diferenças adaptativas entre plantas de savanas e...
TRANSCRIPT
Maíra Figueiredo Goulart
Diferenças adaptativas entre plantas de savanas e florestas:
o caso das populações de Plathymenia reticulata
(Leguminosae-Mimosoideae) do Cerrado e da Mata Atlântica
Tese apresentada ao programa de pós-graduação em Ecologia, Conservação e Manejo de Vida Silvestre da Universidade Federal de Minas Gerais como requisito parcial para obtenção do título de Doutor.
Orientador: Prof. Dr. José Pires de Lemos Filho
Co-orientador: Profa. Dra. Maria Bernadete Lovato
Belo Horizonte
2008
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Aos que plantam árvores.
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“São seis ou são seiscentas
distâncias que se cruzam, se dilatam
no gesto, no calar, no pensamento?
Que léguas de um a outro irmão.
Entretanto, o campo aberto, os mesmos copos,
o mesmo vinhático das camas iguais.
A casa é a mesma.
Igual, vista por olhos diferentes?”
Carlos Drummond Andrade
(Irmão, Irmãos)
“Mas se deu que, certo dia, nosso pai mandou fazer para si uma canoa.
Era a sério. Encomendou a canoa especial, de pau de vinhático, pequena,
mal com a tabuinha da popa, como para caber justo o remador.
Mas teve de ser toda fabricada, escolhida forte e arqueada em rijo,
própria para dever durar na água por uns vinte ou trinta anos.
(...) E esquecer não posso, do dia em que a canoa ficou pronta”.
João Guimarães Rosa
(A Terceira Margem do Rio)
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ÍNDICE
Agradecimentos ............................................................................................................
Resumo .........................................................................................................................
Abstract ........................................................................................................................
Apresentação
- Introdução ............................................................................................................
- Espécie estudada ..................................................................................................
- Contextualização dos estudos ..............................................................................
- Estrutura da tese ..................................................................................................
- Referências bibliográficas ...................................................................................
Capítulo 1: Evidences for local adaptations from early developmental
responses to light and soil fertility in the tropical tree Plathymenia reticulata
along a forest-savanna boundary
- Introduction .........................................................................................................
- Material and methods
- Studied tree species ....................................................................................
- Studied populations, seeds collection and germination .............................
- Non destructive experiment .......................................................................
- Destructive experiment ..............................................................................
- Analysis of data .........................................................................................
- Results .................................................................................................................
- Discussion
- Differences among populations .................................................................
- Ecotypic differentiation .............................................................................
- Evolutionary trends in savanna-forest boundary .......................................
- References ...........................................................................................................
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Capítulo 2: To which extent phenotypic plasticity in response to light is
involved in the ecotypic differentiation of a tree species from savanna and
forest habitats?
- Introduction .........................................................................................................
- Material and methods
- Studied populations ...................................................................................
- Experimental design ..................................................................................
- Morphological and physiological measurements ......................................
- Analysis of data .........................................................................................
- Results .................................................................................................................
- Discussion
- Phenotypic plasticity in response to light ..................................................
- Functional heterogeneity of the light environment ....................................
- Functional traits and ecotypic differentiation ............................................
- References ...........................................................................................................
Capítulo 3: How important is soil fertility in driving ecotypic differentiation of
a tropical tree species from savanna and forest habitats?
- Introduction .........................................................................................................
- Material and methods
- Studied populations ...................................................................................
- Nursery experiment and data collection ....................................................
- Field experiment and survival censures .....................................................
- Analysis of data .........................................................................................
- Results .................................................................................................................
- Discussion
- Responses to soil ........................................................................................
- Habitat specialization .................................................................................
- Phenotypic plasticity ..................................................................................
- Survival in field conditions ........................................................................
- Concluding remarks ...................................................................................
- References ..................................................................................................................
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AGRADECIMENTOS
Ao Prof. José Pires e à Profa. Bernadete, orientadores com os quais iniciei minha
formação acadêmica já faz muitos anos, serei eternamente grata por terem me proporcionado
incontáveis oportunidades de aprendizado, por toda a confiança em mim depositada e por serem
sempre um estímulo a seguir em frente.
Ao Fernando Valladares, meu orientador durante um estágio “sanduíche” no Centro de
Ciencias Medioambientales do Consejo Superior de Investigaciones Científicas em Madri,
Espanha. Inicialmente, o objetivo do estágio foi aprender a usar índices para estimativa de
plasticidade fenotípica e sua interpretação, mas a orientação do Fernando transcendeu os
objetivos e suas boas idéias foram muito importantes para a interpretação e discussão de todos
dados, bem como para redação dos manuscritos. Portanto, agradeço muitíssimo a enormidade de
coisas que aprendi. Agradeço ainda a todos do Grupo de Ecología Mediterránea pelo empenho
em compreender meu “portunhol” e por me proporcionarem um ambiente de trabalho tão
amigável. Agradeço, em especial, as amigas Teresa Gimeno e Virginia Gancedo, por
transformarem a casa em lar.
Fernanda Barros esteve presente desde os primeiros rascunhos dos experimentos,
auxiliando-me em todas as etapas dos trabalhos e sempre foi uma verdadeira amiga. Sérgio
Teles apresentou-se como um estagiário incansável, sempre disposto a aprender. A disposição
dos dois em medir centenas de plantas em situações nada confortáveis foi para mim um grande
estímulo e, definitivamente, não teria chegado até aqui sem essa valiosa ajuda, pela qual sou
muito grata. Agradeço às outras pessoas que contribuiram para a coleta dos dados: Alex,
Letícia, Eugênio, Ana Clara, Marcos, Renan, Viviane, Carmem e Socorro, e ao Sr. José dos
Reis, que cuidou diariamente das plantas no viveiro, sempre de forma muito atenciosa.
Sou grata às instituições que me apoiaram financeiramente: CAPES (Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior) e CNPq (Conselho Nacional de
Desenvolvimento Científico e Tecnológico), pelas bolsas concedidas, respectivamente de
doutorado e doutorado-sanduíche; FAPEMIG (Fundação de Amparo à Pesquisa do Estado
de Minas Gerais) financiadora do projeto de pesquisa “Abordagem filogeográfica e
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ecofisiológica em populações de leguminosas arbóreas da Mata Atlântica e do Cerrado”, cujos
recursos possibilitaram a coleta de muitos dos dados abordados na tese.
