properties of the former conferring the species certain adaptive advantages (e.g.

highly negative water potential, accumulations of certain protective solutes and mild inhibition of

photosynthetic activity; Sandoval-Gil et al. 2012; Sandoval-Gil et al. in press; Chapters 3 and 4).

Further to interspecific variation, intraspecific divergences in physiological plasticity between

plants from separated populations (in space and/or time) could operate as another source of

variation in the mechanisms which both species can use to cope with stress conditions (Dudley

2004; Ghalambor et al. 2007; Baquedano et al. 2008). In this sense Mediterranean populations

of both P. oceanica and C. nodosa span contrasting environmental conditions throughout their

respective distributional areas (e.g. depth and geographical gradients) and annual production

cycles (e.g. seasonal variations), which seems to drive structural and physiological plant varia-

tions related to ecotypic and genotypic differentiation (Dennison 1987; Duarte 1991; Dalla Via

et al. 1998; Olesen et al. 2002; Migliaccio et al. 2005; Procaccini et al. 2007; Serra et al. 2010).

Thus, for instance, depth-associated variation in photosynthetic-related traits have been des-

cribed in both species (Drew 1978b; Dalla Via et al. 1998; Olesen et al. 2002). However, it

is unknown at which point the plasticity of these and other relevant ecophysiological aspects

could lead to intraspecific differences in the capacity of the species to tolerate hypersaline stress.

144

In the present study, we analysed species-specific ecophysiological responses of the Medi-

terranean seagrasses P. oceanica and C. nodosa to hypersaline stress, taking into consideration

the possible influence of intraspecific variation associated with the depth gradients along which

these species are adapted for growth. We specifically hypothesize that the capacity of each se-

agrass species to cope with hypersaline stress can be a function of the differential plastic expres-

sion of ecophysiological and vegetative plant traits between shallow and deep meadows. To this

end, several physiological (i.e. photosynthesis, leaf pigmentation, leaf-water relations, and leaf

optical properties) and vegetative traits were examined in both seagrass species and meadows

were exposed to chronic hypersaline stress simulated in a highly controlled mesocosm system.

5.3. MATERIAL AND METHODS

5.3.1. PLANT COLLECTION

In April (spring) 2009, large fragments of rooted P. oceanica and C. nodosa rhizomes

bearing apical growth meristems and 7–10 connected shoots (i.e. maintaining clonal plant

integrity) were collected by scuba divers in shallow (5–7 m) and deep (18–20 m) meadows

of dense and healthy meadows of the Murcia Region on the south-eastern coast of Spain.

Shallow and deep P. oceanica meadows had, respectively, a shoot density of 687 ± 16.5

and 321 ± 20.7 shoots m-2 and were located in Isla Plana (Mazarrón Bay, 37º 33´14.4´´N 1º

10´35.5´´SO); the shallow meadow of C. nodosa was adjacent to the shallow P. oceanica

meadow and had a shoot density of 347.8 ± 9.4 shoots m-2, while the deep C. nodosa meadow

was located in a different locality at 25 km distance (Calblanque, Cartagena; 37º 34´53´´N

0º 44´ 5.4´´ SO. All these seagrass meadows are classified with a favourable conservation

status within the Habitat Directive and with none or very low influence from human acti-

vities. Shoot density values correspond to spring–summer when this and other structural

variables (e.g. biomass, shoot size) of these species reach their seasonal maximum (Marbà

and Duarte 2001; Vasapollo and Gambi 2012; see Annex 1). Continuous underwater records

of salinity, temperature and irradiance were available for these meadows (see Annex 1, sec-

tion 6). These environmental variables followed the characteristic seasonal pattern descri-

145

bed for Mediterranean regions (Margalef 1985; Hofrichter 2004), except that mean salinity is

very constant across all sites, depths and seasons (37.38 ± 0.059 psu). In the period from mid-

spring to the end of summer (when the experiment was performed), ranges of daily incident

irradiance (measured at the top of the leaf canopy) and temperature were 19.9–20.9 mol

quanta m-2 day-1 and 19–24.8 ºC in shallow meadows, and 8.55–11.8 mol quanta m-2 day-1 and

16.8–21.6 ºC in deep meadows. Data for these environmental variables were obtained using CT

and PAR irradiance recorders Compact-CT and MDS-MkV/L of Alec Electronics (Japan).

Collected plants were transported to the laboratory in large coolers within two hours of

their collection, and then introduced into the aquaria of the mesocosm system. To this end

and in order to standardize the experiment, plant fragments of each species with a simi-

lar size and morphology were carefully selected and mounted on plastic pots filled with clea-

ned sediment. Total shoot number per pot was 40 to 60 for both species, and a total of 24

pots were produced for each species and then introduced in the mesocosm system. We as-

signed each of these pots with transplanted plants to be an operative experimental unit (EU).

5.3.2. MESOCOSM SYSTEM AND EXPERIMENTAL DESIGN

The experiment was performed in the mesocosm system, developed in the fa-

cilities of the marine laboratory of the Oceanography Centre of Murcia, as des-

cribed in the previous chapters and in the Annex 2 of this thesis. In this stu-

dy we used a modified, improved version of the mesocosm system which basically

consisted of 12 independent 500 L aquaria, each one with its own illumination and seawater

circulation system and a precise control of water flow, temperature, pH and nutrients.

Two EUs of each species from the same meadow (i.e. depth) were introduced per

aquarium. Six aquaria were randomly assigned to EUs with plants from deep meadow

(D) and the other six aquaria to EUs with plants collected in the shallow meadow (S).

For acclimation prior to the onset of salinity experimental treatments, all EUs were main-

tained in the aquaria for one week at the same environmental conditions, which corres-

146

ponded to mean values of field records obtained for natural meadows during the same

season in which the experiment was carried out. These were a temperature of 19 ± 0.1ºC,

a salinity of 37 ± 0.1 psu, and irradiances of 10.19 ± 0.5 and 20.47 ± 1.64 mol quanta m-2

day-1 for the deep and shallow meadows, respectively. The photoperiod was 12/12 h.

In natural meadows, shelf-shading of the leaf canopy strongly reduces light availability

and modifies its spectral quality at the level of mature photosynthetic tissues located inside

the meadow in the basal two-thirds of each shoot (Dalla Via 1998; Olesen et al. 2002; Collier

et al. 2008). In order to obtain results for ecophysiological variables that would be compara-

ble with natural conditions (in particular those related to photosynthesis), we were especially

concerned simulating this light climate in our aquaria. This was possible because plant frag-

ments mounted in the EUs had comparable shoot densities and the leaves formed a dense

canopy. To assess to what extent this artificial arrangement simulated shelf-shading of the

natural meadows, we performed measurements of light availability and determined the light

attenuation coefficient (Kd, m-1) inside the leaf canopy of the shallow and deep P. oceanica

meadows, and in experimental populations of the aquaria. To this end, vertical profiles of

irradiance were obtained using underwater PAR sensors each placed 5 cm between the top

and the base of the leaf canopy (n = 6, replicated profiles); Kd was derived from the Lambert–

Beer equation fitted to each light vertical profile (Kirk 1994). Mean Kd values obtained in

aquaria leaf canopies were 6.59 ± 0.38 m-1 in D EUs and 9.98 ± 0.82 m-1 in S EUs; differences

in Kd between experimental populations were significant (t-test, P < 0.05) and were very simi-

lar to those obtained in the natural deep (5.59 ± 04 m-1) and shallow ( 9.17 ± 0.66 m-1) mea-

dows. Daily integrated photon flux densities inside the leaf canopy (in the basal third) were

also very similar between natural and EU leaf canopies and showed a pattern of variation

that was negatively correlated with that observed for Kd between shallow and deep plants:

2.21 ± 0.09 and 0.77 ± 0.04 mol quanta m-2 day-1 for deep and shallow natural meadows,

respectively; and 1.97 ± 0.2 and 1.01 ± 0.13 mol quanta m-2 day-1 for EUs with plants from

the deep and shallow meadows, respectively. These values represented only 7–10% of the

light available at the top of the leaf canopy, both in the natural meadows and in the aquaria.

147

After the acclimation period, salinity was adjusted to experimental levels (37 and

43 psu), all other parameters remaining constant throughout the experimental period.

Six aquaria were randomly selected (three with D EUs and three with S EUs), maintai-

ned with ambient salinity (37 psu) and used as the control treatment of shallow (CS) and

deep (CD) populations. In the other six aquaria, salinity was increased to obtain the hy-

persaline treatment for shallow (43S) and deep (43D) populations. This hypersaline level

was selected on the basis of the upper thresholds of salinity tolerance assumed for these

seagrass species in the field and mesocosm (e.g. Fernández-Torquemada and Sánchez-

Lizaso 2005, 2006; Ruiz et al. 2009; Marín-Guirao et al. 2011; Sandoval-Gil et al. 2012),

and also in accordance with the maximum mean values measured in hypersaline plumes

created by brine effluents from desalination plants on the Mediterranean shoreline (37

to 42; Fernández-Torquemada et al. 2005a; Gacia et al. 2007). Plants were maintained

for 62 days under these experimental conditions, a time period demonstrated to be long

enough to induce physiological plant stress responses (Marín-Guirao et al. 2011; Sando-

val-Gil et al. 2012; Sandoval-Gil et al. in press), but not so long as to induce more se-

vere metabolic impairment and damage which could severely compromise shoot survival.

5.3.3. PLANT TRAITS

All plant traits were determined at the end of the experimental period. Within each EU,

1–3 replicated measures of each plant variable were performed on different randomly selected

shoots (except for photosynthesis measurements, see below). Only healthy shoots (i.e. without

wounds or herbivore damage) of a similar age (3–5 years old) were selected. The use of apical

and neighbour shoots was avoided to prevent the effects of spatial dependency associated with

the clonal structure of the plant fragments (Marbá et al. 2002; Olivé et al. 2009). Within each

shoot, measurements and analyses were always performed on mature leaf tissues of leaf rank

2–3 for P. oceanica and rank 1–2 for C. nodosa. These leaf tissues are positioned in the mid-lower

region of the leaf canopy and are hence exposed to low light levels (see above). Analyses on

rhizomes were performed using the first 2–3 cm of the rhizome apex. The aquarium is the true

experimental unit for each seagrass species and variable, so that measurements performed

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on shoots of the same pot and in different pots of the same aquarium are ‘pseudorepli-

cates’, which were therefore averaged to obtain an independent replicated value. This

means that the number of replicates used in statistical tests were n = 3 (total N = 12).

5.3.3.1. LEAF-WATER RELATIONS

For the determination of leaf-water relations variables (i.e. water potential–Ψw, osmotic

potential–Ψπ and turgor pressure–Ψp), 12 different shoots of each species and treatments

(i.e. two measurements per each EU in each aquarium) were employed. For each shoot,

measurements of leaf-tissue osmolality (mmol kg-1FW) were performed in fresh and free-

ze-thawed leaf tissues using a Wescor Vapor Pressure Osmometer 5520 (Logan, Utah)

and following the methods described in Chapter 4. Osmolality measurements were ex-

pressed in megapascals (MPa), using the van’t Hoff relation (Tyerman 1982; Nobel 2009),

and the leaf-tissue turgor pressure (Ψp) of each leaf segment was then calculated as the

absolute difference between Ψw and Ψπ for each measurement (Kramer and Boyer 1995).

5.3.3.2. ORGANIC COMPOUNDS AND ELEMENTAL COMPOSITION

Non-structural carbohydrates (soluble sugars and starch) and free amino acids were deter-

mined following the methods detailed in Chapter 4. Leaf and rhizomatic tissues were collec-

ted from six different shoots from each species and treatment (i.e. one shoot per each EU in

each aquarium) to determine soluble sugars and starch using anthrone assay (see Methods in

Chapter 4). Leaf tissue of another six different shoots were collected in the same way for the

analysis of free amino acids (FAAs). Because the initial analyses using this method indicated

that the amino acid proline was either not detected or was only detectable at concentrations

very close to the analytical detectable limits in leaf tissues of P. oceanica, we decided to analyse

proline separately (12 measurements per species and treatment; two shoots per each EU in each

aquarium) using an acidic ninhydrin reagent colorimetric assay also described also in Chapter 4.

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Total carbon and nitrogen content and the signature of isotopic carbon (δ13C) were determi-

ned in leaf and rhizomatic tissues (previously dried and finely ground) from another six different

shoots of both species collected in EUs as described above. For the analytical process, we used

an Elemental Analyzer FlashEA1112 (ThermoFinnigan) connected to an isotopic relations mass-

spectrometer (ThermoFinnigan). Elemental C and N composition was expressed as percentage

per unit dry weight and as the molar ratio C/N. Isotopic values were reported in the δVPDB notation

relative to the standard Vienna Pee Dee Belemnite (δsample = 1000 [ ( Rsample/Rstandard) - 1], R = 13C/12C).

5.3.3.3. CHLOROPHYLL FLUORESCENCE

Measurements of chlorophyll a fluorescence emission were performed once a week

during the experiment following the protocol described in the Material and Methods sec-

tions of Chapters 2 and 3, using a diving-PAM portable fluorometer (Walz, Germany).

The maximum quantum yield of photosystem II (PSII; Fv/Fm) was measured early in the

morning in all night dark-adapted leaves (Schreiber 2004; Larkum et al. 2006) of 12 diffe-

rent shoots per species and treatment (i.e. two shoots per each EU in each independent

aquarium).

5.3.3.4. PHOTOSYNTHETIC PARAMETERS DERIVED FROM P-E CURVES

The photosynthesis–irradiance (P vs. E) curves of P. oceanica and C. nodosa leaves were

determined from six different shoots per species and treatment (i.e. one per each EU in each

independent aquarium) (see Material and Methods in Chapters 2 and 3). The following varia-

bles were determined from each P vs. E curve: net and gross photosynthetic rates (net-P and

gross-P; μmol O2 cm-2 h-1); respiration (Rd; μmol O2 cm-2 h-1); and photosynthetic efficiency (α;

μmol O2 cm-2 h-1/ μmol quanta m-2 s-1). A P:R ratio was obtained from each curve and used as

a proxy of the daily leaf carbon balance; P was estimated as the integration of net-Pmax during

the light period (12 h) and R was the total consumption during the night period (Rd x 12 h).

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5.3.3.5. PIGMENT CONTENT AND LEAF OPTICS

For the determination of photosynthetic pigments and leaf optical properties, 12 shoots

per species and treatment were collected (i.e. two shoots per each EU in each aquarium).

Photosynthetic pigment density (Chla, Chlb, carotenoids) and pigments ratios (Chlb:a,

carotenoids:Chla) and leaf optical descriptors (absorbance-D; absorptance-A; pigment spe-

cific coefficients-a*, m2 mg-1 pigments; mass-specific absorption coefficients-aw*, cm2 mg-1 leaf

DW) were determinied according to the methods and protocols described in Chapters 2 and

3. In order to determine the contribution of leaf morphology and/or packaging to the variation

in leaf optical properties, the leaf mass per area index (LMA; mg DW cm-2) was estimated by

measuring the area and weight of each leaf segment employed for pigment determinations.

5.3.3.6. SHOOT MORFOLOGY, GROWTH AND SURVIVAL

Further to physiological measurements, a set of vegetative variables were considered in

order to address the acclimative and adaptative consequences of ecophysiological respon-

ses at the level of whole plant fitness and survival. To determine leaf growth rates, three di-

fferent shoots of each species were marked in each EU at the beginning of the experimental

period following methods described in Ruiz et al. 2009 (Chapter 1). Marked shoots were har-

vested at the end of the experimental period to determine mean values of shoot leaf growth

(i.e. newly formed tissue below the mark; cm2 of new tissue shoot-1 day-1), number of leaves

per shoot, production of new leaves per shoot and percentage of the necrotic leaf surface.

All shoots in each pot were counted at the beginning and at the end of the experimental pe-

riod, and the differences were normalized to initial shoot numbers and expressed as a percen-

tage of net change; negative values indicated a net decline from initial shoot numbers due to

higher mortality rates compared to shoot recruitment (i.e. shoot division or rhizome branching).

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5.3.4. REFERENCE FIELD VALUES

A set of plant variables were also determined in natural meadows of sampling sites

during the experimental period (Table 5.1, see Annex 1), except for the deep C. nodo-

sa meadow which could not be studied due to logistic constraints. Mean values (and

ranges) of these variables were used as reference values with which we could compa-

re values obtained for each seagrass species in the mesocosm system to address how

representative the measurements performed under laboratory conditions were in re-

lation to those observed in the natural meadows. The detection of possible deviations

between laboratory and field measurements also allowed us to evaluate the existence

of any additional stress, caused by experimental manipulation and the artificial condi-

tions of the mesocosm system, in specific plant responses to experimental treatments.

Table 5.1. Mean values (SE) of plant physiological and vegetative descriptors determined in reference to P. oceanica and C. nodosa meadows selected for this study (see Material and Methods section).

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5.3.5. DATA ANALYSES

5.3.5.1. UNIVARIATE ANALYSIS

Statistical analysis of each individual variable was performed using the statistical pac-

kage STATISTICA (StatSoft INC. 2001, version 6.0). Two-way analysis of variance

(ANOVA) was used to test the effect of two fixed factors on response variables: `salini-

ty´ (with two levels: control vs. hypersalinity) and `depth´ (with two levels: deep vs. sha-

llow), this last involving both plants (i.e. the experimental population) and environmen-

tal conditions of each depth level. For the analysis of the temporal evolution of Fv/Fm, a

three-way analysis of variance was employed (sampling time x salinity treatment x depth).

A post-hoc mean comparison test (Student–Newman–Keuls, SNK; Zar 1984) was per-

formed when significant differences were found (P < 0.05). Prior to the analysis, data

were checked for normality and homocedasticity, and transformed when necessary.

5.3.5.2. PLASTICITY INDEX

The phenotypic plasticity index was used to compare the plasticity of physiological

responses developed for each seagrass species under the experimental treatment, as pro-

posed by Valladares et al. (2006). This index was calculated from the absolute differen-

ce (i.e. xi´j´- xij or distance, dij→i´j´) between all pairs of values (j and j´) for each trait (x)

of the same species and meadow exposed to the different treatments (i and i´, control

and hypersaline). Relative distances were calculated by dividing these differences by the

sum (xi´j´ + xij). A relative distance plasticity index (RDPI) for each trait, ranging from 0

(no plasticity) to 1 (maximal plasticity), was calculated as RDPI = Σ(dij→i´j´/(xi´j´ + xij))/n.

With the aim simplifying the results, the plasticity indexes calculated for vegetative des-

criptors were not shown due to the lack of significant differences between treatments.