Sinto-me privilegiada em agradecer a três universidades: à Universidade Federal de
Minas Gerais por fazer parte da minha vida há 10 anos. Ao curso de graduação em Ciências
Biológicas e ao curso de pós-graduação em Ecologia, Conservação e Manejo da Vida Silvestre,
todos os professores e colegas, pelo imensurável aprendizado. Agradeço à Universidade Federal
de Ouro Preto, que me proporcionou uma experiência didática tão profunda. Aos professores do
Departamento de Ciências Biológicas, agradeço por terem confiado em mim tantas
responsabilidades, e agradeço ainda aos meus alunos dos cursos de Ciências Biológicas,
Turismo e Engenharia Ambiental, com quem tanto aprendi. Também sou grata à Universidade
Federal dos Vales do Jequitinhonha e Mucuri, na qual ingressar como professora foi a
realização de um sonho, reconheço que a redação desta tese ainda não estaria finalizada se não
fosse o apoio que recebi do Departamento de Ciências Biológicas.
Apenas uma parte do meu aprendizado durante o doutorado está apresentada nessa tese.
A outra parte não pode ser expressa em números e nem ser divulgada em artigos científicos,
mas representa um aprendizado fundamental na minha vida. Deixo registrado um agradecimento
muito especial ao Instituto Biotrópicos de Pesquisa em Vida Silvestre por tantas oportunidades
de crescimento humano e profissional. Aos colegas da Biotrópicos, por serem amigos e
companheiros empenhados em continuar sonhando com um futuro de mais harmonia com a
natureza, apesar da dura rotina que a ciência nos impõe.
Agradeço a todos que compartilham da minha alegria em ver as plantas crescendo
verdinhas. Aos meus amigos e às minhas queridas famílias, pela alegria de viver. Aos meus
avós, que são as pessoas mais amorosas que conheço, em especial, agradeço ao vô Thadeu por
ter deixado tantas boas lembranças. Ao Nando, agradeço por compartilhar comigo a vida de
biólogo desde a nossa infância. Aos meus pais, corujas incondicionais, minha mais profunda
gratidão por todo o apoio, o aprendizado e o maior amor do mundo. Ao Alex que, enquanto
colega, compartilhou comigo todas as angústias e as alegrias do desenrolar dos nossos
doutorados, e que, enquanto marido, traz flores para o jardim. Obrigada por ser meu melhor
amigo, meu atencioso confidente e meu maior incentivador.
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RESUMO
Esta tese aborda diferenças adaptativas entre populações da arbórea Plathymenia reticulata
(Leguminosae-Mimosoideae) no Cerrado e na Mata Atlântica. Indivíduos de quatro populações
naturais desta espécie (uma em área “core” de cada bioma e duas em região ecotonal entre eles,
a primeira com fisionomia de Cerrado e a segunda de Mata Atlântica) foram crescidos em
viveiro e submetidos a quatro tratamentos de luz (100, 53, 36 e 22% da luz solar plena) e dois
tratamentos de solo (solo de Cerrado e de Mata Atlântica). Em cada tratamento, a avaliação de
características como morfologia, crescimento, fotossíntese e pigmentos foliares, forneceu uma
indicação da extensão das diferenças genéticas entre as populações. Para uma mesma população
a comparação entre os tratamentos permitiu uma avaliação da plasticidade fenotípica. Os
resultados mostraram diversas diferenças entre populações de P. reticulata do Cerrado e da
Mata Atlântica durante o crescimento inicial. Freqüentemente, os indivíduos das populações em
região de ecótone apresentaram valores intermediários para as características avaliadas. Quanto
à morfologia e ao crescimento, comparativamente, as plantas do Cerrado mostraram
características de resistência à estresses, enquanto as plantas da Mata Atlântica apresentaram
características de tolerância à sombra e maior habilidade competitiva. A avaliação dos
pigmentos foliares apontou para um padrão de que as plantas do Cerrado tendem a apresentar
um maior investimento em fotoproteção e as de Mata Atlântica em captação de luz. Os
caracteres relacionados à fotossíntese, avaliados pela fluorescência da clorofila, mostraram-se
conservados entre as populações. Foi encontrada menor plasticidade fenotípica em resposta à
luz na população de Cerrado do que na de Mata Atlântica, bem como um padrão geral de maior
plasticidade em reposta ao solo nas populações de ecótone do que nas de área “core” dos
biomas. Após um ano de crescimento em condições de viveiro, os indivíduos foram
transplantados para o campo. Censos de sobrevivência não mostraram diferenças significativas
entre as populações nos primeiros vinte meses. Coletivamente, os resultados dos experimentos
no viveiro mostram que as populações de P. reticulata são adaptadas ao ambiente de luz e às
condições de solo do seu bioma de origem, caracterizando a existência de ecótipos de savana e
floresta nesta espécie. É discutido como a seleção natural está atuando na promoção de
diferenças entre os ecótipos, como fatores luz e solo estão envolvidos neste processo, quais são
as tendências evolutivas na região de ecótone e como investigações futuras dos indivíduos
transplantados podem acrescentar a este conhecimento.
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ABSTRACT
This thesis investigates adaptive differences between populations of the tree Plathymenia
reticulata (Leguminosae-Mimosoideae) in the Cerrado and the Atlantic Forest. Individuals from
four natural populations (one from each biome core area and two from ecotonal region, the first
with Cerrado physiognomy and the other with Atlantic Forest physiognomy) were grown in a
nursery with four different light treatments (100, 53, 36 and 22% of full sunlight) and two soil
treatments (Cerrado and Atlantic Forest soils). At each treatment, comparisons of traits such as
morphology, growth, photosynthesis and leaf pigments, provided an indication of the extent of
genetic differences; for individual populations, comparisons of different treatments allowed the
assessment of the phenotypic plasticity. The results showed several differences in P. reticulata
from Cerrado and Atlantic Forest core populations during the seedling and sapling phases.
Frequently, individuals in the ecotonal populations showed intermediate values. Comparatively,
concerning morphology and growth, plants from Cerrado were characterized by more stress
resistance traits while plants from Atlantic Forest showed more evident shade avoidance traits
and higher competitive ability. Leaf pigment evaluation showed that plants from Cerrado
invested comparatively more in maximizing photoprotection and plants from Atlantic Forest in
improving light interception. Photosynthesis traits related to chlorophyll fluorescence were very
conserved among populations. We found lower levels of plasticity in response to light in
Cerrado than in Atlantic Forest core populations, and a general pattern of higher levels in
plasticity in response to soil in ecotonal populations than in core populations. After one year of
growth in the nursery, individuals were transplanted into field conditions. Survival censors
showed no significant differences among populations in the first twenty months. Collectively,
the results from the nursery experiments showed that populations of P. reticulata are locally
adapted to the light environment and the soil properties from their home habitat, which
characterizes the existence of distinct savanna and forest ecotypes in this species. It is discussed
how natural selection is promoting the differences between ecotypes, how light and soil is
involved in this process, which are the evolutionary trends in the biomes’ boundary and how
further evaluations of individuals in the field should add to this knowledge.
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APRESENTAÇÃO
Introdução
A ecologia funcional é o ramo da Ecologia que busca explicar a distribuição das
espécies ou genótipos baseado nas características funcionais dos mesmos, como
atributos fisiológicos, morfológicos, anatômicos e de história de vida (Poorter e Garnier
2007). Normalmente os estudos de ecologia funcional são realizados por meio de
comparações, buscando compreender, por exemplo, diferenças entre plantas pioneiras e
plantas clímax (Westoby 2007). A maioria dos estudos compara o desempenho médio
de indivíduos de diferentes grupos, porém a avaliação da plasticidade das respostas dos
indivíduos em um mesmo grupo vem ganhando importância nos últimos anos. Tem sido
demonstrado que esta plasticidade desempenha um papel crítico na resposta dos
indivíduos aos fatores ambientais, influenciando, portanto, a ecologia e a distribuição
dos mesmos (Valladares et al. 2000a, b).
Estudos sobre ecologia funcional podem ser conduzidos em escalas
macroevolutiva e microevolutiva. Na primeira, é enfocada a comparação entre espécies,
buscando compreender padrões gerais. Na escala microevolutiva são comparadas
populações ou genótipos de uma única espécie, permitindo o estudo na escala real em
que a evolução atua, uma vez que o processo evolutivo inicia-se com a alteração de
freqüências gênicas entre populações (Ridley 2004).
Nos estudos que compõem esta tese, microevolução de plantas na Mata
Atlântica e no Cerrado é abordada com objetivo geral de acrescentar dados ao
conhecimento sobre ecologia funcional e plasticidade fenotípica das mesmas. Os
resultados são discutidos do ponto de vista evolutivo, buscando contribuir para a
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compreensão das características que evoluem de forma diferenciada entre as plantas que
colonizam savanas e florestas.
Espécie estudada
Foram avaliadas populações de Plathymenia reticulata (Leguminosae-
Mimosoideae) uma leguminosa arbórea conhecida popularmente como vinhático, nome
que se refere à coloração vinho da madeira (Silva Júnior 2005). Esta espécie é
amplamente distribuída pelo território brasileiro, ocorrendo em pelo menos 15 estados,
especialmente em áreas de Cerrado e Mata Atlântica. Há também registro de sua
ocorrência na Bolívia, Paraguai e Suriname (Warwick e Lewis 2003).
A classificação do gênero Plathymenia foi proposta por G. Bentham em 1842
contendo duas espécies: P. reticulata de ocorrência no Cerrado e P. foliolosa, de
ocorrência na Mata Atlântica. Ao longo do século passado algumas modificações na
sistemática do gênero ocorreram e a existência de apenas uma espécie de Plathymenia
chegou a ser proposta, mas logo refutada (Heringer 1956). Em 2002, os resultados de
um estudo com marcadores moleculares sugeriram fluxo gênico entre as duas espécies
(Lacerda et al. 2002). Em seguida, em 2003, uma revisão taxonômica do gênero foi feita
e apenas P. reticulata passou a ser reconhecida desde então (Warwick e Lewis 2003).
Indivíduos de P. reticulata do Cerrado e da Mata Atlântica diferem-se em
termos de porte. Geralmente os do Cerrado são mais baixos (6 a 12 m), com tronco
retorcido e ramificado, enquanto os da Mata Atlântica são mais altos (15 a 30 m) e
alongados (Figura 1A e B). Indivíduos do Cerrado e da Mata Atlântica compartilham
características gerais como o hábito decíduo, inflorescências formadas por pequenas
flores de coloração esbranquiçada visitadas por pequenos insetos, e sementes aladas
dispersas pelo vento (Figura 1C, D e E).
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B A
C
E D 1 cm
Figura 1. Planthymenia reticulata, o vinhático. A) indivíduo do Cerrado; B) indivíduo da Mata Atlântica;
C) inflorescências; D) frutos maduros; E) frutos, sementes com e sem artículo endocárpico. (Fotos: M. F. Goulart)
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Contextualização dos estudos
Esta tese representa uma continuidade aos estudos que venho desenvolvendo
sobre o vinhático desde 2002, tendo sido também tema da minha dissertação de
mestrado a comparação de populações desta espécie nos diferentes biomas (Goulart
2004). Na ocasião, foram realizadas avaliações baseadas na observação e mensuração
de caracteres dos indivíduos em populações naturais, sobre a fenologia e a morfologia e
dispersão de frutos e sementes. Os resultados indicaram que populações do vinhático
apresentam fenologia semelhante nos diferentes biomas, tendo sido encontrada uma
maior diversidade de comportamentos fenológicos entre indivíduos de uma mesma
população do que entre biomas distintos. Diferenças na fenologia entre populações de
Cerrado e Mata Atlântica são basicamente restritas ao processo de perda de folhas
durante a estação seca, que se inicia com mais antecedência nos indivíduos do Cerrado,
provavelmente em resposta à maior carência de água neste ambiente (Goulart et al.
2005). As sementes dos indivíduos da Mata Atlântica, apesar de apresentarem tamanho
e massa semelhante às do Cerrado, possuem estruturas mais desenvolvidas para
dispersão pelo vento. Este padrão foi interpretado como uma resposta à maior
dificuldade de dispersão por vento imposta pelos ambientes florestais do que os
savânicos (Goulart et al. 2006). Ambos os estudos revelaram um padrão geral de
características intermediárias em populações localizadas em regiões ecotonais entre os
biomas (Goulart et al. 2005, Goulart et al. 2006).
Como uma etapa seguinte a estes trabalhos baseados na observação, iniciei
estudos de experimentação, buscando avaliar a extensão da adaptação das populações de
vinhático ao seu bioma de origem. Conduzi experimentos in situ (em condições de
campo) e ex situ (em viveiro, sob condições ambientais controladas), com o propósito
de avaliar diferenças entre populações de Cerrado, Mata Atlântica e de região ecotonal
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entre os biomas, durante as fases iniciais do desenvolvimento. Experimentos in situ de
longo prazo também foram iniciados para avaliação dos indivíduos em estágios mais
avançados do desenvolvimento. Grande parte dos resultados obtidos até então compõe
esta tese. Para uma melhor contextualização, apresento a seguir algumas informações
gerais sobre os experimentos desenvolvidos.
Experimentos in situ
Foram realizados experimentos de transplante recíproco para testar a hipótese de
que seleção natural é responsável por diferenças entre as populações de vinhático. A
predição foi de que as plantas do Cerrado se estabeleceriam com mais sucesso na área
de Cerrado e as de Mata Atlântica em um sítio de Mata Atlântica, o que demonstraria a
existência de diferenças genéticas relacionadas à adaptação dos indivíduos ao seu
ambiente de origem.