A proxy of some general physiological plasticities, related to the species, meadows or

group of related variables (mentioned in the Discussion), was obtained by the mean of

the RDPIs calculated for each physiological trait relevant to each situation. Differen-

ces between RDPI values between species and for each physiological trait, or between

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plants of each species from different meadows, were tested by one-way analysis of va-

riance (ANOVA) and a post-hoc mean comparison test (Student–Newman–Keuls, SNK).

5.3.5.3. MULTIVARIATE ANALYSIS

In order to identify and characterize differential patterns of ecophysiological responses

to experimental treatments between seagrass species and populations depths, a principal

components analysis (PCA) was applied using the program CANOCO 4.5 (ter Braak and

Ŝmilauer 2002). Previously, in order to avoid colinearity between variables, three groups of

highly intercorrelated ecophysiological variables were identified and summarized as ‘water

relations’ (water and osmotic potential and turgor pressure), ‘leaf optical properties’ (absorp-

tance PAR-averaged; specific absorption coefficients a* and aw*) and ‘photosynthetic rates’

(net and gross Pmax), applying PCA to each group (ter Braak 1995). The resulting compo-

nents of each analysis could be treated as traditional variables for further statistical analysis,

with the benefits of reducing many variables to a few components and including all original

variables in each component so that further analysis was not based on only a single variable

but on all variables of the data group, and generation of orthogonal, non-correlated, com-

ponents (Jenerette et al. 2002; Marín-Guirao et al. 2005). The new variables, labelled as

‘optical properties’, ‘water relations’ and ‘photosynthetic rates’, were obtained from these

analyses by selecting the first axis of each PCA which explained > 95% of the variability

of these groups of variables, and were included as independent variables in the final PCA.

5.4. RESULTS

5.4.1. INDIVIDUAL PLANT TRAITS

Under control conditions, C. nodosa showed higher mean values than P. oceanica of net

and gross photosynthetic rates and efficiency (α), P:R ratios and δ13C (Fig. 5.1). These va-

riables were clearly affected by the hypersaline conditions, although the type and intensity

of the observed responses depended on the species and the depth considered. Alterations

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in all photosynthetic descriptors of both species were significant for both salinity and the

salinity x depth interaction (two-way ANOVA, P < 0.001, degrees of freedom (d.f.) = 1, 8).

Net and gross photosynthesis (Fig. 5.1a, c) were significantly reduced in saline-stressed P.

oceanica plants by 20% and 40% in both the deep (43D) and the shallow (43S) experimental

populations, respectively, relative to the control treatments (CD and CS). Respiration was

enhanced by hypersaline stress but only in plants of the deep meadow of P. oceanica (Fig.

5.1b). The P:R ratio of P. oceanica leaves significantly decreased (from 40 to 20%) in 43S and

43D plants and also in 43S C. nodosa plants due to a significant reduction in the photosyn-

thetic rates in combination with an abrupt increase in respiratory activity (Fig. 5.1d). In both

seagrass species, photosynthetic efficiency showed a similar, but opposite response pattern

to hypersaline stress depending on the depth, increasing in 43D plants and decreasing in 43S

plants (Fig. 5.1e). The carbon isotopic signal (δ13C; Fig. 5f) of P. oceanica followed a response

pattern similar to that of α in P. oceanica, which had leaves more isotope-depleted in the

43D plants and less isotope-depleted in the 43S plants; the opposite pattern was observed

in C. nodosa for this variable, although no significant differences were found in this case.

For both species the temporal evolution of maximal photochemical quantum effi-

ciency (Fv/Fm) (Fig. 5.2) had values of between 0.78 and 0.8 throughout the experi-

ment in both the control and the hypersaline treatments. The statistical analysis revea-

led a slight but significant reduction of Fv/Fm values for P. oceanica plants of both depths

exposed to the hypersaline treatment (three-way ANOVA, P < 0.05, d.f. = 6, 56). Varia-

tion in this fluorescence variable was only observed in C. nodosa in relation to the popula-

tion depth, being higher in deep plants (CD and 43D) than in shallow ones (CS and 43S).

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Figure 5.1. Variation in photosynthetic parameters of P. oceanica (solid bars) and C. nodosa (open bars) plants obtained from shallow (S) and deep (D) meadows, and maintained under experimental treatments (C = control, 43 = hypersaline). (a) Net-P = net photosynthetic rates (μmol O2 cm-2 h-1); (b) Rd = respiration rates (μmol O2 cm-2 h-1); (c) gross-P = gross photosynthetic rates (μmol O2 cm-2 h-1); (d) P:R = proxy to the leaf carbon balance; (e) α = photosynthetic efficiency (μmol O2 cm-2 h-1/ μmol quanta m-2 s-1); (f ) δ13C = leaf carbon isotopic discrimination. Values are reported as means ± standard errors.

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The leaf pigment analysis generally revealed higher content and lower molar ratios in P.

oceanica than for C. nodosa (Fig. 5.3). The hypersaline treatment caused significant modifica-

tions in the content of chlorophylls and carotenoids (and their ratios) in both species depths.

More specifically, saline-stressed P. oceanica plants from 43D and 43S treatments showed a

consistent and significant increase in leaf chlorophyll concentrations (Fig. 5.3a, b), but oppo-

site variation of its molar ratios between D and S plants (Chlb:a; Fig. 5.3d), in a similar way

to that described earlier for the photosynthetic efficiency. Saline-stressed C. nodosa plants

also showed a significant reduction in chlorophyll content and the Chlb:a molar ratio in

plants from the shallow meadow (43S) (Fig. 5.3a-d). Total carotenoids and carotenoid:Chla

generally increased in plants from S and D meadows of both seagrass species (Fig. 5.3e).

The leaf pigment analysis generally revealed higher content and lower molar ratios in P. oce-

anica than for C. nodosa (Fig. 5.3). The hypersaline treatment caused significant modifica-

tions in the content of chlorophylls and carotenoids (and their ratios) in both species depths.

More specifically, saline-stressed P. oceanica plants from 43D and 43S treatments showed a

consistent and significant increase in leaf chlorophyll concentrations (Fig. 5.3a, b), but oppo-

Figure 5.2. Maximum photochemical efficiency (Fv/Fm) determined for P. oceanica and C. nodosa leaves in the different experimental treatments. Values are reported as means ± standard errors.

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site variation of its molar ratios between D and S plants (Chlb:a; Fig. 5.3d), in a similar way

to that described earlier for the photosynthetic efficiency. Saline-stressed C. nodosa plants

also showed a significant reduction in chlorophyll content and the Chlb:a molar ratio in

plants from the shallow meadow (43S) (Fig. 5.3a-d). Total carotenoids and carotenoid:Chla

generally increased in plants from S and D meadows of both seagrass species (Fig. 5.3e).

Figure 5.3. Leaf pigment content (a–c) and molar ratios (d, e) determined in P. oceanica (solid bars) and C. nodosa (open bars) leaves in the different experimental treatments. Values are reported as means ± standard errors.

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Leaf optical properties are shown in Figure 5.4. It is noteworthy that very high values of

leaf absorptance and the specific-mass absorption coefficient (aw*) were measured in P. oce-

anica in all experimental treatments, relative to C. nodosa. All leaf optic properties showed sig-

nificant effects in the interaction term salinity x population depth for P. oceanica and for the

pigment-specific absorption coefficient a* in C. nodosa (two-way ANOVA, P < 0.001, d.f. =

1, 8). Hypersaline stress caused a significant increase in how efficiently pigments and leaf mass

of P. oceanica leaves from the deep meadow (43D) were able to absorb light (and hence the

leaf absorptance) , but efficiency clearly decreased in leaves of shoots from the 43S treatment

(Fig. 5.4a-c). In the case of C. nodosa leaves, the capacity to absorb light consistently in-

creased under hypersaline conditions in both S and D plants. In respect of the leaf mass per

area (LMA or biomass packing), the hypersaline treatment had significant effects only in the

shallow plants, increasing in the case of P. oceanica and decreasing for C. nodosa (Fig. 5.4d).

Figure 5.4. Leaf optical propertiesmeasured in P. oceanica (solid bars) and C. nodosa (open bars) leaves exposed to the different experimental treatments. Upper panels: light absorbance spectra of both species in the PAR range (values are mean of six replicates). Lower panels: (a) average of leaf absorptance (A), (b, c) pigment-specific absorption coefficients (a*, aw*) and (d) leaf mass per area (LMA). Values represented in the lower panels are reported as means ± standard errors.

159

160

Figure 5.5. Leaf water relations determined in P. oceanica (solid bars) and C. nodosa (open bars) leaves exposed to the experimental treatments. Ψw = water potential, Ψπ = osmotic potential Ψp = turgor pressure The lines inside the bars represent the seawater osmolality of each treatment expressed in pressure units. Values are reported as means ± standard errors.

Hypersaline treatment significantly affected water relations in leaf tissues of shallow and

deep plants of both seagrass species (two-way ANOVA, P < 0.01, d.f. = 1, 8; Fig. 5.5). Chan-

ges consisted of a general reduction in their leaf-water potential (Ψw) and osmotic poten-

tial (Ψπ), leading to significant increments in turgor pressure, except in the deep C. nodosa

plants. In agreement with this reduction in leaf Ψπ, leaf tissues of saline-stressed plants showed

a significant increase of organic osmolytes (soluble sugars and proline concentrations; Fig.

5.6a, e). Leaf starch content also increased in saline-stressed plants, except in those of the

shallow C. nodosa meadow (Fig. 5.6b). Carbohydrate content in rhizomes was also signi-

ficantly affected by hypersaline treatments, but followed a more complex behaviour depen-

ding on the species and depth (Fig. 5.6c, d). Meanwhile, total free amino acid concentrations

showed a generalized reduction as a hypersaline effect (Fig. 5.6f). Similarly, the C/N ratio sig-

nificantly increased in leaves of the 43S and 43D plants, but only in P. oceanica (Fig, 5.6g).

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Figure 5.6. Concentration of non-structu-ral carbohydrates (starch and soluble frac-tion) measured in leaf and rhizome tissues (a–d), leaf concentration of proline (e) and total free amino acids (FAAs; f ), and C/N molar ratio of leaves (g) and rhizomes (h), obtained in P. oceanica (solid bars) and C. nodosa (open bars) plants exposed to the experimental treatments. Values are reported as means ± standard errors.

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Figure 5.7. Vegetative variables (shoot morphology and growth) and net shoot change determined in experimental plants of P. oceanica (solid bars) and C. nodosa (open bars) at the end of the experimental period. Values are reported as means ± standard errors.

Differences in shoot size and productivity between both species are notable, as is cha-

racteristic of this seagrass species (Table 5.1; Annex 1; Olesen et al. 2002). Major effects of

treatment on shoot variables were associated with depth, while hypersalinity had no or only

mild effects. Shoots of the deep C. nodosa meadow showed a slight (but only significant

in plants from the deep meadow, P < 0.05) increment in shoot size (Fig. 5.7a) and produc-

tivity (Fig. 5.7b, c, e). Shallow and deep P. oceanica plants showed a slight (but not signi-

ficant) decreasing trend in the rate of production of new leaves and a significant shoot de-

cline (two-way ANOVA, P < 0.01, d.f. = 1, 8) in response to hypersalinity (Fig. 5.7c, f).

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5.4.2. PLASTICITY INDEX (RDPI)

In general, and considering all variable groups (Figure 5.8), global RDPI values were higher

in deep P. oceanica plants (0.12) than in shallow plants (0.09), in particular due to the relative and

significant contribution of variables belonging to the ‘photosynthesis’, ‘water relations’ and ‘or-

ganic compounds’ groups in plants of the deep meadow (one-way ANOVA, P < 0.001). None-

theless, RDPI values calculated for some individual variables (net-P, Chla, LMA and FAAs) did

not follow this general pattern and were significantly higher in the shallower plants (P < 0.001).

In contrast, the global RDPI value obtained for C. nodosa was lower in the deep plants (0.07)

when compared to the shallow ones (0.17), with variables related to ‘photosynthesis’ (net-P, Rd

and P:R), soluble carbohydrates in rhizomes and ‘pigments and leaf optics’ (Chlb and Chb:a)

being the major contributors to this pattern (P < 0.001). RDPI values obtained for proline and

carbohydrate concentrations in leaves (soluble and starch) and starch in rhizomes followed a

significant inverse pattern between the shallow and deep plants (P < 0.01).

5.4.3. MULTIVARIATE ANALYSIS (PCA)

Multivariate scaling through a two-dimensional PCA plot (Fig. 5.9) showed two clear eigen-

vectors: a) the PC1 axis, which explained the highest percentage of variance (75.5 %) and mainly

represented differences in inherent physiological faculties between P. oceanica and C. nodosa

(i.e. interspecific variation); and b) the second PC2 axis, which explained the variance of 16.9

%, revealing differences between shallow and deep experimental populations (i.e. intraspecific

variation), and the effect of hypersaline treatments on each seagrass species. The PC1 axis was

positively and highly correlated (scores > 0.75) with variables related to water relations, organic

compounds (proline, free amino acids, leaf-soluble sugars and starch) and photosynthetic-rela-

ted descriptors (P:R, δ13C and photosynthesis rates), and negatively correlated with ‘optical pro-

perties descriptors’, Chlb:a ratio and Chlb (scores ≤ −0.7). For its part, the PC2 axis was mainly

correlated with leaf dark respiration (score = −0.69), indicating increased respiratory activity in

saline-stressed plants. Other variables were also correlated with this axis both negatively (water

relations, compatible solutes and light absorption) and positively (net-P, P:R and chlorophylls),

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but with lower scores (−0.3 to −0.6). Based on the distance between objects along the PC2 axis,

the results showed: 1) higher intraspecific variation for C. nodosa than P. oceanica at ambient sali-

nity; and 2) that analyzed variables were more responsive to hypersaline treatments in plants from

the deep P. oceanica meadow, but in C. nodosa the larger effects corresponded to shallow plants.

Figure 5.8. Relative distances between control and hypersaline conditions given by the plasticity index (RDPI) calculated for physiological traits measured in P. oceanica and C. nodosa experimental populations.

165

Figure 5.9. Ordination diagram of principal component analysis (PCA) performed with physiological variables measured in P. oceanica (●) and C. nodosa (○) experimental populations: CD = control-deep, CS = control-shallow, 43D = hypersaline-deep and 43S = hypersaline-shallow.

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5.5. DISCUSSION

5.5.1. INTER- AND INTRASPECIFIC VARIATION

Under normal salinity conditions (i.e. control treatments), major differences in physiolo-

gical variables were observed between species (i.e. 76% of the variance explained by PC1 axis

of the PCA, Fig. 5.9). This interspecific variation consisted of a substantially higher photosyn-

thetic capacity and carbon balance of C. nodosa relative to P. oceanica, which is characteristic

of these seagrass species in summer (Drew 1978b; see also Table 5.1 and Fig. 3 of Annex 1);

the high δ13C values of C. nodosa leaves reflected the high photosynthetic rates reported for

this species, which is in accordance with the different strategies for carbon metabolism sug-

gested for both species (Beer et al. 1980a; Hemminga and Mateo 1996). P. oceanica leaves

had a higher pigment content and capacity to absorb light than C. nodosa, while this latter

species showed higher photosynthetic efficiency (α) and photoprotection capacity, as indi-

cated by the greater investment in antenna pigments (carotenoids:Chla). These differences

probably reflect the different strategy of each species to capture and use light, which could

in part be determined by their respective differences in plant size, clonal architecture and

complexity of meadow structure (Pérez and Romero 1992; Marbà et al. 1996; Dalla Via et

al. 1998; Olesen et al. 2002; Collier et al. 2008). Large and highly pigmented P. oceanica

shoots form typically dense canopies, which strongly reduce available light (e.g. Dalla Via

et al. 1998), as reported in this study for natural and laboratory leaf canopies (see Material

and Methods and Fig. 12 of Annex 1). In comparison, C. nodosa shoots are smaller, with

thinner and less pigmented leaves, forming leaf canopies that exert a lower control of light

availability (40% of light transmission; Raniello et al. 2004); this in turn involves a higher ex-

posure to light of photosynthetic tissues and hence a reduced absorption cross section and

a higher investment in the photoprotection characteristic of sun-adapted plants (Demmig-

Adams and Adams 1993; Ralph et al. 2002; Falkowsky and Raven 2007). One other remarka-

ble interspecific difference was the higher content of soluble carbohydrates and amino acids

(in particular proline) in C. nodosa leaves relative to P. oceanica, which is consistent with in-

terspecific differences in photosynthetic rates, but could also reflect differences in nitrogen

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metabolism or in other relevant metabolic processes (Pirc and Wollenweber 1988; Touchet-

te and Burkholder 2007); for instance, these are known organic osmolites in aquatic marine

macrophytes (Kirst 1989; Tyerman 1989), concentrations of which in C. nodosa tissues have

been associated with osmoregulatory and osmoprotective functions (Drew 1978a; Sandoval-

Gil et al. 2012-Chapter 4). Accordingly, interspecific differences in water and osmotic po-

tentials reported in this study have been interpreted as an adaptive advantage of C. nodosa

in resistance to sudden and chronic salinity increments (Sandoval-Gil et al. 2012-Chapter 4).

Intraspecific (i.e. between-depths) physiological variation had a minor relative impor-

tance (overall in P. oceanica, see Fig. 5.9), but was significant for most variables. In both

seagrass species, photosynthetic rates and efficiencies, carbon balance, pigment content,

proline and water potential were consistently higher in plants from the shallower meadow

relative to those from the deeper one. Such intraspecific variation was less consistent for the

remaining selected variables, and more complex between species. Also, some of this intras-

pecific variation is consistent with depth-related patterns described in natural meadows of

both seagrass species (e.g. Drew 1978b), but not with evidence obtained in other studies

(Olesen et al. 2002). The lack of a general pattern for morphological and physiological plas-

ticity along depth gradients seems to be a common feature of seagrass meadows, attributable

to a number of scale-related considerations (Pirc 1986; Dalla Via et al. 1998; Larkum et al.