Para estes experimentos, sementes foram coletadas em quatro populações
(Cerrado, Mata Atlântica, ecótone com características de Cerrado e ecótone com
características de Mata Atlântica) e, após escarificação mecânica, foram plantadas em
uma área de Cerrado (município de Lagoa Santa, Minas Gerais) e outra de Mata
Atlântica (município de Ipatinga, Minas Gerais) (Figura 2). Estes experimentos, porém,
se mostraram inconclusivos. Na área do Cerrado, o pisoteamento por gado e a
herbivoria por formigas causou a morte de grande parte das plântulas ou a remoção das
etiquetas com a marcação da procedência. Na área de Mata, o sub-bosque foi alagado no
período de chuvas, causando os mesmos problemas. Por fim, três meses após o plantio,
97% das plântulas provenientes do total de 1600 sementes plantadas estavam mortas ou
foram descartadas por serem de procedência desconhecida. A hipótese foi então testada
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em experimentos ex situ, de forma que as condições controladas e a ausência de
herbívoros pudessem reduzir a mortalidade dos indivíduos.
A
B
Figura 2. Experimento desenvolvido no campo.
A) plantio de sementes escarificadas; B) plântula. (Fotos: M. F. Goulart)
Experimentos ex situ
Sementes das mesmas procedências foram cultivadas em viveiro, submetidas a
diferentes combinações de tratamentos de luz e de solo (Figura 3). Ao longo de sete
meses, medidas morfológicas destrutivas e não destrutivas, medidas de fotossíntese e de
pigmentos foliares foram realizadas. Os resultados destes experimentos estão descritos
nos artigos que compõe esta tese.
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A
B C
Figura 3. Experimentos desenvolvidos em viveiro. A) bancadas com diferentes níveis de sombreamento (100, 53,
36 e 22% da luz solar plena); B) plântula em solo de Cerrado e C) em solo de Mata Atlântica. (Fotos: M. F. Goulart)
Experimentos in situ de longo prazo
Após a finalização dos experimentos em viveiro, os indivíduos foram
transplantados para três áreas, dando início a experimentos de longo prazo (Figura 4).
Alguns dos dados de sobrevivência destas plantas no campo integram o terceiro artigo
desta tese. Porém, de maneira geral, um maior tempo de acompanhamento é necessário
para que sejam alcançadas conclusões sobre a adaptação destes indivíduos em diferentes
condições de campo.
Figura 4. Indivíduo com 2,5 anos, crescendo em condições de campo. (Foto: M. F. Goulart)
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Estrutura da tese
Esta tese é composta por três capítulos redigidos em formato de artigo científico,
em inglês. Os três artigos contrastam a biologia de populações do vinhático de
diferentes biomas, Cerrado e Mata Atlântica, e de regiões ecotonais. O primeiro deles
tem como hipótese principal a existência de ecótipos de savana e floresta nesta espécie.
Esta hipótese foi confirmada com a avaliação do crescimento dos indivíduos ao longo
de seis meses iniciais. O segundo artigo dá ênfase na resposta morfológica e fisiológica
de indivíduos com seis meses de idade submetidos á diferentes tratamentos de luz. Este
artigo tem como hipótese principal a de que os diferentes ecótipos apresentam
diferenças nos níveis de plasticidade fenotípica em resposta a luz. O terceiro artigo
apresenta dados sobre a resposta dos indivíduos com seis meses de idade ao solo de
Mata Atlântica e ao solo de Cerrado. Novamente, respostas morfológicas, fisiológicas e
plasticidade fenotípica são avaliadas, bem como dados de sobrevivência dos indivíduos
em condições de campo. Este artigo tem como hipótese principal a de que os diferentes
ecótipos apresentam diferenças nos níveis de plasticidade fenotípica em resposta ao
solo. A abordagem de cada artigo está resumida na Tabela 1.
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Tabela 1. Resumo das abordagens dos três artigos que compõem esta tese.
Título dos artigos
Evidences for local adaptations from early developmental responses to light and soil fertility in the tropical tree
Plathymenia reticulata along a forest-savanna boundary
To which extent phenotypic plasticity in response to light
is involved in the ecotypic differentiation of a tree
species from savanna and forest habitats?
How important is soil fertility in driving
ecotypic differentiation of a tropical tree species
from savanna and forest habitats?
Dados analisados:
Morfológico não destrutivo
Morfológico destrutivo
Fotossíntese
Pigmentos foliares
Plasticidade fenotípica
Sobrevivência no campo
Idade dos indivíduos analisados:
de 1 a 7 meses (crescimento)
6 meses
32 meses*
Experimentos desenvolvidos:
Tratamentos de luz**
Tratamentos de solo**
Sobrevivência no campo
* indivíduos crescidos em viveiro por 12 meses e em condições de campo por 20 meses;
** experimento em viveiro
19
Referências bibliográficas
Goulart MF. 2004. Variação morfológica e na fenologia de Plathymenia (Leguminosae-
Mimosoideae) em áreas de Cerrado, Mata Atlântica e de transição entre biomas, no estado
de Minas Gerais, Brasil. Dissertação de Mestrado. Universidade Federal de Minas Gerais.
Goulart MF, Lemos Filho JP, Lovato MB. 2005. Phenological variation within and among
populations of Plathymenia reticulata in Brazilian Cerrado, Atlantic Forest and transitional
sites. Annals of Botany 96: 445-455.
Goulart MF, Lemos Filho JP, Lovato MB. 2006. Variability in fruit and seed morphology
among and within populations of Plathymenia (Leguminosae - Mimosoideae) in areas of the
Cerrado, the Atlantic Forest, and transitional sites. Plant Biology 8: 112-119.
Heringer E P. 1956. O gênero Plathymenia. Anais da Sociedade Botânica do Brasil 7: 55-64
Lacerda DR, Acedo MDP, Lemos Filho JP, Lovato MB. 2002. Molecular differentiation of two
vicariant neotropical tree species, Plathymenia foliolosa and P. reticulata (Mimosoideae),
inferred using RAPD markers. Plant Systematic and Evolution 235: 67-77.
Poorter H, Garnier E. 2007. Ecological significance of inherent variation in relative growth rate
and its components. In: Functional Plant Ecology (Pugnaire FI, Valladares F, eds). CRC
Press – Taylor and Francis Group: New York.
Ridley M. 2004. Evolution. Blackwell Publishing: New York.
Silva Júnior MC. 2005. Cem árvores do Cerrado: guia de campo. Rede de sementes do Cerrado:
Brasília.
Valladares F, Martinéz-Ferri E, Balaguer L, Peréz-Corona E, Manrique E. 2000a. Low leaf-
level response to light and nutrients in Mediterranean evergreen oaks: a conservative
resource-use strategy? New Phytologist 148: 79-91.
Valladares F, Wright JS, Lasso E, Kitajima K, Pearcy RW. 2000b. Plastic phenotypic response
to light of 16 congeneric shrubs from a Panamanian rainforest. Ecology 81(7): 1925-1936.
Warwick MC, Lewis GP. 2003. Revision of Plathymenia (Leguminosae – Mimosoideae).
Edinburgh Journal of Botany 60: 111-119.
Westoby M. 2007. Generalization in functional plant ecology: the species-sampling problem,
plant ecology strategy schemes and phylogeny. In: Functional Plant Ecology (Pugnaire FI,
Valladares F, eds). CRC Press – Taylor and Francis Group: New York.
20
CAPÍTULO 1:
Evidences for local adaptations from early developmental responses
to light and soil fertility in the tropical tree Plathymenia reticulata in a
forest-savanna boundary
21
Introduction
Plant populations occurring in contrasting habitats may evolve in two different
ways: populations may specialize to a fraction of the environment or may generalize
some kind of adaptation to a broad range of the environment (Bazzaz 1996). The
specialization strategy is related to the selection of distinct, genetically-controlled
character states in different populations, giving rise to ecotypes (Nagy and Rice 1997).
Ecotypes are likely to be observed in species that colonize contrasting altitudinal,
moisture, salinity, light and nutrients habitats (Hogan 1996). In the tropics, boundaries
between forest and savanna vegetations may be characterized by adjacent contrasting
habitats concerning several of those environmental characteristics.
In tropical regions, forests usually dominate sites of greater nutrient and water
availability, while savannas are more associated to poor, deep and well drained soils,
seasonal climate and the effect of intermittent fires (Furley 1992, Haridasan 1992). For
plants in forest, light is the major limiting resource for growth, survival and
reproduction (Chazdon et al. 1996, Pearcy 2007), while at savannas, nutrient and water
are much more limiting (as reviewed by Jackson et al. 1999). Because of these major
environmental differences, forest and savanna plant species typically conform very
distinct functional types, differing in physiological, morphological and life history
attributes (Hoffmann et al. 2005).
In the present work we approached the microevolution of a tropical tree by
focusing on early-life adaptations of different populations to forest and savanna
environmental conditions. Considering that evolutionary changes begin with a shift of
gene frequencies in populations (Ridley 2004), the microevolution perspective is of
great importance to understand how populations evolve in forest and savanna ecotones.
22
This issue is of great importance as boundaries between these vegetation types are
highly dynamic and, although under present climatic conditions forests are in a phase of
natural expansion over savannas, human activities, especially fires, are strongly
changing this process (Favier et al. 2004).
In South America, a corridor of xeric savanna vegetation runs between the two
main areas of moist tropical forest: the Amazonian Forest in the northwest and the
Atlantic Forest in the east and southeast (Oliveira-Filho and Ratter 2002). In central
Brazil, the corridor is dominated by the Cerrado biome which comprises widely varying
physiognomic types of vegetation that ranges from treeless grasslands to dense
woodlands. The most common physiognomy is named Cerrado sensu stricto (opposed
to Cerrado sensu lato, which is a generic term for all physiognomies of the biome) and
originally occupied more than 65% of the biome (Haridasan 1992). It is a xeromorphic
savanna characterized by a community of trees and shrubs, usually about 2-8 m in
height with contorted trunks, thick corky bark, sclerophyllous leaves and crown cover
of 10-60%, below which there is a well developed grassy ground layer (Ratter et al.
1997). The Cerrado forms an extent boundary with the Atlantic Forest biome, a typical
tropical forest, showing a more mesic environment. Although physiognomically very
different, the biomes may occur under the same latitude, under the same major climatic
condition and show some floristic similarity (Oliveira-Filho and Fontes 2000, Oliveira-
Filho and Ratter 2002).
In the present work we tested the hypothesis that the differentiation of ecotypes
adapted to forest or savanna conditions is involved in the colonization of Atlantic Forest
and Cerrado by a single species. Further, we hypothesized that the ecotypes should
show similar traits to those reported for forest and savanna species, although the
differences in traits across populations should be less contrasting than when comparing
23
different species. We also hypothesized that populations from ecotonal areas between
forest and savanna should show traits similar to populations on their respective core
area.
In order to test the hypotheses, we assumed that earlier stages of life cycle are
the phases at which selective pressures are highest (Reich et al. 2003, Silvera et al.
2003). We predicted that habitat-based selection should lead to different set of traits in
seedlings and saplings from populations from different habitats. These hypotheses were
investigated in populations of the woody species Plathymenia reticulata (Leguminosae,
Mimosoideae) located in core sites of each biome and in ecotonal area between them.
Individuals were grown in a common garden and submitted to different light and soil
treatments mimicking the range of conditions experienced in the natural habitats.
Materials and methods
Studied tree species
Plathymenia reticulata, known as “vinhático”, can be commonly found in both
Cerrado and Atlantic Forest. Originally, the different populations were considered
vicariant species, P. reticulata and P. foliolosa, occurring respectively at Cerrado and
Atlantic Forest (Mendes and Paviani 1997). In 2002 the possibility of gene flow
between Plathymenia from Cerrado and Atlantic Forest was pointed out by a molecular
markers study (Lacerda et al. 2002). In the following year, a taxonomic revision of this
genus was made and only P. reticulata was recognized as taxonomic characters occur in
a continuum or there is no geographic correlation (Warwick and Lewis 2003). So far, no
ecotypes or any infra specific taxa is recognized for this species (Warwick and Lewis
2003).
24
Studied populations, seeds collection and germination
We evaluated four populations of P. reticulata from Minas Gerais state,
southeast Brazil. Two populations are characterized as the Atlantic Forest semi-
deciduous physiognomy and in them individuals of P. reticulata reach 20 m in height.
The first population is located in the biome core area (19°45’S 43°31’W) and the other
one is located in its periphery, in an ecotone with the Cerrado (19°56’S 46°56’W). The
two other evaluated populations are in the Cerrado biome, both characterized as Cerrado
stricto sensu physiognomy and in them P. reticulata trees are shorter and tortuously
branched. One Cerrado population is located in the core area of the biome (18°43’S
45°03’W) and the other one in its periphery, in an ecotone with the Atlantic Forest
(19°49’S 43°48’W). The approximate location of each population are shown in Figure
1.