2006; Ralph et al. 2007; Collier et al. 2008). For instance, some photosynthetic variables

(e.g. α, Rd and pigment content) suggested shade adaptation of leaves in the shallow ex-

perimental population just where light availability was higher, and a similar pattern was ob-

served under natural conditions in their respective reference meadows (see Table 5.1 and

Annex 1). This result can only be explained by a differential effect of canopy structure on

self-shading, as indicated by the reported differences in light attenuation coefficients (Kd)

and light levels (two- to three-fold higher in shallow canopies) between shallow and deep

P. oceanica plants in both natural and mesocosm conditions (see Material and Methods).

This consistency in results between experimental plants and reference meadows (Table

5.1) was in general found for most of the physiological and vegetative variables and was within

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the ranges for these variables reported in the literature for the same season (e.g. Drew 1978b;

Pirc and Wollenweber 1988; Pirc 1989; Pérez and Romero 1994; Alcoverro et al. 1998; Alcove-

rro et al. 2001; Olesen et al. 2002; Enríquez et al. 2004). This strongly supports the notion that

the patterns of both intraspecific and interspecific variation observed in our mesocosm system

can be considered highly representative of real natural conditions, at least of the meadow

locations and season considered in this study. Furthermore, net shoot mortality under control

conditions was always lower than 4% throughout the experimental period, supporting the as-

sumption that no additional stress was caused by artificial conditions. These are crucial aspects

determining the reliability of this type of experimental study, in particular through avoiding

artefacts or confounding effects that could mask specific plant responses to experimental

manipulations (Marín-Guirao et al. 2011-Chapter 2; Sandoval-Gil et al. 2012-Chapter 4).

5.5.2. PHYSIOLOGICAL RESPONSES TO HYPERSALINE STRESS

The overall physiological plasticity indexes calculated at the species level indicated that P.

oceanica and C. nodosa had similar plastic physiological capacities in response to hypersaline

stress (RDPI = 0.13 and 0.12, respectively), contrary to what might be expected on the basis of

their known differences in biological attributes and ecological strategies (Drew 1978b; Marbà

et al. 1996; Olesen et al. 2002) and differential tolerance to chronic hypersaline conditions

(e.g. Fernández-Torquemada and Sánchez-Lizaso 2005, 2011; Sandoval-Gil et al. 2012). This

result is mainly explained by the high variability of the RDPI index between plants from di-

fferent depths within each seagrass species, with higher values obtained in deep P. oceanica

and shallow C. nodosa plants (as also illustrated in the PCA results, Fig. 5.9). As documented

for terrestrial plants in relation to stress tolerance (DeWitt and Scheiner 2004; Dudley 2004;

Pugnaire and Valladares 2007), the true functional sense of this variability was based in the di-

fferential capacity of species and populations to modify particular plant traits that were closely

related with key acclimation processes. The specific physiological alterations and adjustments

responsible for the reported patterns of physiological plasticity are summarized as follows.

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Photosynthesis inhibition, as reported for 43S and 43D P. oceanica plants, is a common

response to chronic salinity increments in this and other seagrass species (Ogata and Matsui

1964; Biebl and McRoy 1971; Kerr and Strother 1985; Fernández-Torquemada et al. 2005b;

Kahn and Durako 2006; Koch et al. 2007b; Marín-Guirao et al. 2011-Chapter 2). Based on

previous similar studies, the authors provided evidence indicating that photosynthesis of C.

nodosa is less sensitive to salinity increments than P. oceanica (Marín-Guirao et al. 2011-Chap-

ter 2; Sandoval-Gil et al. in press-Chapter 3), which supports the hypothesis of a higher ca-

pacity of the former for growth in hypersaline conditions; this contention was corroborated in

the case of plants from the deep meadow, but not for those of the shallower one, in which

the photosynthetic rate was reduced by 48% from the control mean. This reduction was even

more intense than that reported in a previous study of C. nodosa plants from the same sha-

llow meadow and using the same experimental approach (17%, Sandoval-Gil et al. in press-

Chapter 3), although some key experimental conditions differed as the lower duration of the

experiment (45 days) and the season (autumn) in which the former experiment was performed.

Salt-induced changes in pigment composition and optic properties of seagrass leaves

have also been reported and have been interpreted as severe deterioration of the structure

and function of the photosynthetic apparatus (Ralph 1998, 1999), but also as adjustments to

optimize light capture and for use in relation to carbon balance (Sandoval-Gil et al. in press-

Chapter 3). The pigment content of P. oceanica leaves was unaffected in a previous experiment

(Marín-Guirao et al. 2011-Chapter 2), but Chla and Chlb content consistently increased in plants

of 43S and 43D treatments in this work. This consistent effect, however, gave rise to opposite

trends in the Chlb:a ratio, which increased in P. oceanica leaves of deep plants and decrea-

sed in those of shallow plants. This variable is indicative of the antenna size of photosystem

units (Kirk 1994; Falkowsky and Raven 2007) and could account for the same response pat-

tern observed in leaf absorptance variables and photosynthetic efficiency (α) of experimental

plants of this seagrass species. Other salt-induced changes in variables related to leaf anatomy,

such as LMA, are able to influence leaf optic properties and hence could also contribute to

explaining the reported pattern in these variables (e.g. the increase in mean LMA and pig-

ment package effect in the shallow plants; Enríquez 2005). As was the case for P. oceanica,

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leaf absorptance and photosynthetic efficiency of C. nodosa also increased in the 43D plants,

but in this case there was a decline in pigment content and antenna size; a possible explanation

could be the slight decrease in LMA observed in these experimental plants, since it is related to a

more efficient distribution of pigments in photosynthetic structures (e.g. reduced package effect;

Enríquez 2005; Enríquez et al. 1992), as already evidenced for this species in a previous study

(Sandoval-Gil et al. in press-Chapter 3). All this complex variety of photosynthetic adjustments

reflects physiological alternatives developed by species and populations to cope with metabolic

alterations induced by hypersaline stress. A reduced capacity to capture and use light can be a

consequence of down-regulation mechanisms resulting from inhibition of photosynthetic carbon

metabolism (Vogelmann 1993; Enríquez and Sand-Jensen 2003), while an enhancement in light

absorptance should be necessary to cope with respiratory demands and/or alternative metabo-

lic pathways enhanced under hypersaline conditions (e.g. photorespiration; Wingler et al. 2000).

Enhancement of respiration rates is another important documented effect of hypersaline

stress on these seagrass species (Marín-Guirao et al. 2011-Chapter 2; Sandoval-Gil et al. in press-

Chapter 3), related to the high cost of metabolic processes activated to cope with increments

in external salinity, such as ion transport, osmoregulation or translocation of internal resources

(Ogata and Takada 1968; Drew 1978a; Munns 2002, Atkin and Macherel 2009). Changes in this

variable represented the major contribution explaining both intraspecific variation and the diffe-

rential response of shallow and deep plants to hypersaline treatments (r = −0.62 on the PC2 axis

of the PCA; Fig. 5.9), and accounted in part for the severe carbon imbalance observed in plants

of P. oceanica in the deep meadow and in those of C. nodosa from the shallow meadow. Based

on the effect of hypersaline stress on gross photosynthesis and δ13C, other mechanisms of pho-

tosynthesis inhibition should be involved in these carbon imbalances, as in shallow P. oceanica

salt-stressed plants. Such mechanisms are probably related to an inhibition of enzymes operating

in carbon assimilation and reduction pathways (Beer et al. 1980a; Parida and Das 2005; Farooq et

al. 2009). In deep plants of P. oceanica, this metabolic imbalance could be compensated by the

higher photosynthetic efficiency, which involves higher photosynthetic rates at the low light levels

inside the leaf canopy simulated in our mesocosm system (which is consistent with the higher car-

bohydrate content founded in these plants). A consequence of the inhibition of photosynthetic

171

carbon assimilation in plants from the shallow meadow can additionally be the down-regulation

of the structure and function of the photosynthetic apparatus (e.g. through decrease of the

Chlb:a ratio and photosynthetic efficiency) and hence the avoidance of overenergizing PSII and

photodamage (Vogelmann 1993; Kirk 1994; Enríquez and Sand-Jensen 2003; Enríquez 2005).

Variables related to leaf carotenoid content showed a relatively low plastic response to

hypersaline stress, but with functional consequences particularly interesting for plant accli-

mation and survival. Total carotenoid concentration and the carotenoid:Chla mass ratio con-

sistently increased in all salt-stressed plants of both seagrass species, and the same response

was recently reported in similar experiments performed with these species (Marín-Guirao et

al. 2011-Chapter 2; Sandoval-Gil et al. in press-Chapter 3). In terrestrial plants, this response

has been demonstrated to be an effective photoprotective mechanism for dissipating excess

energy (as heat) from photosystems and for avoiding the toxic effects of accumulation of reac-

tive oxygen species on photosynthetic membranes (Demmig-Adams and Adams 1996; Niyogi

2000; Atkin and Macherel 2009). The operation of the xantophyll cycle has been evidenced

in seagrasses (Ralph et al. 2002; Collier et al. 2008; Trevathan et al. 2011; García-Sánchez et

al. 2012), and in these Mediterranean seagrasses, further experimental evidence also demons-

trates enhancement of thermal dissipation (NPQ) under hypersaline conditions (Marín-Guirao

et al. 2011a). This mechanism seems to be effective in C. nodosa, since the maximum photo-

chemical efficiency (Fv/Fm) of which remained unaffected by hypersaline conditions. This was

not so clear in P. oceanica, since the mean Fv/Fm decreased below control values, indicating

some accumulation of photodamage on the PSII of leaves exposed to hypersaline treatments.

Other relevant physiological effects of hypersaline stress showing a consistent response

pattern between population depths were those indicated by key variables for water relations.

In all cases, C. nodosa and P. oceanica leaves reduced their osmotic potential (Ψπ) to main-

tain favourable water balances (i.e. more negative Ψw). The accumulation of organic osmoli-

tes, as indicated by the consistent increment of soluble sugars and proline in leaf tissues, pro-

bably contributed to the reported Ψπ adjustments and strongly supported the activation of

osmoregulatory processes (Kramer and Boyer 1995; Verslues et al. 2006; Sandoval-Gil et al.

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2012-Chapter 4). Another consistent salt-induced metabolic alteration was the reduction in

total free amino acid content in leaves of both seagrass species, reported also in a previous

similar experiment but only for C. nodosa (Sandoval-Gil et al. 2012-Chapter 4), that has been

suggested as a result of other alterations in the nitrogen metabolism (Joshi et al. 1962; Pu-

lich 1986; Invers et al. 2004; Gacia et al. 2007). Reduced N content (and higher C/N) in salt-

stressed leaves supported this notion for P. oceanica, but not for C. nodosa in our experiment.

Evidence of osmotic adjustment has been obtained for seagrasses exposed to hypersali-

ne conditions (Brock 1981; Pulich 1986; Tyerman 1989; Murphy et al. 2003; Koch et al. 2007)

and in P. oceanica, but not for C. nodosa, which has developed an alternative dehydration

avoidance strategy, probably based on cell-wall hardening processes (Sandoval-Gil et al.

2012-Chapter 4). Intraspecific variation was also found in the magnitude of these responses

and in other related aspects. Those plants that developed more negative osmotic potentials

were just those in which respiration was enhanced (i.e. deep P. oceanica and shallow C. nodo-

sa), probably reflecting the high metabolic cost associated with active osmoregulation (Munns

2002); also in these plants turgor pressure increased, suggesting the activation of a different

type of dehydration avoidance strategy based on some turgor-dependent processes, as evi-

denced in terrestrial plants and other marine macrophytes (e.g. turgor-sensing mechanisms,

ionic fluxes and downstream signalling cascades: Zimmermann 1978; Bisson and Kirst 1995).

The mechanisms by which metabolites are concentrated in salt-stressed tissues in response

to osmoregulatory demands also appears to differ among experimental populations. For ins-

tance, as mentioned earlier, the increase in soluble carbohydrates in deep P. oceanica plants

exposed to hypersaline conditions could be explained by a higher assimilation of photosyn-

thetic carbon in these plants; in contrast, in salt-stressed shallow P. oceanica plants, transloca-

tion from rhizome reserves is more probable, since their photosynthetic performance redu-

ced more severely and starch concentration was significantly reduced in their rhizome tissues.

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5.5.3. FUNCTIONAL CONSEQUENCES FOR PLANT GROWTH AND SURVIVAL

The integration of physiological plasticity expressed in response to stress can result in

different functional consequences for plant performance and survival, and hence in the de-

termination of differential tolerance capacities between species and/or populations (deWitt

and Scheiner 2004). In this study, based on plant responses at the vegetative level, the

physiological plasticity developed by P. oceanica to cope with hypersaline stress did not

seem to interfere with shoot morphology and growth, but shoot mortality did significantly

increase relative to the control plants. Reduction in shoot density has been demonstra-

ted to be the most important plastic acclimatory response of seagrass species in relation

to natural (e.g. depth; Dalla Via et al. 1998; Olesen et al. 2002; Collier et al. 2008) and

human-disturbed (Ruiz et al. 2001; Ruiz and Romero 2003; Ruiz et al. 2009-Chapter 1)

environmental gradients, relative to acclimation capacities at the morphological and phy-

siological level. Similarly, the P. oceanica shoot decline reported in this experiment seems to

be a consequence of maladaptive physiological plasticity which was unable to compensate

for the high metabolic cost of acclimation to hypersaline conditions (e.g. osmoregulation;

Sandoval-Gil et al. 2012-Chapter 4). Furthermore, these shoot losses significantly differed

between P. oceanica populations, suggesting a role of the reported intraspecific physiologi-

cal plasticity in determining stress tolerance. In plants from the deep meadow, for instance,

a higher physiological plasticity probably involves a high metabolic cost in acclimating to

hypersaline stress, but also provides compensating photosynthetic adjustments and enables

a higher shoot survival than in the shallow experimental population. Such compensatory

mechanisms did not occur only in the shallow plants and resources needed to be transloca-

ted from other plant parts, as evidenced in a previous study in which a shoot density of P.

oceanica declined in response to simulated hypersaline stress (Ruiz et al. 2009-Chapter 1).

Contrary to the results for P. oceanica, metabolic alterations caused by hypersaline stress

did not interfere with shoot morphology, growth and survival of C. nodosa, regardless of the

depth of origin and the great differences in physiological plasticity between experimental

populations (e.g. RDPI = 0.07 and 0.17 in deep and shallow plants, respectively). The re-

174

markable productive capacity of C. nodosa, together with its high levels of internal resour-

ces, suggests that the metabolic cost associated with the physiological acclimation to hy-

persaline stress can be easily assumed by the plant; these species-specific characteristics are

also indicative of its high turnover rate and ability to respond to disturbance, as suggested

by the apparent (but not significant) increase in the production of new leaf tissue and shoots

in the deep C. nodosa plants. Our results are therefore consistent with the ecological stra-

tegy of this seagrass species in marine coastal environments of the Mediterranean Sea and

its higher capacity for growth across a wider range of salinity regimes than the slow-growing

P. oceanica, which is more sensitive to salinity increments and is restricted to open coastal

waters with an almost constant salinity (Fernández-Torquemada and Sánchez-Lizaso 2005,

2011; Marín-Guirao et al. 2011-Chapter 2; Sandoval-Gil et al. 2012-Chapter 4; Sandoval-Gil

et al. in press-Chapter 3). Thus, the results obtained in this study provide robust evidence

for the importance of species-specific characteristics in determining tolerance capacities to

hypersaline stress for P. oceanica and C. nodosa, but also suggest that intraspecific plasticity

can play an important, although secondary, role at least in P. oceanica. This intraspecific

physiological plasticity has been related to ecotypic and/or genotypic differentiation of

plant populations associated with geographic or environmental gradients (Dudley 2004;

Ghalambor et al. 2007; Baquedano et al. 2008) and, based on our results, should be consi-

dered a relevant factor in the determination of tolerance mechanisms and the capacity of

seagrasses to respond to hypersaline stress. However, based on this and previous similar

experimental studies, few generalizations can be made about the type and strength of accli-

matory physiological mechanisms developed in response to hypersaline conditions in these

Mediterranean seagrass species, since they are complex and differ in function between spe-

cies and plants from different depths, and also in stress characteristics (e.g. intensity and du-

ration of the salinity increment), growth patterns, seasonality and environmental variability.

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Shallow Posidonia oceanica meadow. Isla Plana (Mazarrón, Murcia Region)

176

Shallow Posidonia oceanica and Cymodocea nodosa meadows. Isla Plana, Mazarrón (Murcia Region)

DISCU

SIÓN

GENE

RAL

2

179 TOLERANCIA DE P. OCEANICA Y C. NODOSA

AL INCREMENTO DE LA SALINIDAD

180 OSMOACLIMATACIÓN

184 FOTOSÍNTESIS Y RESPIRACIÓN

189 PIGMENTOS Y PROPIEDADES ÓPTICAS FOLIARES

192 CONSECUENCIAS A NIVEL VEGETATIVO

194 PLASTICIDAD FISIOLÓGICA DE P. OCEANICA Y

C. NODOSA EN RESPUESTA AL ESTRÉS HIPERSALINO

197 PERSPECTIVAS DE INVESTIGACIÓN

Jose Miguel Sandoval-Gil, Juan Manuel Ruiz, Lázaro Marín-Guirao, Jaime Bernardeau-Esteller, Jose Luis Sánchez-Lizaso, Lia Piro, Ilia Anna Serra, Antonia Spadafora, Silvia Mazzuca.Responses of the Mediterranean seagrasses (Posidonia oceanica and Cymodocea nodosa) to hypersaline stress at the

whole plant, physiological and molecular levels.

En preparación para su publicación en Frontiers

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1. TOLERANCIA DE P. OCEANICA Y C. NODOSA AL INCREMENTO DE LA SALINIDAD

Por su distribución y características ecológicas, las praderas de P. oceanica y C. nodosa estan consideradas entre

las comunidades bentónicas más vulnerables al impacto ambiental derivado de los vertidos hipersalinos de plantas

desalinizadoras (Fernández-Torquemada et al. 2005a; Ruiz 2005; Palomar y Losada 2010). El desarrollo de la in-

dustria desalinizadora en las costas mediterráneas españolas a principio de la pasada década, motivó la puesta en

marcha de los primeros estudios diseñados para determinar los límites de tolerancia al incremento de la salinidad

de estas especies, con el fin de establecer unos criterios de control de los vertidos de salmuera al medio marino

que garantizaran la conservación de estos valiosos hábitats (Sánchez-Lizaso et al. 2008; Fernández-Torquemada

y Sánchez-Lizaso 2011). Estos primeros trabajos se basaron en diferentes aproximaciones experimentales que ana-

lizaron, por un lado, los efectos reales a largo plazo de vertidos de salmuera sobre praderas de P. oceanica (Gacia

et al. 2007), y por otro parte, sus efectos a corto plazo (2-3 semanas) sobre plantas de P. oceanica y C. nodosa,

simulados en condiciones de laboratorio (Fernández-Torquemada y Sánchez-Lizaso 2005, 2006; Fernández-Tor-

quemada y Sánchez-Lizaso 2011). De forma complementaria a estos estudios, se llevó a cabo un tercer tipo de

aproximación que consistió en la simulación de un incremento de la salinidad a medio plazo (3 meses), pero in situ,

es decir, desviando un vertido de salmuera experimental sobre una pradera intacta, y distribuyéndolo en una serie

de parcelas experimentales controladas. Los resultados de este complicado e inusual experimento son los presen-

tados en el capítulo 1 de esta tesis; dichos resultados permitieron corroborar la elevada sensibilidad de P. oceanica

a pequeños incrementos de la salinidad por encima de la salinidad media ambiente (ca. +1 ups; Ruiz et al 2009), y

por tanto fijar el límite de tolerancia de esta especie, a partir del cual se reduce su abundancia, vitalidad y supervi-

vencia. Si bien en estos primeros trabajos, y en otros pioneros más tempranos (Drew 1978a), se aportaba ya alguna

evidencia de las posibles alteraciones fisiológicas inducidas por el incremento de la salinidad en ambas especies,

el conocimiento básico de los mecanismos de respuesta ecofisiológica al estrés hipersalino y su relación con los

límites de tolerancia a dicho estrés, era prácticamente ausente. Los trabajos presentados en la presente tesis, jun-

to con otros más recientes (p.e. Serra et al. 2011, 2012), suponen un primer avance significativo en este campo.