AmazoniaForest
Cerrado
Caatinga
AtlanticForest0 1000 Km Minas Gerais state
0 160 Km
Cerrado
Cerrado in ecotoneForest in ecotone
Forest
AmazoniaForest
Cerrado
Caatinga
AtlanticForest0 1000 Km Minas Gerais state
0 160 Km
Cerrado
Cerrado in ecotoneForest in ecotone
Forest
Figure 1. Brazil’s and Minas Gerais state’s main biomes, with the approximate
location of the studied populations of P. reticulata.
25
Seeds were collected randomly as mixed samples from 10 individuals from each
population in September 2004. In late December 2004, seeds were submitted to
mechanical scarification and germination was conducted at 28oC in darkness as in
Lacerda et al. (2004). After six days of germination, seedlings were planted in pots and
placed in a nursery at a site characterized as ecotone between the two biomes (19°56’S
46°56’W). Most individuals were submitted to a non destructive experiment. By being
evaluated only by non destructive measurements, these individuals could be later
transferred to field conditions as part of a long term experiment. Fewer individuals were
submitted to a destructive experiment due to low seed number.
Non destructive experiment
A total of 120 seedlings from each one of four populations were planted in
numbered pots (18 cm of diameter and 32 cm of depth). Half of the pots were filled
with soil collected from Atlantic Forest and another half with soil from Cerrado.
Chemical analyses of soils are shown in Table 1 and revealed a higher organic matter
and nutrients content in soil from Atlantic Forest and higher levels of exchangeable
aluminium in Cerrado, a typical pattern found while comparing soil between these
biomes (Ruggiero et al. 2002).
Seedlings were equally and randomly arranged in four growing houses (1 x 8
m), each of them representing a different light environment: sun and three levels of
shade, produced with the use of layers of neutral shade cloth. The shade cloths were
supported by wood frames and stood 1 m above the tables and covered also its sides.
Measures of photosynthetic photon flux density were performed in three locations in
each house during a clear summer day, with quantum sensors (Licor, LI-189) at 30
minutes interval. These measures showed that the houses provided 50.3, 26.5, 18.2 and
26
11.0 mol.m-2.day-1, respectively 100%, 53%, 36% and 22% of full sunlight. The blocks
were established to test for homogeneity of irradiance in each light treatment, however
analysis of variance (ANOVA) showed the block effect on the response variables
studied was not significant.
Table 1. Chemical analysis of soil samples collected in a Cerrado location and in a
Atlantic Forest, used as different soil treatments in the nursery experiment (V = base
saturation, m = aluminum saturation).
Soil treatments
Chemical parameters Cerrado Atlantic Forest
pH in water 5.2 5.6
Organic matter (g/Kg) 18.0 28.4
N (g/Kg) 1.0 1.4
C (g/Kg) 1.0 1.4
P (mg/Kg) < 1 2.2
K (mg/Kg) 48 128
Al 3+ (cmolc/Kg) 0.86 0.27
Ca 2+ (cmolc/Kg) 0.36 1.65
Mg 2+ (cmolc/Kg) 0.13 0.68
V% 17.01 44.01
m% 58.39 9.21
After 30 days of growth (late January 2005), each individual had the cotyledon
area estimated by doubling the multiplication of the maximum length and maximum
width of a single cotyledon. Cotyledon retention time was estimated by evaluating
presence or absence of cotyledon in each individual at 30, 60 and 90 days of growth.
Morphology of individuals was evaluated at 30 day interval during 7 months (from
January to July 2005). With the use of a digital paquimeter (0.01 mm precision) and
27
common rule, plants total height (cm) and base diameter (mm) were measured and
slenderness index estimated as height / base diameter.
Destructive experiment
A total of 60 seedlings from each population were used, excluding Cerrado in
transition population due to low seed number. Seedlings were planted in numbered pots
(25 cm of diameter and 40 cm of depth), filled with 3:1 nutrient enriched peat and sand
mixture. Growth responses to two light treatments were evaluated during six months
(from January to June 2005). For each population, 30 seedlings were placed in full
sunlight growing house and the other 30 in 22% of full sunlight growing house.
At 30 day interval, five individuals from each population at each light treatment
were harvest. Seedlings were separated into leaves, stems and roots and dry mass of
each fraction was weighted after three days at 70oC. Leaf area was measured on a
flatbed scanner with computer software (Easy Quantify), before drying. We calculated
the total dry mass, shoot:root ratio (shoot dry mass / root dry mass), specific leaf area
(SLA = leaf area / leaf dry mass) and leaf area ratio (LAR = leaf area / total dry mass).
We also calculated monthly relative growth rate by the paring method (Hoffmann and
Poorter 2002) where 1212 /)]ln()[ln( ttMMRGR −−= being M1 and M2 plant dry
masses at times t1 and t2 and net assimilation rate as
)ln)(ln/()( 121212 AAttMMNAR +−−= being M1 and M2 plant dry masses and A1
and A2 leaf area and at times t1 and t2.
28
Analysis of data
All data were logarithmically transformed and the assumptions of normality and
homoscedasticity were met. Concerning non-destructive experiment, one-way ANOVA
followed by post hoc Tukey test were made in order to compare cotyledon’s area among
P. reticulata populations. Comparisons of height and slenderness index among
populations were made for each combination of soil and light treatment separately.
Repeated measures ANOVAs were conducted using day of measure as within subject
variable (Potvin et al. 1990) followed by post hoc Tukey tests to rank populations
according to mean traits values at each survey.
For traits obtained in the destructive experiment, ANOVAs were conducted
considering as source of variance: population, light treatment, day of measure and the
interactions population x light, population x day, light x day and population x light x
day. For each trait, post hoc Tukey tests were made to rank populations being done
separately for each light treatment at each survey.
Results
The duration of the seedling phase (estimated by the maintenance of cotyledons)
differed among populations. Independently of light and soil treatments, individuals from
Atlantic Forest core and ecotone had already lost the cotyledons sixty days after
germination while the majority of the individuals from Cerrado core and ecotone only
lost the cotyledons one month later (Figure 1). The area of the cotyledon did not show
significant differences among populations (F=2.50; p=0.07).
29
Figure 2. Percentage of individuals of P. reticulata from four different populations (Cerrado, Cerrado
in ecotone, Atlantic Forest in ecotone and Atlantic Forest) with cotyledons present at different ages.
Concerning non destructive evaluations, populations of P. reticulata showed
several differences on the initial growth phase. In relation to height, differences among
populations tended to increased over time in all light and soil treatments, especially
once cotyledons were lost. Atlantic Forest core population showed a tendency towards
higher values than Cerrado core and frequently populations from ecotonal region
showed intermediated values. Overall differences were stronger in plants submitted to
lower irradiance and to Atlantic Forest soil treatment. Under these conditions, in the last
survey, plants from Atlantic Forest core area showed almost three times the mean height
than individuals from Cerrado core population (Table 2 and Figure 3).