Los resultados obtenidos en esta tesis doctoral, ponen en evidencia los efectos inducidos por estrés hipersalino

a distintos niveles de organización de las especies estudiadas (i.e. población, individuo, fisiológico/metabólico).

Dichos efectos, resumidos esquemáticamente en la Fig. 3, se han discutido con detalle por capítulo, y se resu-

men en los siguientes apartados en un contexto más general. Los principales procesos y funciones con los que

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se relacionan las respuestas observadas son: i) procesos de osmo-aclimatación de las relaciones hídricas foliares,

ii) reducción de las capacidades fotosintéticas, iii) alteración de las tasas respiratorias, y iv) modificaciones de las

propiedades ópticas foliares debido a cambios a nivel de pigmentos fotosintéticos, ultraestructura y anatomía foliar.

Por otro lado, y como se dicute en el capítulo 5, los rangos de tolerancia o sensibilidad al estrés hipersalino específi-

cos de cada especie, dependerán en general de i) su capacidad fisiológica inherente resultante de su adaptación al

rango salino del hábitat que ocupa (i.e. osmoadaptación) y ii) de su plasticidad fisiológica y capacidad aclimatativa

a dicho estrés (Kirst 1989). En general, especies como C. nodosa, capaces de sobrevivir y desarrollarse en ambientes

con distintos regímenes salinos, presentarán ciertas ventajas adaptativas y, presumiblemente, mayores capacidades

de aclimatación a condiciones de hipersalinidad, que aquellas como P. oceanica, cuya distribución se encuentra

limitada a rangos de salinidad más reducidos. Pero los mecanismos de aclimatación, y su grado de expresión en con-

diciones de estrés hipersalino (plasticidad fisiológica), también pueden estar condicionados por fuentes de variación

intra-específica. En efecto, determinados límites de tipo ecológico (p.e. herbivoría, competencia, sinergia con otros

tipos de estrés, etc) o internos (p.e. integración fenotípica, estados fisiológico y ontogénico relacionados con ciclos

estacionales, etc), junto con divergencias ecotípicas y/o genotípicas, son consideradas en la actualidad como impor-

tantes factores condicionantes de la variación en la potencial plasticidad de las plantas frente a condiciones de estrés

(Dudley 2004; Valladares et al. 2007). Así pues, la influencia de estos factores debe ser también tenida en cuenta para

explicar la variación en las respuestas documentadas en los diferentes experimentos de esta tesis, incluso cuando se

trata de la misma especie y condiciones experimentales.

2. OSMOACLIMATACIÓN

Al aumentar la salinidad disminuye el potencial hídrico/osmótico del medio. Como respuesta aclimatativa inme-

diata, se pueden producir desequilibrios hídricos y alteraciones de la homeostasis iónica en los tejidos (Verslues et al.

2006). Sin embargo, y a más largo plazo, las plantas son capaces de desarrollar respuestas de aclimatación con el fin de

restablecer las condiciones iniciales de equilibrio osmótico. En este sentido, P. oceanica y C. nodosa han demostrado

ser capaces de activar este tipo de mecanismos conocidos como de evitación de la deshidratación tisular (i.e. dehydra-

tion avoidance strategies, sensu Verslues et al. 2006), que pueden resumirse en dos : i) reducción del potencial hídrico

(Ψw) mediado por disminución del potencial osmótico (Ψπ), como consecuencia de la activación de procesos osmo-

rregulatorios, y ii) procesos de endurecimiento de pared celular, relacionados con reducciones de Ψw mediadas por

pérdidas de presión de turgor celular (Ψp) pero no de agua simplástica (Kramer y Boyer 1995; Verslues et al. 2006).

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Como consecuencia de las capacidades osmorregulatorias mostradas por P. oceanica y C. nodosa frente a incremen-

tos de salinidad, ambas especies mostraron la capacidad de mantener valores de Ψp similares, o incluso más elevados,

que plantas no estresadas. De forma semejante a lo observado en otras especies de angiospermas marinas, la reduc-

ción de Ψπ fue posible, al menos en parte, por el incremento en la concentración de solutos orgánicos intracelulares

como azúcares solubles y prolina (Brock 1981; Pulich 1986; Tyerman 1989; Murphy et al. 2003; Koch et al. 2007b). En

este sentido, cabe destacar que dichos solutos no sólo son considerados como los principales agentes osmóticos orgá-

nicos, sino también como eficientes solutos compatibles, cuya capacidad osmoprotectora ha sido demostrada frente

a los efectos de toxicidad por la acumulación de iones (Flowers et al. 1977). Sin embargo, también se obtuvieron evi-

dencias de la activación de procesos alternativos a la osmorregulación en C. nodosa, como los derivados de un posible

endurecimiento de pared celular (cap. 4). Desde el punto de vista metabólico, éstos son mecanismos menos costosos

que la osmorregulación y podrían ser efectivos como estrategias conservativas de la planta en situaciones de escasez

de recursos. Esta situación podría ser el caso del experimento del capítulo 4 realizado en el periodo otoño-invierno,

donde esta especie presenta un estado natural caracterizado por reducidas capacidades fotosintéticas, menor con-

centración de carbohidratos, tasas respiratorias más elevadas y valores menos negativos de Ψw (y Ψπ) (ver Anexo 1).

A diferencia de los azúcares solubles y el aminoácido prolina, el contenido de almidón y la concentración foliar de

aminoácidos libres totales, mostraron patrones de variación poco consistentes con el incremento de la salinidad. En

el caso del almidón, se observó una reducción significativa de su contenido en los tejidos rizomáticos de plantas de P.

oceanica de praderas naturales expuestas in situ a estrés hipersalino (capítulo 1), aunque sin embargo, no se observó

una pauta de respuesta general (ni en tejidos rizomáticos ni foliares) de este descriptor en los experimentos de meso-

cosmos realizados posteriormente con ambas especies (ver capítulos 4 y 5). No obstante, esta falta de consistencia

podría ser explicada atendiendo a la variedad de funciones metabólicas en las que los carbohidratos de reserva pueden

verse implicados. En efecto, y aunque el almidón ha sido sugerido como agente osmótico (p.e. Parida y Das 2005), la

mayoría de estudios llevados a cabo con plantas terrestres y angiospermas marinas indican que la alteración en sus ni-

veles puede encontrarse relacionada con la demanda metabólica de reservas energéticas, translocación entre tejidos y

ramets, o incluso, con ajustes osmorregulatorios indirectos como la regulación de volumen osmótico celular (Ackerson

y Hebert 1981; Ogata y Takada 1968; Hsiao,1973; Drew 1978a; Alcoverro et al. 2001; Marbá et al. 2002). Adicionalmen-

te, podrían intervenir procesos de compartimentación de recursos internos que no supongan una modificación de la

concentración total de almidón en tejidos foliares y rizomas. De hecho, si bien los análisis de carbohidratos no fueron

capaces de detectar diferencias entre tratamiento control e hipersalino a nivel de tejido foliar en P. oceanica (capítulo

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4), microfotografías obtenidas por microscopía electrónica de transmisión realizadas en cortes de las mismas hojas

analizadas en dicho experimento, mostraron la presencia de gránulos de almidón en los cloroplastos de hojas de plan-

tas control, pero no en las de plantas expuestas a estrés hipersalino (Fig. 1). No obstante, este aspecto no ha sido tra-

tado con profundidad en esta tesis y debería ser analizado de forma más rigurosa en futuros estudios más específicos.

Figura 1. Microfotografías de células epidérmicas foliares de plantas de P. oceanica procedentes de los tratamientos control (con salinidad de 37 ups, columna de la izquierda) e hipersalino (43 ups, columna de la derecha), correspondientes a los capítulos 2 y 4, obtenidas mediante microscopía electrónica de transmisión (MET, x 21000). Las flechas negras indican los gránulos de almidón acumulados en el estroma de cloroplastos, presentes únicamente para el caso de plantas control.

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Por otra parte, las distintas variaciones observadas en la concentración de ciertos aminoácidos libres de-

tectados en hojas de P. oceanica y C. nodosa, pudieron estar relacionados con diversas alteraciones metabóli-

cas derivadas de la exposición al estrés hipersalino. Entre ellas, posibles modificaciones de las tasas fotorrespira-

torias, así como posibles perturbaciones que pudieran darse a distintos niveles del metabolismo del nitrógeno

(e.g. asimilación, biosíntesis aminoacídica, síntesis y degradación de proteínas) han sido sugeridas en ambas es-

pecies (ver discusión al respecto en cap. 4), atendiendo a su probada activación en plantas terrestres y otras es-

pecies de angiospermas marinas en respuesta a distintas alteraciones del medio e incrementos de salinidad (Jos-

hi et al. 1962; Jørgensen et al. 1981; Pulich 1986; Dey y Harborne 1997; Invers et al. 2004; Gacia et al. 2007).

Los iones (principalmente las especies Na+, Cl-, K+ y Ca2+) desempeñan un papel clave para interpretar los

efectos de los cambios de la salinidad externa en las variaciones de las relaciones hídricas de macrófitos marinos y

plantas terrestres (Kirst 1989; Tyerman 1989; Hasegawa et al. 2000; Touchette 2007). Aunque éste ha sido un as-

pecto no tratado en esta tesis, se dispone ya de cierta información al respecto obtenida en investigaciones paralelas

desarrolladas por el equipo investigador. Tan sólo a efectos de dar una idea actualizada del estado del conocimiento

en este tema, a continuación se exponen, resumidamente, algunos resultados todavía inéditos (Marín-Guirao et al.

en revisión y Garrote et al. en preparación). En primer lugar, y de forma similar a lo descrito en halófitas terrestres

y angiospermas marinas (Tyerman 1989; Hasegawa et al. 2000), se dispone de evidencia experimental acerca de la

implicación de Na+ y Cl- en procesos de ajuste osmótico en P. oceanica y C. nodosa. Por su parte, las variaciones de

los iones K+ y Ca2+ parecen estar relacionados con mecanismos de homeostasis iónica celular (asimilación, exclusión

o compartimentación/translocación), cuyo estado viene indicado por los ratios K+:Na+ y Ca2+:Na+. El mantenimiento

de una homeostasis iónica adecuada es considerada como condición indispensable para el mantenimiento óptimo del

metabolismo celular (p.e. rutas enzimáticas y rutas de transducción de señal dependientes de los cofactores iónicos

de K+ y Ca2+)(Bisson y Kirst 1995; Hasegawa et al. 2000). Los valores de estos ratios sugieren que P. oceanica no

es capaz de mantener una homeostasis iónica bajo exposiciones crónicas a condiciones hipersalinas (43 ups), hecho

que ha sido relacionado con una reducción de su crecimiento vegetal (Greenway y Munns 1980; Hasegawa et al.

2000; Cramer 2002) y en general, con la baja tolerancia que presenta esta especie frente a incrementos de salinidad.

De acuerdo con el conocimiento disponible para el caso de otros grupos vegetales (Bisson y Kirst 1995; Hasegawa

et al. 2000; Läuchli y Lüttge 2002; Kahn y Weber 2006), el análisis de las relaciones hídricas sugiere que C. nodosa

presenta importantes ventajas adaptativas respecto a P. oceanica para tolerar el incremento de la salinidad; dichas

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propiedades se pueden resumir en: 1) C. nodosa mantiene menores valores (i.e. más negativos) de Ψw, propiedad

generalmente asociada a mayores rangos de tolerancia frente a estrés hídrico o salino; 2) C. nodosa presenta mayor

concentración de ciertos solutos, con funciones tanto osmóticas como osmoprotectoras; 3) C. nodosa es capaz de

adoptar estrategias de endurecimiento de pared celular de menor coste metabólico.

3. FOTOSÍNTESIS Y RESPIRACIÓN

El incremento de la salinidad causa, por lo general, una inhibición de las tasas fotosintéticas de las angiospermas

marinas, aunque el grado de alteración varía no sólo con la intensidad del estrés, sino también en función de la especie

y ecotipo/población (Ogata y Matsui 1964; Biebl y McRoy 1971; Kerr y Strother 1985; Fernández-Torquemada et al.

2005b; Kahn y Durako 2006; Koch et al. 2007b). De acuerdo con estos resultados previos y los obtenidos aquí en los

capítulos 2 y 5, las tasas de fotosíntesis bruta de P. oceanica se redujeron significativamente a salinidades ligeramente

superiores a la salinidad media ambiente (39 ups), mientras que en C. nodosa (y sólo para el caso de plantas de la

pradera más somera) no se observaron alteraciones de las mismas hasta niveles de salinidad más extremos (43 ups).

Esta distinta expresión de sensibilidad fotosintética puede estar relacionada con diferencias a nivel de metabolismo

fotosintético, sugeridas ya anteriormente entre ambas especies (Drew 1978b; Beer et al. 1980b; Hemminga y Mateo

1996; Invers et al. 1997), aunque también puestas de manifiesto a partir de algunos resultados obtenidos en esta tesis.

En relación a éstos últimos, tanto en las poblaciones experimentales de mesocosmos (capítulo 5) como en las prade-

ras de referencia (Anexo 1, sección 1), C. nodosa mantiene tasas fotosintéticas más elevadas y señales isotópicas del

carbono (δ13C) menos negativas, respecto a los valores de estas variables medidos en P. oceanica. Estas diferencias

han llevado incluso a sugerir que C. nodosa tiene un metabolismo fotosintético más eficiente, basado en algún tipo

de mecanismo de concentración de carbono, más cercano al de plantas tipo C4 (Larkum et al. 2006). De acuerdo

con estas observaciones, la distinta sensibilidad enzimática observada in vitro entre las enzimas principales de ambos

mecanismos (i.e. C3: RuBisCo y C4: fosfoenolpiruvato carboxilasa) (Beer et al. 1980a) podría ser determinante, junto

con otros factores (p.e. mayor presencia de solutos osmoprotectores, como prolina), de la mayor resistencia fotosin-

tética detectada en C. nodosa frente a incrementos de salinidad.

Al contrario que lo descrito para las tasas fotosintéticas, las tasas respiratorias de P. oceanica y C. nodosa no mos-

traron un patrón general de respuesta frente al incremento de la salinidad a lo largo de los experimentos realizados

en esta tesis. Conclusiones similares pueden extraerse de revisiones recientes de las alteraciones de la respiración

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en plantas terrestres sometidas a condiciones de estrés hídrico (Atkin y Macherel 2009), así como de los traba-

jos previos con angiospermas marinas (incluyendo C. nodosa) expuestas a estrés salino (Ogata y Takada 1968;

Drew 1978a; Khan y Durako 2006; Fernández-Torquemada et al. 2005b). En este sentido, se han identificado

tipos muy variados de alteraciones de las tasas respiratorias, incluyendo respuestas opuestas y de “no-respuesta”

(i.e. respuesta de canalización, Valladares et al. 2002), dependiendo de la especie considerada, de la duración e

intensidad de la exposición al estrés o incluso, de la madurez o del tipo de tejido. Esta diversificación de respuestas

es, a su vez, el reflejo de la variedad de procesos metabólicos alterados por el estrés hipersalino con los que puede

estar relacionada la actividad respiratoria, como la actividad de enzimas implicadas en procesos catabólicos, las

alteraciones en la concentración de sustratos respiratorios, reducciones de las tasas de fijación de carbono y sín-

tesis de azúcares, y activación de procesos con altos requerimientos energéticos (p.e. producción y/o transporte

de osmolitos) (Kramer y Boyer 1995; Atkin y Macherel 2009). Aunque los resultados obtenidos aquí no permiten

discernir qué alteraciones metabólicas están implicadas en los cambios respiratorios observados en plantas de P.

oceanica y C. nodosa en respuesta a incrementos de salinidad, sí que sería posible especular sobre la posible na-

turaleza de las mismas. Así, por ejemplo, el hecho de que las plantas que desarrollaron mayores respuestas osmo-

rreguladoras fueron las que exhibieron mayores incrementos en las tasas respiratorias, apoya la existencia de una

estrecha relación entre ambos procesos en plantas sometidas a este tipo de estrés (Munns 2002; Kahn y Durako

2006). Por otro lado, y como se observó en plantas C. nodosa expuestas a estrés hipersalino, la reducción de las

tasas respiratorias podría considerarse una respuesta compensatoria a la disminución de la actividad fotosintéti-

ca, y el subsiguiente desequilibrio del balance de carbono (capítulo 3). En experimentos más recientes aplicando

exposiciones más prolongadas a niveles similares de estrés hipersalino (3 meses; Marín-Guirao et al. enviado), la

severa reducción de las tasas fotosintéticas y concentraciones de carbohidratos de P. oceanica, sugiere que la re-

ducción de la respiración puede ser también consecuencia de limitación de disponibilidad de sustrato respiratorio.

Los posibles mecanismos por los que el estrés hipersalino puede afectar a la actividad fotosintética bruta de las

angiospermas marinas estudiadas son: a) modificando la actividad enzimática relacionada con la rutas metabólicas

implicadas en la fijación y asimilación fotosintética del carbono y, b) alterando la estructura y función del aparato

fotosintético en las membranas tilacoidales. Respecto al primer tipo de mecanismo, la evidencia directa o indirecta

disponible en éste y otros trabajos (p.e. Beer et al. 1980a) es nula o muy escasa, y se relaciona con el efecto tóxico

de la acumulación de iones en las células o la pérdida de la homeostasis iónica. Respecto al segundo nivel, se han

documentado en angiospermas marinas diversos efectos del estrés hipersalino relacionados con alteraciones de la

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composición pigmentaria, cambios conformacionales de la ultraestructura de los cloroplastos y tilacoides, así como

desacoplamientos del flujo electrónico entre fotosistemas y complejos antena (Ralph 1998, 1999; Kamermans et al.

1999; Kahn y Durako 2006; Koch et al. 2007b). Aunque en esta tesis se han obtenido evidencias de alteraciones en la

composición pigmentaria de P. oceanica y C. nodosa inducidas por el estrés hipersalino (tratadas específicamente en

el siguiente apartado), desde el punto de vista fotoquímico no parecen observarse alteraciones del funcionamiento

del aparato fotosintético de estas especies, tal y como se deduce de los valores poco o nada alterados de la eficiencia

fotoquímica máxima del PSII (Fv/Fm). Sin embargo, reducciones significativas de Fv/Fm han sido documentadas en

algunas especies de angiospermas marinas sometidas a estrés hipersalino (Ralph 1998; Khan y Durako 2006; Koch

et al. 2007a,b; Pagés et al. 2010), aunque a niveles de estrés mucho más extremos que los empleados en los expe-

rimentos de esta tesis. Por tanto, y al menos bajo las condiciones experimentales empleadas, la inhibición de la tasa

fotosintética no parece explicarse por alteraciones en la funcionalidad del aparato fotosintético a nivel de PSII.

En base a la presumible correspondencia documentada entre los descriptores obtenidos a partir de medidas de

fluorescencia, y las tasas fotosintéticas basadas en métodos de medida de la evolución de oxígeno en angiospermas

marinas (Beer et al. 1998; Silva y Santos 2004; Ralph y Gademann 2005), resulta notablemente llamativo el hecho de

que en P. oceanica y C. nodosa se hayan observado valores inalterados Fv/Fm, bajo las mismas condiciones de estrés

hipersalino que han provocado reducciones significativas de la actividad fotosintética en ambas especies. En tal situa-

ción, la disponibilidad de aceptores terminales de electrones (CO2 y NADP+) se reduce, lo que puede resultar en una

acumulación en exceso de energía de excitación en los fotosistemas, y su desviación a la producción de especies reac-

tivas del oxígeno (Reactive Oxigen Species o ROS). La actividad y acumulación de ROS, puede dar lugar a la acumu-

lación de fotodaño en los fotosistemas y daño oxidativo en las membranas fotosintéticas (Niyogi 2000), con la consi-

guiente disminución de Fv/Fm. Así pues, una posible explicación a los valores casi inalterados de Fv/Fm obsevados en

P. oceanica y C. nodosa bajo condiciones de estrés, podría ser la inducción de rutas metabólicas que funcionaran como

sumideros alternativos de electrones (e.g. flujos cíclicos de electrones en PSI y PSII, fotorrespiración, ciclo de Me-

hler), mecanismos antioxidantes y procesos de apagamiento no fotoquímico (non-photochemical quenching, NPQ).

Se ha sugerido que estos mecanismos son activados o intensificados en plantas terrestres bajo similares condiciones

de estrés (Demmig-Adams y Adams 1996; Niyogi 2000; Morales et al. 2006). En la presente tesis no se aportan evi-

dencias directas de la existencia de estos mecanismos bioquímicos, pero sí se dispone de cierta información adicional,

obtenida durante la realización del experimento correspondiente al capítulo 5, mediante el empleo de un fluorómero

Imaging-PAM; parte de estos datos todavía inéditos se exponen en la Fig. 2, y muestran un claro incremento de la di-

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sipación térmica (NPQ) en hojas de plantas de P. oceanica procedentes de praderas someras y profundas expuestas a

condiciones hipersalinas (43 ups) (Alfonso et al. 2010). También en estudios más recientes, se ha comprobado un in-

cremento de la actividad de ciertas enzimas antioxidantes en P. oceanica (p.e. catalasa) y la ausencia de daño oxidativo

(i.e peroxidación lipídica de membranas) bajo estas mismas condiciones experimentales (Marín-Guirao et al. 2011a).

Estos resultados (y otros no presentados aquí; Marín-Guirao et al. en revisión), sugieren que el empleo de des-

criptores obtenidos mediante técnicas de fluorescencia como indicadores o proxies de las tasas fotosintéticas de

angiospermas marinas, puede llevar a conclusiones erróneas en situaciones de estrés hipersalino (y probablemente

de estrés en general). El estrés hipersalino puede inducir rutas metabólicas que actúan de sumideros alternativos de

electrones (mecanismos “aliviadero” o safety valves, sensu Niyogi 2000) que permiten mantener valores normales

(o incluso superiores) de tasas de transporte electrónico (ETR) o de máxima eficiencia cuántica (Fv/Fm), aunque las

tasas fotosintéticas (en términos de evolución de oxígeno) se encuentren inhibidas (e.g. Morales et al. 2006). Esta

situación impide o distorsiona la relación lineal entre medidas de fluorescencia y de evolución de oxígeno descrita en

angiospermas marinas (Beer et al. 1998; Silva y Santos 2004; Ralph y Gademann 2005) y por tanto, ambas técnicas

deben ser aplicadas de forma complementaria cuando se estudian situaciones en las que las plantas estén sometidas

a algún tipo de estrés ambiental. No obstante, se requieren estudios específicos más detallados que corroboren la

operación de dichos mecanismos en las especies estudiadas, sus relaciones entre los diferentes niveles del proceso fo-

tosintético y con las características del estrés, aspectos por otro lado, muy poco conocidos en angiospermas marinas.

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Figura 2. Resultados de apagamiento no fotoquímico obtenidos mediante medidas de fluorescencia de Chla con Imaging-PAM correspondientes al Cap. 5. Arriba: imágenes de apagamiento no fotoquímico en hojas enteras (desde la base hasta el ápice) de P. oceanica correspon-dientes a irradiancias de 20 y 185 μmoles de fotones m-2 s-1 y obtenidas a partir de curvas rápidas de luz. Abajo: variación de la Media ± Error Estándar de apagamiento no fotoquímico obtenido a partir de curvas rápidas de luz y a lo largo de hojas (base, zona intermedia y ápice) de P. oceanica. CD y CS = tratamientos control de plantas de poblaciones profundas y someras respectivamente; 43D y 43S = tratamien-tos hipersalinos de plantas de poblaciones profundas y someras respectivamente.

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4. PIGMENTOS Y PROPIEDADES ÓPTICAS FOLIARES

En angiospermas marinas se han documentado reducciones de las concentraciones de pigmentos fotosintéticos,

aunque inducidas por condiciones hipersalinas más severas que las empleadas en los experimentos de esta tesis (Mc-

Millan y Moseley 1967; Ralph 1998).

De forma general, las modificaciones en el contenido de clorofilas y sus ratios molares (Chlb:a), están asociadas

a respuestas relacionadas con cambios en la eficiencia de las hojas para capturar y procesar luz. Estos ajustes parecen

ir dirigidos a modificar la densidad y concentración de centros de reacción y el tamaño de antena de los mismos

(absorption cross section; Kirk 1994; Falkowsky y Raven 2007). La plasticidad de la morfología foliar y sus efectos en

el grado de empaquetamiento de pigmentos, indicados por los descriptores SLA (Specific Leaf Area) y LMA (Leaf

Mass per Area), desempeñan también un papel crucial en el contexto de respuestas foto-aclimatativas (Olesen et al.

2002; Enríquez 2005; Cayabyab y Enríquez 2007), aunque su implicación frente a condiciones de estrés no ha sido

estudiada con anterioridad en angiospermas marinas.

En los experimentos realizados en esta Tesis, P. oceanica y C. nodosa exhibieron diferentes tipos de al-

teraciones pigmentarias en respuesta a las mismas condiciones de estrés hipersalino, aunque dichos efec-

tos no parecieron indicativos, en ningún caso, de un deterioro del aparato fotosintético causado por la sali-

nidad. Más concretamente, el estrés hipersalino causó, en ciertas ocasiones, incrementos en el contenido de

pigmentos en hojas de P. oceanica y C. nodosa, probablemente responsables de un aumento de la eficiencia fo-

tosintética (α), y de la absorbancia (D) y absortancia foliares (A) (capítulos 2, 3 y 5). Puesto que en estas mis-

mas circunstancias las tasas fotosintéticas se encontraron reducidas y/o la respiración intensificada, estos

cambios del aparato fotosintético podrían interpretarse como mecanismos compensatorios del importante des-

equilibrio del balance de carbono. En otras ocasiones no se observaron ni cambios en la composición pigmentaria

ni en la capacidad de las hojas de absorber luz en condiciones de estrés hipersalino (p.e. P. oceanica, capítulo 2).

Con independencia de los patrones de respuesta pigmentaria, los procesos de expansión y empaquetamiento

de la biomasa foliar (i.e. menores y mayores valores de LMA, respectivamente) pueden resultar en modificaciones

del área foliar fotosintéticamente activa y en el grado de empaquetamiento de pigmentos (Enríquez 2005), que a su

vez, pueden derivar en cambios en la eficiencia de absorción y procesamiento de luz. Este tipo de respuestas fueron

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observadas de forma distinta entre P. oceanica y C. nodosa en relación al estrés hipersalino. Plantas de C. nodosa pro-

cedentes de las praderas más someras, experimentaron reducciones en LMA que resultaron en mayores eficiencias

fotosintéticas, absorbancias, absortancias y coeficientes específicos de absorción, entendidas éstas como posibles

respuestas compensatorias ante condiciones de capacidades fotosintéticas reducidas. De forma contraria, las hojas

de P. oceanica mostraron un incremento de LMA en respuesta al incremento de salinidad, y sólo en las poblaciones

experimentales procedentes de las praderas someras (capítulo 5). Dichos incrementos de LMA, dieron lugar a dismi-

nuciones de los coeficientes específicos de absorbancia (a*, aw*) y eficiencias fotosintéticas (incluso si el contenido de

pigmentos aumentaba). Teniendo en cuenta que en estas condiciones P. oceanica mostró su capacidad fotosintética

reducida, esta respuesta podría desempeñar un papel importante como mecanismo fotoprotector; al disminuir la

incidencia de energía lumínica en exceso, se reduce la probabilidad de fotodaño en los fotosistemas (Vogelmann

1993; Enríquez y Sand-Jensen 2003), lo que permitiría el mantenimiento del Fv/Fm. Esta hipótesis estaría también de

acuerdo con el incremento de las concentraciones de carotenoides (contenido total y ratio carotenoides:Chla) en las

hojas de P. oceanica y C. nodosa expuestas a condiciones de hipersalinidad. Los pigmentos de la familia de los caro-

tenoides (i.e. xantofilas) juegan un importante papel en la fotoprotección del aparato fotosintético a través de meca-

nismos de disipación térmica (i.e. ciclo de las xantofilas, Demmig-Adams y Adams 1996), cuya existencia se ha puesto

en evidencia en angiospermas marinas (Ralph et al. 2002; Trevathan et al. 2012), y en las especies mediterráneas P.

oceanica y C. nodosa (Marín-Guirao et al. 2011a; García-Sánchez et al. 2012; Marín-Guirao et al. datos no publicados)

En resumen, bajo condiciones de capacidades fotosintéticas reducidas por el estrés hipersalino, ambas es-

pecies mostraron diferentes respuestas basadas en cambios en la concentración de pigmentos y de las pro-

piedades ópticas de sus tejidos foliares, resultantes en tres posibles estrategias y consecuencias funcionales:

i) estrategias compensatorias del balance de carbono, a través del aumento del contenido en pigmentos en P. oceani-

ca y C. nodosa, ii) estrategias de optimización de recursos, a través la expansión de la biomasa fotosintética y aumento

de eficiencia fotosintética (sólo en C. nodosa) y, iii) estrategias de protección, a través de la reducción de la capta-

ción de luz mediada por un incremento en el empaquetamiento de la biomasa fotosintética (sólo en P. oceanica).

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Figura 3. Modelo conceptual de las principales alteraciones observadas a nivel fisiológico en respuesta al incremento de la salinidad y las relaciones entre ellas. Las flechas sólidas indican relaciones con consecuencias maladaptativas (ver punto 6 de esta Discusión General) y las flechas punteadas indican relaciones que pueden resultar en respuestas de tipo compensatorio. Bajo condiciones de incremento de salinidad, el potencial hídrico del medio (Ψext) se reduce y se activan los procesos de aclimatación de las relaciones hídricas (principalmente osmorregulatorios) para reducir el potencial hídrico foliar (Ψw). La posible toxicidad por acumulación de osmolitos (iones) y los altos requerimientos energéticos de los procesos potencialmente involucrados, pueden resultar en reducciones de la actividad fotosíntética e incrementos de la respiración. Situaciones de reducidas capacidades fotosintéticas pueden causar sobre-excitación del fotosistema II (i.e. PSII) y la aparición de especies reactivas del oxígeno (i.e. ROS) que prevengan la acumulación de fotodaño en el fotosistema (mantenimineto de Fv/Fm). Mecanismos alternativos de disipación térmica y sumideros alternativos de electrones, así como la activación del sistema antioxidante, pueden participar como procesos de mitigación y protección frente a dichas condiciones de elevada presión sobre el PSII. Asimismo, alteraciones a nivel de pigmentos fotosintéticos y propiedades ópticas del tejido foliar pueden jugar un papel fundamental como mecanismos compensatorios fotosintéticos y de fotoprotección, a consecuencia de su influencia en la capacidad de absorber y procesar luz (i.e. modificaciones en el empaquetamiento o LMA).

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5. CONSECUENCIAS A NIVEL VEGETATIVO

Los ajustes fisiológicos descritos para P. oceanica y C. nodosa en respuesta al estrés hipersalino pueden traducirse

en cambios a nivel del organismo entero (haces, pradera), ya que parte de los recursos de la planta para mantener su

biomasa y crecimiento es desviado para satisfacer el coste metabólico de dichos ajustes a la condición de estrés. Al-

gunos de estos cambios habitualmente documentados en angiospermas marinas expuestas a estrés hipersalino, con-

sisten en incrementos de la proporción del tejido foliar necrosado, disminución del tamaño de los haces (superficie,

biomasa, número de hojas) y del crecimiento foliar, y aumento de la mortalidad de la población de haces (Walker y

McComb 1990: van Katwijk et al. 1999; Tomasko et al. 2000; Koch et al. 2007b; Fernández-Torquemada y Sánchez-

Lizaso 2011).

El aumento de la superficie de las marcas de necrosia en las hojas de P. oceanica, ha sido descrito inicialmente

como un síntoma característico de la influencia del estrés hipersalino (Gacia et al. 2007; Fernández-Torquemada

y Sánchez-Lizaso 2005), y fue observado también en praderas naturales expuestas a un incremento de la salinidad

simulado in situ (capítulo 1, Ruiz et al. 2009). Normalmente, esta respuesta se interpreta como la pérdida de super-

ficie fotosintéticamente activa por el efecto tóxico de las sales, pero en realidad se ha descrito también en praderas

sometidas a otros tipos de estrés antrópico (González-Correa et al. 2005; Boudouresque et al. 2009). De hecho, este

“envejecimiento” acelerado de los tejidos fotosintéticos es una de las respuestas más comunes descritas en plantas

bajo situaciones de estrés, y se relaciona con procesos de reabsorción o translocación de recursos internos , activados

con el fin de mantener la actividad de los meristemos de crecimiento, producir nuevas hojas aclimatadas a la condi-

ción de estrés o para el mantenimiento de los costes metabólicos de los ajustes aclimatativos. El hecho de que esta

respuesta no fuera observada en los experimentos de mesocosmos realizados en el marco de esta tesis no tiene una

explicación sencilla y aparente, ya que las condiciones experimentales de los diferentes estudios (los anteriores y los

aquí propuestos) son muy diferentes. Así, por ejemplo, experimentos de mesocosmos de corta duración (2 semanas;

Fernández-Torquemada y Sánchez-Lizaso 2005) mostraron un incremento de la superficie foliar necrosada de haces

de P. oceanica a partir de 42.5 ups, aunque en este caso otros factores bajo control experimental (p.e. irradiancia)

podrían haber sido limitantes y actuar sinérgicamente con el efecto del estrés hipersalino.

La reducción en el tamaño de haz es otro de los efectos documentados en praderas naturales de P. oceanica en

respuesta a exposiciones a medio y largo plazo a estrés hipersalino causado por vertidos de salmuera (capítulo 1;

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Gacia et al. 2007; Ruiz et al. 2009-capítulo 1). Esta reducción del haz puede producirse por reducción de las tasas de

crecimiento, aceleración de la caída de hojas y también a cambios en la morfología foliar, respuestas a su vez, descri-

tas en relación al efecto del incremento de la salinidad en las plantas de P. oceanica estudiadas aquí y en estudios pre-

vios (Gacia et al. 2007; Fernández-Torquemada y Sánchez-Lizaso 2005; Fernández-Torquemada y Sánchez-Lizaso

2011). La reducción del tamaño de las plantas o “enanismo” es una respuesta habitual descrita en plantas terrestres

sometidas a situaciones de estrés crónico (Lichtenthaler 1996; Valladares y Niinemets 2007); algunas plantas some-

tidas a un estrés prolongado pueden sobrevivir a costa de presentar tasas de crecimiento y tamaño más reducidos,

con requerimientos metabólicos mucho menores. Esta estrategia conservativa explicaría la supervivencia de angios-

permas marinas sometidas a los efectos de condiciones hipersalinas a largo plazo, como en el caso de las praderas de

P. oceanica sometidas a la influencia prolongada de vertidos de plantas desalinizadoras (Gacia et al. 2007; y capítulo

1), o como las praderas de esta especie (genotípicamante adaptadas) que se desarrollan en ambientes hipersalinos

naturales extremos (48 ups en Stagnone di Marsala, Sicilia, Tomasello et al. 2009).

El incremento de la mortalidad de haces supone otro tipo de respuesta que, a este nivel, puede ser interpre-

tada en el contexto de estrategias conservativas. La pérdida de haces es considerada una de las principales res-

puestas plásticas desarrolladas por P. oceanica (Olesen et al. 2002; Ruiz y Romero 2001) y otras especies del

mismo porte (Collier et al. 2008) frente a la variación en la disponibilidad de luz. En una situación de estrés, y

de acuerdo con la naturaleza clonal de estos organismos, la disminución del número de haces permitiría el re-

parto de recursos internos entre la población de haces supervivientes, contribuyendo a ajustar el balance de

carbono a nivel del organismo entero, clon o genet. En los experimentos presentados en esta tesis, el estrés hi-

persalino provocó, consistentemente, incrementos suaves o moderados de la mortalidad de haces de P. oceani-

ca; así pues, esta respuesta, más que un efecto letal de la salinidad, bien podría ser considerada como uno de los

principales mecanismos empleados por esta especie para ajustar las importantes alteraciones del balance de

carbono, causadas por los efectos de dicho estrés en el metabolismo fotosintético y las demandas respiratorias.