0
20
40
60
80
100
30 60 90
Days
% in
divi
dual
s with
cot
yled
ons
Cerrado Cerrado in ecotone
Forest in ecotone Forest
0
20
40
60
80
100
30 60 90
Days
% in
divi
dual
s with
cot
yled
ons
Cerrado Cerrado in ecotone
Forest in ecotone Forest
Slenderness indexes were always higher in Atlantic Forest core population when
compared to Cerrado, showing that individuals from Atlantic Forest have a tendency
towards investing proportionately more in height than in stem diameter. In similar way
to height, frequently, populations from ecotonal region showed intermediated values for
slenderness index, especially Atlantic Forest in ecotone. In a general way, the
slenderness indexes were higher in individuals submitted to lower irradiance treatments
and decreased overtime (Table 2 and Figure 4).
30
Table 2. Analysis of variance of repeated measures of height and slenderness index recorded P. reticulata
from four populations (Cerrado, Cerrado in ecotone, Atlantic Forest in ecotone and Atlantic Forest)
submitted to different combinations of soil (soil from Cerrado and from Atlantic Forest) and light (100,
53, 36 and 22% of full sunlight) treatments. Day of measurement was considered as a repeated variable.
DF stands for degrees of freedom. Variance ratios (F values) are reported with associated level of
significance (* = p<0.05; ** = p<0.01; *** = p<0.001; ns = not significant).
Soil treatment Light treatment Source of variation DF Height Slenderness index
Cerrado 100% Population 3 4.72 * 10.76 ***
Day 6 46.75 *** 10.61 ***
Day x population 18 2.43 ** 1.78 *
Cerrado 53% Population 3 30.65 *** 54.22 ***
Day 5 73.17 *** 47.11 ***
Day x population 15 5.65 *** ns
Cerrado 36% Population 3 11.70 *** 37.53 ***
Day 6 28.60 *** 62.16 ***
Day x population 18 4.51 *** 2.02 **
Cerrado 22% Population 3 11.72 *** 37.53 ***
Day 6 91.84 *** 62.15 ***
Day x population 18 11.04 *** 2.02 **
Forest 100% Population 3 ns 13.02 **
Day 6 65.66 *** 13.30 ***
Day x population 18 2.54 ** ns
Forest 53% Population 3 7.15 *** 20.21 ***
Day 5 130.21 *** 80.18 ***
Day x population 15 4.00 *** 2.12 **
Forest 36% Population 3 3.16 * 6.53 **
Day 6 50.86 *** 17.92 ***
Day x population 18 3.16 *** ns
Forest 22% Population 3 12.00 *** 15.53 ***
Day 6 173.09 *** 68.71 ***
Day x population 18 18.77 *** 2.88 ***
31
Figure 3. Mean values of height obtained in individuals of P. reticulata from four different populations
(Cerrado, Cerrado in ecotone, Atlantic Forest in ecotone and Atlantic Forest) during early phase of
growth. Individuals where submitted to different combinations of light treatments (100, 53, 36 and 22%
of full sunlight) and soil treatments (Cerrado soil and Atlantic Forest soil). At each time interval, letters
indicate significant comparisons among populations by Tukey test with p<0.05, alphabetical order
corresponds to ranking mean value and absence of letters indicates no significant differences among
populations.
0
4080
120160
200240
280320
360
30 60 90 120 150 180 210
Days
Hei
ght (
mm
)
0
4080
120
160200
240
280320
360
Hei
ght (
mm
)
0
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160200
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320
360
Hei
ght (
mm
)
0
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80
120
160
200
240
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320
360
Hei
ght (
mm
)
30 60 90 120 150 180 210
Days
Cerrado soil Atlantic Forest soil22
% o
fful
lsun
light
36%
off
ulls
unlig
ht53
% o
fful
lsun
light
100%
off
ulls
unlig
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a
bb
ab
Cerrado Cerrado in ecotone Forest in ecotone Forest
a
b
b
ab
aab
bb
a
ab
bb
a
b
bb
0
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200240
280320
360
30 60 90 120 150 180 210
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Hei
ght (
mm
)
0
4080
120
160200
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280320
360
Hei
ght (
mm
)
0
40
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160200
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320
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Hei
ght (
mm
)
0
40
80
120
160
200
240
280
320
360
Hei
ght (
mm
)
30 60 90 120 150 180 210
Days
Cerrado soil Atlantic Forest soil22
% o
fful
lsun
light
36%
off
ulls
unlig
ht53
% o
fful
lsun
light
100%
off
ulls
unlig
ht
a
bb
ab
Cerrado Cerrado in ecotone Forest in ecotone Forest
a
b
b
ab
aab
bb
a
ab
bb
a
b
bb
32
0123456789
10
30 60 90 120 150 180 210
Days
0123456789
10
0123456789
10
0123456789
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30 60 90 120 150 180 210
Days
Cerrado soil Atlantic Forest soil
fsu
n6
ull
light
% o
fsu
t10
n
Slen
dern
essi
ndex
(cm
.mm
-1)
Sl
ende
rnes
sind
ex(c
m.m
m -1
)
S
lend
erne
ssin
dex
(cm
.mm
-1)
Sle
nder
ness
inde
x(c
m.m
m -1
)
Figure 4. Mean values of slenderness indexes obtained in individuals of P. reticulata from four different
populations (Cerrado, Cerrado in ecotone, Atlantic Forest in ecotone and Atlantic Forest) during early
phase of growth. Individuals where submitted to different combinations of light treatments (100, 53, 36
and 22% of full sunlight) and soil treatments (Cerrado soil and Atlantic Forest soil). At each time interval,
letters indicate significant comparisons among populations by Tukey test with p<0.05, alphabetical order
corresponds to ranking mean value and absence of letters indicates no significant differences among
populations.
22%
ofu
lllig
ht3
% o
ffsu
n53
full
nlig
h0%
off
ulls
ulig
ht
a
a
ab ab
b bbb
b
ab
a
b
b
Cerrado Cerrado in ecotone Forest in ecotone Forest
a
bb
0123456789
10
30 60 90 120 150 180 210
Days
0123456789
10
0123456789
10
0123456789
10
30 60 90 120 150 180 210
Days
Cerrado soil Atlantic Forest soil
fsu
n6
ull
light
% o
fsu
t10
n
Slen
dern
essi
ndex
(cm
.mm
-1)
Sl
ende
rnes
sind
ex(c
m.m
m -1
)
S
lend
erne
ssin
dex
(cm
.mm
-1)
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nder
ness
inde
x(c
m.m
m -1
)
a
a
ab ab
b bbb
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ab
a
b
b
Cerrado Cerrado in ecotone Forest in ecotone Forest
a
bb
light
full
su0%
of
nlig
hfu
ll53
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% o
ff3
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full
22%
o
33
Concerning destructive evaluations, individuals’ dry mass increased over time
with higher values in plants submitted to full sunlight treatment. In the two final surveys
differences were found among populations with significant higher values in Atlantic
Forest core and ecotone. At the age of 210 days, plants from Atlantic Forest core area
showed around twice the mean dry mass than individuals from Cerrado, at both light
treatments (Table 3 and Figure 5).