En el caso de C. nodosa los efectos del estrés hipersalino a nivel vegetativo no fueron tan aparentes como

en P. oceanica. Bajo las condiciones experimentales empleadas, no se detectaron efectos de la hipersalini-

dad en la proporción de tejidos necrosados, en el tamaño y crecimiento de los haces ni en su superviven-

cia. Contrariamente, efectos a este nivel sí han sido detectados en esta especie en estudios anteriores (Pa-

gés et al. 2010; Fernández-Torquemada y Sánchez-Lizaso 2011), aunque bajo condiciones salinas algo más

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severas, claramente superiores a los niveles de salinidad causantes de dichos efectos en P. oceanica. Esto sugiere

que los ajustes a nivel fisiológico desarrollados por C. nodosa en respuesta al estrés hipersalino son suficientes para

compensar las demandas metabólicas impuestas por dicho estrés, y apoya la hipótesis de que es una especie fisio-

lógicamente mejor adaptada que P. oceanica para crecer y sobrevivir sobre rangos más amplios de hipersalinidad.

6. PLASTICIDAD FISIOLÓGICA DE P. OCEANICA Y C. NODOSA EN RESPUESTA AL ESTRÉS HIPERSALINO

De forma general, las diferencias inter-específicas en el grado de plasticidad fisiológica que las plantas pueden

desarrollar frente a los cambios de las condiciones del medio, están de acuerdo con la distribución y requerimientos

ecológicos específicos (deWitt y Scheiner 2004). Se considera que los atributos y plasticidad de especies llamadas

generalistas, les confieren una posición ventajosa ante los cambios del medio respecto a especies especialistas, de re-

querimientos ecológicos más estrictos (van Tyenderen 1991; Dudley 2004). En este contexto, las diferentes respues-

tas fisiológicas mostradas por P. oceanica y C. nodosa al incremento de la salinidad en los experimentos de esta tesis y

otros trabajos, parecen estar de acuerdo con las características biológicas inherentes de ambas especies y su ecología

en los hábitats infralitorales mediterráneos (Drew 1978b; Terrados y Ros 1991; Pérez y Romero 1994; Cancemi et al.

2002; Boudouresque et al. 2009).

Las evidencias experimentales obtenidas en esta tesis demuestran que C. nodosa, especie de estrategia tipo ge-

neralista, posee ciertas propiedades fisiológicas inherentes que, como se ha discutido en capítulos anteriores, parecen

proporcionarle ciertas ventajas adaptativas bajo condiciones de estrés hipersalino respecto a P. oceanica, de estrate-

gia ecológica más especialista. En este sentido, y a modo de ejemplo, cabe destacar las características particulares de

la capacidad osmótica y metabolismo fotosintético de C. nodosa descritas en los capítulos 3, 4 y 5, y en qué medida

podrían explicar la mayor tolerancia fisiológica de esta especie al incremento de la salinidad. Sin embargo, y como

se ha visto en el capítulo 5, estas diferencias inter-específicas no se reflejaron de forma cuantitativa en el grado de

plasticidad fisiológica determinado para ambas especies por el índice RDPI (relative distance plasticity index). Por

tanto, la tolerancia diferencial al estrés hipersalino no viene determinada por la cantidad de variación plástica que cada

especie es capaz de desarrollar en términos globales, sino más bien por el mayor o menor potencial aclimatativo y/o

adaptativo de la misma (deWitt y Scheiner 2004; Dudley 2004; Ghalambor et al. 2007). Generalmente, se considera

que una respuesta fisiológica plástica es adaptativa, cuando directa o indirectamente, es capaz de mantener o imple-

mentar la adecuación biológica del individuo a nivel vegetativo (es decir su abundancia, crecimiento y supervivencia)

bajo condiciones de estrés; por el contrario, las respuestas de índole mal-adaptativo (o no-adaptativas), son aquellas

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que pueden surgir como consecuencias de la alteración metabólica causada por el propio factor estresante, o alter-

nativamente, como respuestas relacionadas con costes metabólicos ligados a la activación de las propias adaptativas

(Ghalambor et al. 2007). De este modo, este tipo de respuestas no-adaptativas, pueden actuar en detrimento de la

adecuación biológica de los individuos, y por tanto, reducir sus posibilidades de aclimatación, adaptación y en defi-

nitiva, de su éxito frente a las condiciones estresantes (Pigliucci 2001; deWitt y Scheiner 2004). Ante una situación

de estrés coexisten ambos tipos de respuestas de forma más o menos integrada y, aunque su identificación es com-

pleja, el desequilibrio metabólico resultante es lo que determinará la sensibilidad específica frente al estrés (Fig. 4).

Como se ha discutido en los diferentes capítulos de esta tesis, P. oceanica y C. nodosa parecen desarrollar ambos

tipos de respuestas fisiológicas (adaptativa y no adaptativa) frente condiciones de estrés hipersalino, condicionadas

por supuesto, a múltiples factores de variación intra- e inter-específicos (Fig. 4). En este sentido, la capacidad de

osmorregulación, y la consecuente reducción del potencial hídrico foliar (Ψw) mostrada por ambas especies frente

a incrementos de salinidad, puede considerarse como respuesta de plasticidad adaptativa a nivel fisiológico. Sin em-

bargo, el mantenimiento de estos mismos procesos, puede tener consecuencias no tan ventajosas en términos estric-

tamente adaptativos o aclimatativos a otros niveles y escalas temporales. Así, por ejemplo, efectos directos como la

posible toxicidad causada por la acumulación citoplasmática de ciertos solutos (principalmente iones) implicados en

la osmorregulación, o indirectos, como los asociados al aumento de la actividad respiratoria, podrían resultar, a medio

y largo plazo, en estados fisiológicos desfavorables o maladaptativos frente a condiciones de incremento de salinidad.

De acuerdo con el modelo conceptual de la Fig. 4, la forma en que ambos tipos de respuestas (adaptativas y

no adaptativas) se relacionan entre sí y se integran metabólicamente es fundamental en la determinación de la to-

lerancia de cada especie, población, ecotipo o genotipo, al estrés. Pero además, el coste metabólico resultante de

dicha integración (o coste derivado de la plasticidad fenotípica), resulta clave para entender las consecuencias de

las respuestas adaptativas o aclimatativas a nivel de la adecuación biológica o fitness a nivel de individuo o pobla-

ción (deWitt and Scheiner 2004; Bradshaw-Sultan effect). Estos costes asociados a plasticidad permitirían explicar

la situación descrita por los resultados del experimento del capítulo 5: en este experimento se documentaron des-

equilibrios en las relaciones hídricas o en el balance de carbono comparables entre P. oceanica y C. nodosa, aunque

variables como el crecimiento, tamaño y supervivencia de la población sólo se mostraron afectados para el caso de

P. oceanica. Por tanto, ante capacidades similares de plasticidad fisiológica, la mayor tolerancia de C. nodosa al in-

cremento de la salinidad, puesta en evidencia en éste y otros trabajos, sugieren que esta especie es capaz de asumir

con mayor eficacia, el coste metabólico de las respuestas aclimatativas desarrolladas para resistir el estrés hipersalino.

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Figura 4. Representación esquemática del complejo proceso global de plasticidad fisiológica que P. oceanica y C. nodosa podrían desarrollar en respuestas a condi-ciones de estrés por incremento de salinidad del medio. El balance global de los procesos activados mediante integra-ción metabólica, a su vez dependientes de fuentes variación inter e intra-específicas, puede desempeñar un papel fundamen-tal en la tolerancia específica frente a incrementos de salinidad, y por tanto, en la adecuación biológica del organismo al estrés.

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7. PERSPECTIVAS DE INVESTIGACIÓN

Los resultados obtenidos en la presente tesis doctoral, suponen un avance significativo en el conocimiento

básico de las respuestas aclimatativas de las angiospermas marinas mediterráneas, P. oceanica y C. nodosa, al es-

trés causado por el incremento de la salinidad. Pero sobre todo, ponen en evidencia la enorme cantidad de va-

riables y aspectos que influyen en las propiedades aclimatativas de ambas especies y en la determinación de sus

límites de tolerancia. A continuación se relacionan y comentan brevemente algunas de las posibles líneas de in-

vestigación que, en el futuro, deberían desarrollarse para seguir avanzando en este campo de conocimiento.

1. Esta tesis se ha centrado, principalmente, en analizar experimentalmente las respuestas ecofisiológi-

cas de las especies de angiospermas marinas mediterráneas bajo condiciones crónicas de estrés hipersalino (ex-

cepto en el capítulo 1). Aunque en estos experimentos se ha considerado el efecto de intensidades crecientes de

estrés, aún se desconoce cómo pueden variar las capacidades aclimatativas y tolerancias de ambas especies en

función de otros parámetros del régimen salino, como la duración y frecuencia de los aumentos de salinidad, y

combinaciones de los mismos. Esto es un aspecto importante, ya que en praderas naturales las alteraciones del

régimen salino causadas por la actividad antrópica (vertidos desaladoras) son de naturaleza más fluctuante.

2. La respuesta al estrés hipersalino (y a cualquier otro tipo de estrés) no se puede evaluar sólo en base a los efec-

tos observados durante la fase de exposición, tal y como se ha contemplado en los experimentos de esta tesis, sino

que es fundamental conocer la evolución y capacidad de resilencia de tales efectos tras la eliminación del factor estre-

sante (fase de recuperación; Fig. 1 en Introducción General) (p.e. González-Correa et al. 2005, 2008). Precisamente

en esta línea, el equipo de investigación en el que se ha desarrollado esta tesis, ha emprendido unos primeros trabajos

en el marco del proyecto OSMOGRASS II ref. CTM2009-08413MAR del Plan Nacional I+D+i; en dichos trabajos se

ha podido comprobar que muchas de las respuestas aclimatativas observadas a nivel fisiológico, se muestran reversi-

bles o no, dependiendo del período de exposición al estrés; de este modo, prolongados periodos de exposición (p.e.

3 meses) llevan a las plantas a una fase de agotamiento (Fig. 1 en Introducción General), a partir de la cual la recupe-

ración y supervivencia de plantas de P. oceanica, son mucho más difíciles (Marín-Guiaro et al. en revisión). Sería inte-

resante conocer así, la reversibilidad de las respuestas aclimatativas bajo diferentes combinaciones de los parámetros

que definen el régimen de salinidad, en condiciones naturales y alteradas, y en el sentido comentado en el punto 1.

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3. Otra línea interesante en la que profundizar, podría estar enfocada en la determinación de la importancia del

genotipo en las respuestas aclimatativas de ambas especies al estrés hipersalino. Como se ha indicado en el capítulo 5,

la existencia de variaciones genotípicas entre poblaciones de P. oceanica y C. nodosa separadas por algún tipo de gra-

diente ambiental (p.e. profundidad o régimen salino; Procaccini y Mazzella 1996; Migliaccio et al. 2005; Procaccini et al.

2003; Tomasello et al. 2009), podría influir en la eficacia y el tipo de respuestas aclimatativas al estrés hipersalino. Esta

hipótesis ha sido sugerida también por otros autores para explicar la presencia de praderas permanentes de P. oceanica

en ambientes hipersalinos extremos (Tomasello et al. 2009), o la tolerancia diferencial al incremento de salinidad en-

tre praderas de C. nodosa presentes en aguas costeras abiertas y las desarrolladas en lagunas hipersalinas (Fernández-

Torquemada y Sánchez-Lizaso 2011). De este modo, se requeriría analizar, por un lado, i) las características ecofisioló-

gicas y genotípicas de praderas naturales de ambas especies desarrolladas en diferentes tipos de régimen salino, y por

otro lado, ii) la influencia del genotipo sobre la variación de las respuestas aclimatativas al estrés hipersalino (incluyendo

su recuperación), en la misma línea desarrollada para otras especies de angiospermas marinas expuestas a otros tipos

de estrés, relacionados con el nivel de nutrientes (Tomas et al. 2011) o el calentamiento del agua (Reusch et al. 2005).

4. Los resultados obtenidos en esta tesis, y en el resto de estudios realizados en este campo, ponen en evidencia

que las capacidades de adaptación y aclimatación de angiospermas marinas ante condiciones de estrés salino, parecen

condicionadas por multitud de respuestas metabólicamente integradas a distintos niveles fisiológicos. En compara-

ción con el conocimiento existente en este campo con plantas terrestres, el correspondiente a angiospermas marinas

es más que insuficiente para comprender la naturaleza de dichas respuestas, los mecanismos que las controlan, la for-

ma en que se integran y se relacionan, etc. Este conocimiento básico es fundamental para una mayor comprensión de

la tolerancia de las diferentes especies y los factores que la determinan. Sólo mediante aproximaciones ecofisiológicas

no es posible alcanzar dicho nivel de conocimiento, y son necesarias aproximaciones interdisciplinares que permitan la

aplicación de técnicas moleculares (transcriptómica, proteómica y metabolómica), aplicadas habitualmente al estudio

de los mecanismos de respuesta de las plantas a estrés ambiental (Fiehn 2002; Viant 2007; Shulaev et al. 2008). Al-

gunas publicaciones recientes muestran ya algunos primeros resultados prometedores, relacionados con la aplicación

de este tipo de aproximaciones novedosas en angiospermas marinas (Zostera marina y P. oceanica) sometidas a estrés

por temperatura, limitación de luz e hipersalinidad (Mazzuca et al. 2009; Serra y Mazzuca 2011; Serra et al. 2011, 2012).

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5. Además de lo comentado en el punto anterior, es necesario seguir profundizando en el conocimiento a nivel

ecofisiológico de las respuestas al estrés hipersalino, que representan los mecanismos aclimatativos fundamentales de

las plantas frente a este tipo de estrés. En esta tesis, se muestra, por primera vez, evidencia empírica y experimental de

las diferentes estrategias osmoaclimatativas de P. oceanica y C. nodosa, aspecto que a pesar de ser fundamental para

entender las diferentes estrategias de tolerancia de las angiospermas marinas a los cambios de la salinidad, tan sólo ha-

bía sido estudiado en profundidad hace 25 años en algunas especies australianas (Tyerman 1989). Así, la necesidad de

avanzar en este campo resulta evidente, aplicando nuevas técnicas como las basadas en métodos electrofisiológicos,

que han permitido demostrar, por ejemplo, la existencia de procesos de regulación, transporte y compartimentalización

iónica en algunas especies de angiospermas marinas y P. oceanica (Pak et al. 1995; Fukuhara et al. 1996; Fernández et al.

1999; Muramatsu et al. 2002; Carpaneto et al. 2004; Rubio-Valverde 2004). En este sentido, también es necesario pro-

fundizar en los componentes principales que rigen los procesos de osmoaclimatación: 1) dispositivos de detección del

estrés (i.e. detector), 2) sistemas de activación de respuestas fisiológicas (i.e. efector) y 3) sistemas de comunicación o

señales de transducción entre ambos (Bisson y Kirst 1995). Para el caso de angiospermas marinas, sólo algunas respues-

tas fisiológicas inducidas por dichas condiciones de estrés han sido estudiadas, mientras que los sistemas de detección

(e.g. turgor sensing mechanism) y de transducción de señales (e.g. activación y/o inhibición de enzimas y ciertos genes

mediados por cofactores como Ca2+), de reconocida importancia en algas marinas, permanecen aún desconocidas.

6. La vertiente aplicada del conocimiento básico obtenido en esta tesis, es el desarrollo y aplicación de bioindi-

cadores de estrés hipersalino, con el fin de evaluar el estado ecológico y funcional de praderas expuestas al impacto

antropogénico (vertidos de plantas desalinizadoras) o de praderas distribuidas a lo largo de gradientes naturales de

salinidad. Algunas de las múltiples respuestas fisiológicas descritas en esta tesis en respuesta al incremento de la sa-

linidad, se manifiestan de forma más o menos consistente en condiciones hipersalinas como síntomas sub-letales de

estrés, lo que les convierte en candidatos idóneos como posibles bioindicadores. Entre dichos síntomas destacan los

siguientes:

• reducción de tasas fotosintéticas brutas y netas (observado a partir de 39 ups en P. oceanica, y de 43 ups en C.

nodosa),

• incrementos en las tasas respiratorias,

• reducción del potencial hídrico foliar (Ψw) en ambas especies, y del potencial osmótico (Ψπ) en P. oceanica (a

partir de 39 ups),

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• acumulación de osmolitos orgánicos (principalmente prolina y azúcares solubles) e inorgánicos (principalmente

iones Na+ y Cl-) en los tejidos foliares de ambas especies, y relacionados con procesos de ajuste osmótico (i.e. re-

ducción de Ψπ),

• incrementos en el ratio carotenoides:Chla, como indicador de la activación de procesos relacionados con la protec-

ción de las membranas fotosintéticas, como la activación de procesos de disipación energética (NPQ).

CONC

LUSIO

NES

GENE

RALE

S

2

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CAPÍTULO 1. EVALUACIÓN IN SITU DE LOS EFECTOS DEL ESTRÉS HIPERSALINO EN POSIDONIA OCEANICA

1. P. oceanica mostró una elevada sensibilidad a la exposición prolongada (3 meses) in situ de incremen-

tos modestos (+1 ups) de la salinidad media ambiente, lo que supone un resultado consistente con estudios an-

teriores y complementarios, que también sitúan el umbral de tolerancia de esta especie ligeramente por enci-

ma del límite superior de su rango natural de salinidad (36-38 ups en la costa mediterránea del sureste español).

2. En respuesta al incremento de la salinidad, las plantas experimentaron diversos cambios significativos, tanto

a nivel vegetativo como fisiológico, cuya intensidad aumentó conforme lo hizo la severidad del estrés hipersalino.

Dichos cambios se manifestaron a diferentes niveles de organización de la pradera, entre los que se destacan las

reducciones del tamaño del haz y del crecimiento foliar, disminución en la concentraciones de carbohidratos no

estructurales de tejidos rizomáticos, reducción en la densidad de haces y aumento de la superficie foliar necrosada.

CAPÍTULOS 2 Y 3. EFECTOS DEL ESTRÉS HIPERSALINO SIMULADO EN MESOCOSMOS SOBRE LA FOTOSÍNTESIS DE POSIDONIA OCEANICA Y CYMODOCEA NODOSA

3. El incremento de salinidad causó la reducción las tasas fotosintéticas brutas de ambas especies. Este

efecto fue evidente en plantas de P. oceanica expuestas a cualquiera de los niveles hipersalinos conside-

rados (≥ 39 ups), mientras que en C. nodosa sólo fue detectado en el tratamiento más severo (43 ups).

4. Las medidas de fluorescencia de la clorofila a (eficiencia fotoquímica máxima, Fv/Fm) no evidenciaron al-

teraciones en la estructura y funcionamiento del aparato fotosintético a nivel de fotosistema II en ninguna de las

dos especies, lo que sugiere que la inhibición fotosintética pudo producirse a nivel del metabolismo del carbono.

5. Las tasas respiratorias de las hojas de P. oceanica mostraron una elevada sensibilidad al estrés, al incrementarse

de forma significativa en todos los tratamientos hipersalinos. En el caso de C. nodosa, las respuestas de la respiración

fueron más complejas, aumentando a niveles moderados de estrés hipersalino pero inhibiéndose a niveles más seve-

ros, lo que permitió compensar el efecto negativo de la reducción de la tasas fotosintéticas en el balance de carbono.

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6. Bajo condiciones de estrés hipersalino, el aparato fotosintético de P. oceanica no mostró cambios en su ca-

pacidad para capturar y procesar luz. En cambio, la hojas de C. nodosa experimentaron incrementos en la concen-

tración de clorofila b, en la relación molar Chlb:a, y una expansión de su biomasa foliar (disminución de LMA),

todas ellas respuestas que contribuyeron a mejorar la capacidad de absorber luz de los tejidos fotosintéticos.

7. Ambas especies respondieron al estrés hipersalino con un incremento en la relación carotenoides:Chla,

comúnmente relacionado con la activación de mecanismos de fotoprotección ante situaciones de fo-

tosíntesis disminuida. Esta respuesta ha sido detectada en todos los experimentos descritos en esta te-

sis (capítulos 2, 3 y 5), y se propone como un síntoma general de estrés hipersalino en ambas especies.

8. El incremento de la salinidad causó reducciones significativas del balance de carbono de ambas es-

pecies, siendo más evidentes para el caso de P. oceanica. Estos fuertes desequilibrios pudieron ser la cau-

sa de la disminución de los carbohidratos de reserva detectada en plantas de P. oceanica en los capítulos 1 y 5,

aunque la implicación de otros procesos como la translocación de recursos internos debería ser considerada.

9. P. oceanica mostró reducciones significativas del crecimiento y supervivencia de haces en res-

puesta al estrés hipersalino. Tales efectos adversos no fueron observados para el caso de C. nodo-

sa bajo las mismas condiciones experimentales, lo que indica su mayor capacidad de tolerar dicho estrés.

CAPÍTULO 4. EFECTOS DEL ESTRÉS HIPERSALINO EN LAS RELACIONES HÍDRICAS DE POSIDONIA OCEANICA Y CYMODOCEA NODOSA

10. En respuesta al incremento de la salinidad externa, ambas especies se mostraron capaces de re-

ducir sus potenciales hídricos foliares (Ψw), mediante la activación de mecanismos relacionados con es-

trategias de «evitación de deshidratación». Esta respuesta ha sido observada de forma consisten-

te en plantas de ambas especies, expuestas a cualquier condición experimental y tratamiento hipersalino.

11. Sin embargo, ambas especies mostraron distintos mecanismos de reducción del Ψw foliar: mien-

tras que en P. oceanica se obtuvieron evidencias de la activación de procesos de osmorregulación, C. nodo-

sa mostró la capacidad de desarrollar procesos relacionados con el endurecimiento de la pared celular.

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12. De acuerdo con la respuesta de osmorregulación observada en P. oceanica, se detectaron au-

mentos significativos en la concentración de solutos orgánicos osmóticamente activos en los tejidos fo-

liares de esta especie (principalmente azúcares solubles y algunos aminoácidos como prolina). Jun-

to con la alteración del metabolismo fotosintético, el coste metabólico asociado a la activación de estos

mecanismos osmorreguladores, explicaría la particular sensibilidad de P. oceanica al incremento de la salinidad.

CAPÍTULO 5. FACTORES QUE DETERMINAN LA PLASTICIDAD FISIOLÓGICA Y LA TOLERANCIA DE LAS ANGIOSPERMAS MARINAS MEDITERRÁNEAS AL ESTRÉS HIPERSALINO

13. En base a los experimentos realizados en éste y anteriores capítulos, P. oceanica se comporta como

una especie menos tolerante al estrés hipersalino que C. nodosa. Esta tolerancia diferencial concuerda con

los atributos fisiológicos y vegetativos inherentes a cada especie, y sus respectivas estrategias ecológicas.

14. De los atributos fisiológicos que confieren a C. nodosa cierta ventaja adaptativa para tolerar el incremento

de la salinidad, destacan los siguientes:

a) valores más negativos de potenciales hídrico y osmótico (Ψw y Ψπ),

b) capacidad de desarrollo de mecanismos de endurecimiento de pared celular,

c) mayores concentraciones de ciertos solutos orgánicos (p.e. carbohidratos no estructurales solubles o prolina)

con reconocidas funciones osmóticas y osmoprotectoras,

d) elevadas tasas fotosintéticas y señal isotópica del carbono (δ13C), que sugieren mecanismos más eficientes

de asimilación de carbono inorgánico (más próximo al metabolismo C4), y menos sensibles al estrés osmótico e

iónico, y

e) capacidad de regulación (inhibición) de las tasas respiratorias, y su posible efecto compensatorio sobre las

alteraciones fotosintéticas inducidas bajo estrés hipersalino severo.

15. A pesar de las claras diferencias a nivel ecológico, vegetativo y fisiológico, ambas especies mostraron un

grado similar de plasticidad global en sus respuestas al estrés hipersalino. Dicho resultado se atribuye a la ele-

vada variabilidad intra-específica de dicha plasticidad, observada tanto entre plantas de ecotipos y ambien-

tes marcadamente distintos (praderas someras vs. profundas), como entre tipos de variables de respuesta.

206

16. P. oceanica mostró una tolerancia diferencial al estrés hipersalino dependiendo del ecotipo y ambiente, mos-

trando una mayor sensibilidad las poblaciones experimentales procedentes de praderas someras respecto a las de

praderas profundas. Por el contrario, la supervivencia de plantas de C. nodosa no se vio afectada por el estrés hi-

persalino en ninguna de las poblaciones experimentales, a pesar de sus diferencias en el grado de plasticidad global.

17. Por tanto, el desarrollo y la supervivencia (adecuación biológica o fitness) mostrado por ambas especies

y poblaciones experimentales bajo condiciones de estrés hipersalino, no estuvieron determinados por el gra-

do de plasticidad fisiológica de las respuestas desarrolladas, sino más bien por el tipo de mecanismos específi-

cos que protagonizaron dichas respuestas, su integración metabólica, sus consecuencias funcionales y sus costes.

SISTEMA EXPERIMENTAL DE MESOCOSMOS DE LABORATORIO

18. El sistema de mesocosmos experimental fue capaz de simular de forma eficiente las condiciones naturales reales,

manteniendo las plantas de P. oceanica y C. nodosa en estados fisiológicos y vegetativos óptimos durante escalas tem-

porales lo suficientemente amplias (1-2 meses) como para permitir la expresión y medición de las respuestas estudiadas.

Dichos estados resultaron comparables y similares a los presentados por plantas de ambas especies en sus respectivas

praderas naturales durante el desarrollo de los experimentos. Este hecho evidencia que el estrés adicional asociado al dis-

positivo y manipulación experimental, así como la existencia de posibles efectos de artefacto y/o confusión que pudieran

enmascarar las respuestas de las plantas debidas específicamente al incremento de la salinidad, fueron minimizados.

19. A pesar de las conclusiones anteriores, cualquier extrapolación o generalización de las conclusiones ob-

tenidas en el sistema experimental a praderas naturales y a escalas espacio-temporales más amplias, debe reali-

zarse con precaución, ya que los experimentos han sido realizados con plantas de praderas de localización y

épocas del año determinadas. Adicionalmente, otros factores ambientales podrían también influir sinérgica o an-

tagónicamente en la variación inter- e intra-específica de la tolerancia al estrés hipersalino, bien en condiciones

naturales o bajo la influencia de situaciones de estrés múltiple características de las perturbaciones antrópicas.

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230

ANEXO1

Con el fin de determinar la variación estacional na-

tural de importantes descriptores fisiológicos y pro-

piedades vegetativas de P. oceanica y C. nodosa,

se llevaron a cabo muestreos específicos en pra-

deras naturales de ambas especies. Estas praderas

de referencia coincidieron con las originarias de las

plantas que fueron empleadas en las diferentes ex-

periencias de mesocosmos planteadas en la presente

Tesis Doctoral. A lo largo de los diferentes capí-

tulos, estos datos han sido empleados como valores

de referencia, con los que comparar los obtenidos en

plantas control mantenidas bajo condiciones experi-

mentales de laboratorio. Dicha comparativa se realizó

con el fin de posibilitar la detección de potenciales

efectos estresantes causados por el propio dispo-

sitivo experimental, capaces de confundir o enmas-

carar las respuestas de las plantas específicamen-

te relacionadas con la manipulación experimental del

factor objeto de estudio (salinidad en este caso).

Las praderas muestreadas se sitúan en distintos

puntos de la costa mediterránea de la Región de

Murcia (ver localidades específicas en la Fig. 1), y

fueron seleccionadas de acuerdo con dos rangos ba-

timétricos: praderas someras (i.e. situadas a entre 5

y 7 metros de profundidad) para el caso de ambas

especies, y otra pradera profunda de P. oceanica (i.e.

entre 18-20 m). Se prestó especial atención a que

éstas correspondieran a praderas bien desarrolladas

y saludables, poco o nada alteradas por el impacto

antrópico. Del mismo modo, y en relación al capítulo

5, se realizaron muestreos similares de una población

mediterránea profunda de C. nodosa, aunque des-

afortunadamente, estos datos no han estado final-

mente disponibles para su presentación en esta tesis.

Además de los datos que se muestran para el caso de

poblaciones mediterráneas de P. oceanica y C. nodosa,

adicionalmente se presentan los obtenidos a partir

de plantas de C. nodosa originarias de una pradera

somera (i.e. entre 2-3 m de profundidad) ubicada en

la laguna costera hipersalina del Mar Menor (Fig. 1).

Aunque dicha información no ha sido objeto de estudio

a lo largo de los diferentes capítulos que conforman

esta Tesis, se muestra aquí como parte del trabajo

de campo realizado, ya que se considera ilustrativa

de la magnitud de la variación de plasticidad intra-

específica de esta especie en el área de estudio.

Los muestreos fueron realizados con una periodicidad

de 3 meses en cada una de las praderas de refe-

rencia seleccionadas, con el fin de que los valores

obtenidos fueran representativos de las cuatro es-

taciones del año: Octubre de 2008 (otoño), Enero de

2009 (invierno), Abril de 2009 (primavera) y Julio de

2009 (verano). Para la recolección de material vege-

tal empleado en las diferentes analíticas fisiológicas

y distintas medidas morfométricas, se seleccionaron

dos zonas en cada estación o pradera (A y B), y

dentro de ellas, 3 puntos distintos (i.e. A1, 2 y 3;

B1, 2 y 3) escogidos al azar, aunque adecuadamente

distanciados entre sí. Básicamente, los criterios y

protocolos empleados para la selección de las plan-

tas y material vegetal empleados para cada uno de

los descriptores, así como el número de medidas

realizadas para los mismos, fueron similares a los ya

apuntados en las distintas secciones de material y

métodos desarrolladas en los distintos capítulos.

VARIACIÓN ESTACIONAL DE LOS DESCRIPTORES FISIOLÓGICOS Y

VEGETATIVOS DE POSIDONIA OCEANICA Y CYMODOCEA NODOSA

MEDIDOS EN PRADERAS NATURALES (VALORES DE REFERENCIA)

3

Las representaciones gráficas de la variación de los

distintos descriptores se muestran a continuación

agrupadas en diferentes apartados: i) parámetros

fotosintéticos, respiración y composición isotópica del

carbono (Figs. 2, 3), ii) pigmentos fotosintéticos y

propiedades ópticas foliares (Figs. 4, 5, 6), iii) rela-

ciones hídricas y concentración de osmolitos (prolina

y carbohidratos no-estructurales) (Figs. 7, 8, 9), iv)

estado nutricional (Fig. 10) y por último, v) descripto-

res vegetativos (Fig. 11). El apartado (6) corresponde

con información de la variación también estacional

que algunos factores ambientales importantes (i.e.

temperatura, irradiancia y salinidad) experimentan

en las mencionadas praderas de referencia (Fig. 12).

Figura 1. Mapa de localización de las estaciones o praderas de referencia de P. oceanica y C. nodosa. Las praderas someras de ambas especies y la profunda de P. oceanica correspondientes a los puntos 1, 2 y 3, respectivamente, se ubican en la Bahía de Mazarrón (1, 2- 37º 34´ N 1º 12´ W-Isla Plana; 3- 37º 33´14.4´´ N 1º 10´35.5´´ W- La Azohía), mientras que la pradera más profunda de C. nodosa se sitúa en la zona de Calblanque cercana a Cabo de Palos (4- 37º 34´N 1º 12´ W). La población de C. nodosa en la laguna costera hipersalina del mar Menor corresponde a las coordenadas (37º 39´41.59´´N 0º44´14.76´´ W). Las líneas punteadas corresponden a isóbatas.

4

5

1. Parámetros fotosintéticos, respiración y composición isotópica del carbono

Figura 2. Tasas de evolución de oxígeno en función del incremento en valores de irradiancia (i.e. curvas de fotosíntesis-irradiancia o P vs. E), obtenidas a partir de plantas originarias de las diferentes praderas de referencia de P. oceanica y C. nodosa (población somera de P. oceanica, población profunda de P. oceanica, población somera de C. nodosa y población de C. nodosa ubicada en la laguna costera hipersalina del Mar Menor). Los diferentes símbolos corresponden a los distintos períodos estacionales: ● = Octubre 08, ○ = Enero 09, ▼= Abril 09, ∆ = Julio 09. Los valores mostrados corresponden a medias ± EE (error estándar).

6

Figura 3. Evolución estacional de parámetros fotosintéticos y tasas respiratorias derivados de curvas P vs. E (i.e. tasas fotosintéticas netas- P-neta (a), Respiración- R (d), productividad diaria (b) y eficiencia fotosintética-α (e)), composición isotópica del carbono en tejido foliar (i.e. δ13C, (c)) y valores de eficiencia fotoquímica máxima (Fv/Fm, (f )), correspondiente a plantas de las distintas poblaciones de P. oceanica (líneas punteadas) y C. nodosa (líneas discontinuas): ●= P. oceanica (5-7 m), ○= P. oceanica (18-20 m), ▼= C. nodosa (5-7 m), ∆= C. nodosa (Mar Menor). Los valores mostrados corresponden a medias ± EE (error estándar).

7

2. Pigmentos fotosintéticos y propiedades ópticas foliares

8

Figura 4. Evolución estacional de la densidad (b-d) y el contenido (f-h) de pigmentos fotosintéticos (Chla, Chlb, Carotenoides) y ratios molares (Chlb:a, Carotenoides:Chla, (a) y (e)) correspondientes a tejido foliar de plantas de las distintas poblaciones deP. oceanica (líneas punteadas) y C. nodosa (líneas discontinuas): ●= P. oceanica (5-7 m), ○= P. oceanica (18-20 m), ▼= C. nodosa (5-7 m), ∆= C. nodosa (Mar Menor). Los valores mostrados corresponden a medias ± EE (error estándar).

Figura 5. Evolución estacional de absorptancia de hojas (A), calculadas a partir de medidas de absorbancia promediadas en el rango de espectro visible (PAR) (a) y las propias del pico de absorción a longitud de onda de 680 nm (b), correspondiente a plantas de las distintas poblaciones de P. oceanica (líneas punteadas) y C. nodosa (líneas discontinuas): ●= P. oceanica (5-7 m), ○= P. oceanica (18-20 m), ▼= C. nodosa (5-7 m), ∆= C. nodosa (Mar Menor). Los valores mostrados corresponden a medias ± EE (error estándar).

9

Figura 6. Espectros de absorción de radiación fotosintéticamente activa (PAR), obtenidos en tejido foliar de plantas originarias de las diferentes praderas de referencia de P. oceanica y C. nodosa (población somera de P. oceanica, población profunda de P. oceanica, población somera de C. nodosa y población de C. nodosa ubicada en la laguna costera hipersalina del Mar Menor). Los diferentes tipos de líneas representan los espectros correspondientes a los distintos períodos estacionales: Línea continua gruesa (Octubre 08), línea punteada (Enero 09), línea discontinua (Abril 09) y línea continua fina ( Julio 09). Los valores mostrados corresponden a medias ± EE (error estándar).

10

3. Relaciones hídricas y concentración de osmolitos (prolina y carbohidratos no estructurales)

Figura 7. Evolución estacional de los descriptores de relaciones hídricas foliares (Ψw= potencial hídrico, Ψπ= potencial osmótico y Ψp= potencial de turgor), correspondientes a plantas originarias de las diferentes praderas de referencia de P. oceanica y C. nodosa (población somera de P. oceanica, población profunda de P. oceanica, población somera de C. nodosa y población de C. nodosa ubicada en la laguna costera hipersalina del Mar Menor). Los valores mostrados corresponden a medias ± EE (error estándar).

11

12

Figura 8. Evolución estacional de la concentración de prolina en tejido foliar de plantas originarias de las distintas poblaciones de P. oceanica (líneas punteadas) y C. nodosa (líneas discontinuas): ●= P. oceanica (5-7 m), ○= P. oceanica (18-20 m), ▼= C. nodosa (5-7 m), ∆= C. nodosa (Mar Menor). Los valores mostrados corresponden a medias ± EE (error estándar).

Figura 9. Evolución estacional de la concentración de carbohidratos no-estructurales (i.e fracción soluble (a,b) y almidón (c, d)) en tejidos foliares y rizomáticos, correspondientes a plantas de las distintas poblaciones de P. oceanica (líneas punteadas) y C. nodosa (líneas discontinuas): ●= P. oceanica (5-7 m), ○= P. oceanica (18-20 m), ▼= C. nodosa (5-7 m), ∆= C. nodosa (Mar Menor). Los valores mostrados corresponden a medias ± EE (error estándar).

13

4. Estado nutricional

Figura 10. Evolución estacional del contenido en nutrientes (i.e. C y N, (b), (c), (e) y (f )), expresado en porcentaje de peso seco, y sus ratios molares (i.e C/N, (a) y (d)), correspondientes a tejidos foliares y rizomáticos de plantas originarias de las distintas poblaciones de P. oceanica (líneas punteadas) y C. nodosa (líneas discontinuas): ●= P. oceanica (5-7 m), ○= P. oceanica (18-20 m), ▼= C. nodosa (5-7 m), ∆= C. nodosa (Mar Menor). Los valores mostrados corresponden a medias ± EE (error estándar).

14

5. Descriptores vegetativos

Figura 11. Evolución estacional de algunos descriptores fenológicos (i.e. tamaño de haz (a), crecimiento foliar por haz (b), tejido foliar necrosado (c) y número de hojas por haz (d)) correspondientes a plantas de las distintas poblaciones de P. oceanica (líneas punteadas) y C. nodosa (líneas discontinuas): ●= P. oceanica (5-7 m), ○= P. oceanica (18-20 m), ▼= C. nodosa (5-7 m), ∆= C. nodosa (Mar Menor). Los valores mostrados corresponden a medias ± EE (error estándar).

15

6. Evolución estacional de factores ambientales

Siguiendo el patrón de evolución temporal de pa-

rámetros descriptivos fisiológicos y vegetativos

propuestos para P. oceanica y C. nodosa, se rea-

lizaron, adicionalmente, medidas de irradiancia, sa-

linidad y temperatura en las praderas o estaciones

de referencia. Estas medidas se realizaron gracias

a la colocación de sensores sumergibles esféricos

(MDS-MKV/L) colocados a dos niveles de altura en

las praderas de P. oceanica: i) sobre el dosel ve-

getal, a la altura de la punta de las hojas, y ii)

dentro de las praderas, a nivel de los rizomas y

tejidos fotosintéticos basales. Dichos sensores per-

mitieron compilar datos a partir de mediciones en

continuo durante al menos 20-30 días, en cada pe-

ríodo de muestreo estacional. La información obte-

nida fue empleada para el ajuste de las variables

ambientales en los experimentos llevados a cabo en

el sistema de mesocosmos (i.e. Caps. 2, 3, 4 y 5).

16

Figura 12. Evolución estacional de los factores ambientales (i.e. temperatura (a), salinidad (b) y dosis lumínica (c)) en las distintas poblaciones de P. oceanica y C. nodosa. Los valores mostrados corresponden a medias ± EE (error estándar).

17

Sensores de irradiancia, conductividad y temperatura instalados en estaciones de muestreo de Posidonia oceanica.

18

ANEXO2

1. Planteamiento general

Para el desarrollo de los trabajos experimenta-

les mostrados en esta tesis (capítulos 2, 3, 4 y

5) resultó imprescindible la instalación y puesta a

punto de un sistema experimental de mesocosmos

de laboratorio. Dicho sistema fue concebido para el

mantenimiento de las especies de angiospermas mari-

nas mediterráneas, Posidonia oceanica y Cymodocea

nodosa, e instalado en los laboratorios húmedos del

Centro Oceanográfico de Murcia (COMU, San Pedro del

Pinatar) del Instituto Español de Oceanografía.

Si bien otras especies de angiospermas marinas han

demostrado una mayor adecuación en respuesta a ma-

nipulaciones experimentales en laboratorio, el mante-

nimiento de P. oceanica presenta grandes dificultades

en este sentido. Así, y en relación a lo publicado en

otras especies, cabe resaltar que los experimentos

realizados con P. oceanica en condiciones de labo-

ratorio son muy escasos, en comparación con los

llevados acabo en praderas naturales. Sin embargo,

y como muestra el capítulo 1, la manipulación in situ

de la salinidad es una tarea altamente compleja y

costosa. Por tanto, y con la colaboración de Emilio

Cortés Melendreras, Director del Acuario Marino de la

Universidad de Murcia, se diseñó un sistema ad hoc

a escala de mesocosmos, dentro de las posibilidades

económicas, logísticas y de espacio que los proyectos

(OSMOGRASS nº ref. 021/SGTB/2007/1.3 Ministerio de

Medio Ambiente y Medio Rural y Marino) y las insta-

laciones del COMU permitieron.

Con el diseño de dicho sistema de mesocosmos, se

procuraron los siguientes aspectos fundamentales:

i) mantenimiento de P. oceanica y C. nodosa en óptimas

condiciones fisiológicas, durante períodos experimentales

lo suficientemente amplios como para permitir el estudio

de las respuestas a las manipulaciones experimentales,

en las escalas de tiempo apropiadas (i.e. mínimo 2 meses),

ii) control estricto de variables ambientales cla-

ve (i.e. luz, pH, temperatura y nutrientes) y fac-

tores objeto de la manipulación experimental (i.e.

salinidad), cuyo control in situ resulta complicado.

iii) elevada capacidad de reproducción de condiciones

ambientales y comportamientos de las plantas, ob-

servadas en las praderas naturales, y por último,

iv) capacidad de permitir la replicación y asignación de

unidades experimentales sujetas a diferentes combina-

ciones de tratamientos experimentales, y de posibilitar

las mayores condiciones de independencia entre ellas.

Los objetivos anteriores son fundamentales para que

los resultados puedan ser, al menos en cierta medi-

da, extrapolables a una situación real, pero también

para que el sistema no introduzca fuentes de estrés

adicionales, que puedan enmascarar o confundir las

respuestas de las plantas a las manipulaciones ex-

perimentales. En cada capítulo se detallan y discuten

los aspectos de cada diseño experimental planteado

en este sistema de mesocosmos, así como su adecua-

ción para evaluar las hipótesis propuestas.

DISEÑO Y DESCRIPCIÓN TÉCNICA DEL

SISTEMA EXPERIMENTAL DE MESOCOSMOS

21

Este anexo se limita a describir los detalles más

técnicos del sistema, que por limitaciones de espacio

no se han podido aportar en los distintos capítulos

de la tesis.

2. Descripción del sistema de acuarios y circulación de agua de mar

El dispositivo experimental de mesocosmos consta de

4 acuarios independientes de dimensiones de 80 x 240

cm, cristal de 16 mm, capacidad hasta 1500L, y dis-

puestos sobre estructuras de acero inoxidable (90 x

240 cm). Cada acuario se subdivide en 3 sub-acuarios

de 500L, conectados mediante rebosadero a una cuba

reservorio de fibra de vidrio (500L), dispuesta en la

parte inferior de la estructura metálica. En la Fig. 1,

se muestra un esquema de cada sistema de acuarios

y el circuito cerrado de circulación de agua de mar.

Para la alimentación de los acuarios con agua de mar,

se instaló un sistema de 4 tanques de fibra de vidrio

de 2000L, conectados con el laboratorio húmedo por

un sistema de 4 bombas auto-aspirantes de 10000 L

hora-1 y tuberías de pvc (Fig. 2). Este sistema des-

empeñó los papeles de reservorio de agua de mar

para los periódicos cambios de agua realizados en los

acuarios durantes los periodos experimentales (ver

más bajo), y de tanques de mezcla de sal con agua

de mar para la producción del agua empleada para

los tratamientos hipersalinos. En este sentido, cada

bomba permite la recirculación interna del agua den-

tro de cada tanque, de manera que se favorece tanto

la oxigenación de agua de reserva, como la disolución

de la sal en los momentos de mezclado.

3. Iluminación

Para el sistema de iluminación se seleccionó el uso

de proyectores de halogenuros metálicos (HQI) de

400W (Aquamedic Aqualight-400) (Fig. 1 y 4a). La

elección de este tipo de iluminación, vino condicio-

nada por las importantes ventajas que su uso su-

pone en el caso de acuarios plantados, entre las

que se destacan: i) un alto índice de reproducción

cromática similar a la solar, ii) el desarrollo de una

larga vida útil, iii) alta capacidad de penetración en

agua de mar, iv) capacidad de alto flujo luminoso

y, v) calidad espectral balanceada entre radiacio-

nes de longitud de onda comprendidas en la zona

del rojo y del azul (i.e. PUR-R/PUR-A≈ 0.93; “pho-

tosynthetic usable radiation”-rojo (600-750 nm)/

“photosynthetic usable radiation”-azul (400-500 nm))

Se dispuso de una lámpara por sub-acuario, separada

ésta del adyacente por paneles de vinilo, coloca-

dos con el propósito de evitar la influencia lumínica

entre sub-acuarios. En este sentido cabe destacar

que, además de las ventajas mencionadas, el uso de

este tipo de proyectores con reflectores de alumi-

nio, permitieron el mantenimiento de campos de ilu-

minación homogéneos en las primeras masas de agua

sub-superficiales (i.e. no afectados por efectos de

atenuación-dispersión) dentro de cada sub-acuario.

Para la adecuación de los fotoperíodos e intensidades

lumínicas requeridos para cada condición experimen-

tal, se instalaron sistemas de ajustes electrónicos

programables y mecánicos. De este modo, las lámpa-

ras fueron ajustadas a distintos niveles de altura, y

22

Figura 1. Representación esquemática del sistema experimental de mesocosmos. (1) acuario de 1500 L subdividido en 3acuarios de 500L (2) sistema de iluminación-lámparas y proyectores de halogenuros metálicos (3) dispositivos electrónico de control de pH conectado a sensor sumergible (4) sistema de filtración mecánica (5) sistema de filtración química o skimmer (6) bomba de circulación de agua (7) cuba reservorio (8) conductos/serpentines del sistema de regulación de temperatura (9) panel electrónico de control de temperatura conectado a sensor sumergible y serpentiners. Las flechas negras indican la dirección de circulación del agua de mar en el circuito cerrado.

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Figura 2. Representación esquemática del sistema experimental de tanques empleados como reserva y para mezclado de sal con agua de mar. (a) 4 tanques con capacidad de 2000L, cada uno con sistema de recirculación mediante bomba. (b) Detalle de un tanque de 2000L donde tiene lugar el mezclado de agua de mar con sal a través de sistema de recirculación de agua. Las flechas negras representan la circulación del agua de mar. Cada uno de los tanques se conecta con las cubas reservorio (7 en Fig. 1) del sistema experimental de mesocosmos.

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se mantuvieron encendidas durante un cierto número

de horas al dia, con el fin de que los valores opteni-

dos de dosis lumínica proporcionados por sensores de

irradiancia sumergibles (LICOR LI-192SA y los esféricos

MDS-MkV/L) dispuestos en el interior de los acuarios

(Fig. 4b), fueran comparables con los obtenidos en las

praderas de referencia (ver apartado 6 en Anexo 1).

4. Filtración

El sistema de filtrado por acuario contó con una fase

de filtración mecánica y una fase de filtración química

(Fig. 1). La filtración mecánica consistió en la reten-

ción de partículas de pequeño tamaño, en suspensión

o disolución, por medio de un material filtrante inerte

(i.e. fibra sintética). Este sistema de filtración se

dispuso en un paso intermedio de la circulación del

agua de los acuarios (i.e. por rebosaderos) a la cuba

reservorio, y en el interior de ésta, en la aspiración

de la bomba de recirculación.

La filtración química consiste en la captación y eli-

minación química de diferentes compuestos de carbo-

no orgánico disuelto (aminoácidos, fenoles, terpenos,

pigmentos, ácidos orgánicos, etc.). La acumulación de

dichos compuestos es detectada normalmente en este

tipo de sistemas marinos de circuito cerrado, y no

pueden ser eliminados mediante otro tipo de técnica

de filtrado. En nuestro caso, el sistema seleccionado

para este fin fue el de skimmer (o separador de pro-

teínas; Turboflotor 5000®, Fig. 4c), cuya instalación es

considerada imprescindible en mesocosmos desarrolla-

dos en el ámbito marino. El funcionamiento de los ski-

mmers, se basa en la afinidad de la parte hidrofóbica

de ciertas moléculas antipáticas (e.g. ácidos grasos,

proteínas) por la zona aérea de las burbujas, produ-

cidas éstas mediante recirculación con bomba venturi

en el cuerpo o cilindro de reacción del dispositivo.

5. Temperatura

Para el estricto control de la temperatura del agua

de mar en los acuarios (±0.1ºC) , la empresa “Pe-

dro Serrano García SL” desarrolló ad hoc un

sistema para el control específico de este pará-

metro, con aplicación independiente en cada sistema

de circuito cerrado. Dicho sistema estuvo basado en

intercambiadores térmicos de acero inoxidable 316

de alta resitencia (i.e. serpentines). Dichos serpen-

tines, instalados en los tanques reservorio (Fig. 1

y 4d), fueron empleados como los dispositivos de

intercambio térmico, debido a la circulación, por su

interior, de agua a una temperatura máxima de 45ºC

y mínima de 4ºC. El paso de agua a una tempe-

ratura u otra estuvo regulado por la variación de

temperatura del agua de los acuarios, dependiendo

de los rangos establecidos mediante termostato y

monitorizados en continuo mediante un cuadro elec-

trónico general (Fig 4e). La descripción detallada del

sistema se presenta esquemáticamente en la Fig.3.

Para evitar la condensación de agua en la superfi-

cie de los acuarios y estructuras anexas, así como

con el fin de reducir el posible aumento de tem-

peratura por efecto del sistema de iluminación, se

contó, adicionalmente, con la instalación de módu-

los de aire acondicionado en el laboratorio húmedo.

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Figura 3. Esquema simplificado del sistema de regulación de temperatura diseñado para el sistema de mesocosmos. La actividad de una enfriadora y un dispositivo de resistencias acoplados a tanques distintos, permite el abastecimiento al sistema de agua fría o caliente. La circulación de agua fría (4ºC) o caliente (45ºC) discurre por circuitos cerrados aislados térmicamente, hasta llegar a los intercambiadores térmicos de acero inoxidable 316 (serpentines) instalados en los 4 tanques reservorio correspondientes a los 4 sistemas de acuarios. El paso de agua fría o caliente a dichos serpentines está regulada por un panel electrónico de termostato, conectado a 4 sensores sumergibles de temperatura también dispuestos en los tanques reservorio.

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6. Control de calidad de agua

Con el fin de mantener el agua circulante del siste-

ma experimental en ópticas condiciones durante los

períodos experimentales, se mantuvo un control ex-

haustivo de importantes variables como pH, oxígeno

disuelto y nutrientes. Para el caso del control de pH,

cuatro sensores sumergibles presurizados (Aqua Me-

dic®) fueron instalados en cada uno de los tanques

reservorio. Dichos sensores proporcionaron informa-

ción en continuo de los valores de pH del agua de los

acuarios, a través de un sistema electrónico compu-

terizado (Aqua Medic AT-Control®) (Fig. 1 y 4f). Para

el caso de acuarios plantados de circuito cerrado, se

considera general el uso de reactores de carbono

acoplados a válvulas solenoides para el mantenimiento

óptimo de los niveles de pH. Considerando esta cues-

tión, dicho sistema de inyección de CO2 fue instalado

en una fase previa de puesta a punto del sistema

experimental. Sin embargo, los resultados obtenidos

a partir de dicha fase fueron concluyentes en la ca-

pacidad de mantenimiento del pH (i.e. entre 8.02-8.18,

con valores máximos y mínimos paralelos a ciclos

de día/noche) aun prescindiendo de dicho sistema.

Además del control de alcalinidad y pH, se realizaron

medidas y analíticas periódicas de oxígeno disuelto

(mediante oxímetro WTW Oxi3205®) y de nutrien-

tes (i.e. nitratos, fosfatos y oligoelementos) (me-

diante test colorimétricos, Merck ®) en el agua del

sistema de acuarios. Tanto los niveles de oxígeno

disuelto (10.3-11.2 mg/L) como la concentración de

nutrientes reflejaron el óptimo mantenimiento de la

calidad del agua (similar a la de aguas bien oxigena-

das y oligotróficas naturales del litoral mediterráneo

español) durante los períodos de experimentación.

Por su parte, especial atención se prestó al factor

salinidad por su calidad de tratamiento experimen-

tal. En este caso, se realizaron medidas diarias de

salinidad mediante conductivímetro (WTW 197i®). La

compensación de las pérdidas de agua por evapo-

ración y la consecuente acumulación de sales, se

llevó a cabo mediante la adición de agua filtrada

por ósmosis. De esta manera se consiguió un es-

tricto control de la salinidad, con mínimas desvia-

ciones de los niveles establecidos (i.e. ±0.1 ups)

De forma adicional a los sistemas de filtración ins-

talados, y a pesar del control riguroso de la ca-

lidad del agua, se impusieron recambios periódicos

del agua del sistema experiemental. Dichos recambios

(de al menos el 50% del agua total de cada circui-

to cerrado), fueron realizados con una periodicidad

aproximada de unos 10 días, con el fin de evitar

la posible acumulación de substancias disueltas o

particuladas, que pudieran no haber sido elimina-

dos por los medios de filtración antes descritos.

7. Circulación de agua

La circulación de agua en cada sistema de acuarios

se consiguió a través de la instalación de bombas

auto-aspirantes de 10000 L h-1 (Fig. 4g) , que per-

mitieron una elevada oxigenación y ciclos rápidos

de recambio de agua en los sistemas (≈ 124 ciclos

por sistema y día). Además, cabe destacar que la

potencia de dichas bombas, fue calculada para el

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mantenimiento de una controlada recirculación y tur-

bulencia del agua dentro de los acuarios. El empleo

de este tipo de bombas, junto con la instalación de

difusores (Fig. 4h), permitió un movimiento de hojas

adecuado, y necesario para la reducción de la capa

limitante fotosintética situada sobre la superficie de

las mismas (Enríquez y Rodríguez-Román 2006). Por

otra parte, cabe destacar que las bombas empleadas

se adquirieron especialmente diseñadas para evitar la

formación de micro-burbujas en los espacios ocupados

para las plantas. En este caso la presencia de dichas

micro-burbujas podría acarrear diversos problemas

comúnmente descritos en este tipo de sistemas (p.e.

disminución de pH, adherencia a tejidos vegetales y

la interrupción del contacto con el medio, reduc-

ción del campo lumínico por efecto de “scattering”).

Figura 4. Detalles de sistema de mesocosmos: (a) iluminación (lámparas de HQI y panelesde vinilo) (b) sensor esférico de irradiancia PAR instalado dentro del acuario (c) filtración (skimmers)(d) intercambiador térmico (serpentín) de tanque reservorio(e) panel electrónico de control de temperatura(f ) dispositivo de control de pH(g) bomba auto-aspirante de cada circuito cerrado de acuarios (h) difusores de agua dentro de los acuarios

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ba

c d

e f

g h

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CRÉDITOS

ARTE

Víctor Imperial López

DISEÑO Y MAQUETACIÓN

Patricio Imperial López imperial.patricio@gmail.com

FOTOGRAFÍA

Juan Manuel Ruiz Fernández José Miguel Sandoval Gil