Significant differences in shoot:root ratio were observed among populations in
plants submitted to shade treatment, while no significant differences were found among
populations in full sunlight treatment. Under shade, higher values of shoot:root were
found in Atlantic Forest core and ecotone, showing that individuals in these populations
allocated proportionately more mass in shoots than roots, in comparison to Cerrado
(Table 3 and Figure 5).
Concerning leaf area ratio and specific leaf area, a general tendency of a
decrease in values over time was observed, as well as of higher values in plants
submitted to shade. Comparisons among populations showed significant differences in
leaf area ratio at several ages at both light treatments, with higher values in Atlantic
Forest core and ecotone. No significant differences among populations in specific leaf
area were observed (Table 3 and Figure 4).
Relative growth rates showed a general tendency to increase overtime and no
significant differences between light treatments were observed. Comparisons among
populations showed that, most frequently, Atlantic Forest core and ecotone had higher
values for relative growth rates than Cerrado population. Net assimilation rate showed a
general tendency to increase overtime and significantly higher values were found in
plants submitted to full sunlight treatment. No significant differences among
populations were found (Table 3 and Figure 5).
34
Figure 5. Mean values of shoot:root ratio, dry mass, leaf area ratio (LAR), specific leaf area (SLA),
obtained individuals of P. reticulata from three different populations (Cerrado, Atlantic Forest in ecotone
and Atlantic Forest) during early phase of growth. Individuals where submitted to two light treatments
(100 and 22% of full sunlight). At each time interval, letters indicate significant comparisons among
populations by Tukey test with alphabetical order corresponding to ranking mean value and absence of
letters indicating no significant differences among populations.
02468
1012
Dry
mas
s (g)
aabb
a
ab
b
aab
b
a
a
b
0
100
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300
400
500
30 60 90 120 150 180
SLA
(cm
2 . g
-1)
30 60 90 120 150 180
050
100150200250300
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(cm
2 .g -1
)
Days Days
100% of full sunlight 22% of full sunlight
aab
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(cm
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-1)
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)
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aab
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01234567
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b
a
c
b
35
-0.01
0.00
0.01
0.02
0.03
0.04
1 2 3 4 5
th -1
)
Figure 6. Mean values of relative growth rates (RGR) and net assimilation rates (NAR) obtained individuals of
P. reticulata from three different populations (Cerrado, Forest in ecotone and Forest) during early phase of
growth. Individuals where submitted to two light treatments (100 and 22% of full sunlight). At each time
interval, letters indicate significant comparisons among populations by Tukey test with alphabetical order
corresponding to ranking mean value and absence of letters indicating no significant differences among
populations.
Table 3. Analysis of variance of dry mass, shoot:root ratio, specific leaf area (SLA), leaf area ratio (LAR),
relative growth rate (RGR) and net assimilation rate (NAR) recorded on P. reticulata from three different
populations (Cerrado, Atlantic Forest in ecotone and Atlantic Forest) and submitted to two light treatments
(100 and 22% of full sunlight). DF stands for degrees of freedom. Variance ratios (F values) are reported with
associated level of significance (* = p<0.05; ** = p<0.01; *** = p<0.001; ns = not significant).
Source of variation DF Dry mass Shoot:root SLA LAR DF RGR NAR
Population 2 8.45*** 10.65*** ns 15.42*** 2 5.71** ns
Light treatment 1 26.87*** ns 47.20*** 10.41*** 1 ns 29.16***
Day of measure 5 65.78*** 16.72*** 15.92*** 30.71*** 4 33.83*** 21.51***
Population*light 2 ns ns ns ns 2 3.62* ns
Population*day 10 3.38*** ns ns 2.67** 8 12.40*** 4.34**
Light*day 5 14.15*** ns ns ns 4 16.53*** 11.34***
Population*light*day 10 ns ns ns ns 8 9.34*** 3.08**
NA
R (g
.on
cm2 .m
1 2 3 4 5
-1.0
0.0
1.0
2.0
3.0
RGR
(g.g
.mon
th -1
)
60 90 120 150 180 60 90 120 150 180
Days Days
100% of full sunlight 22% of full sunlight
a
b
a
b
bb
a
c
b
a
b
a
b
b
a
aa b
Cerrado Forest in ecotone Forest
-0.01
0.00
0.01
0.02
0.03
0.04
1 2 3 4 5
(g.
onth
-1)
cm2 .m
NA
R
1 2 3 4 5
-1.0
0.0
1.0
2.0
3.0
RGR
(g.g
.mon
th -1
)
60 90 120 150 180 60 90 120 150 180
Days Days
100% of full sunlight 22% of full sunlight
a
b
a
b
bb
a
c
b
a
b
a
b
b
a
aa b
Cerrado Forest in ecotone Forest
36
Discussion
Differences among populations
Individuals from all populations invested in growing taller by elongating stems
when exposed to shade, a known shade escape response related to the shade avoidance
syndrome (Ballaré et al. 1997, Smith and Whitelan 1997, Kurepin et al. 2006). These
shade responses, however, were much more evident in Atlantic Forest than in Cerrado
populations. In general, forest habitats are more limited by light than by any other
resource (Chazdon et al. 1996, Pearcy 2007) and our results show that Atlantic Forest
populations of P. reticulata are more adapted to cope with it than the Cerrado
populations.
Among populations of P. reticulata, no significant differences were observed in
SLA although higher LAR was found in Atlantic Forest. While comparing congeneric
species of Cerrado and Forests, Hoffman and Franco (2003) reported a general tendency
of higher values of SLA and LAR in species from Forests. Some authors have pointed
to higher values of SLA and LAR in shade plants as a strategy to maximize light
capture (Poorter 1999). On the other hand, at Cerrado, lower values may be
advantageous as plants from dryer habitats reduce the transpiration surface area and
minimize the water loss by reducing SLA and LAR (Dudley 1996, Gonzalez-Astorga et
al. 2003, Silvera et al. 2003).
During the early phase of growth, individuals acquire most of necessary
resources from seed reserves, being seedling mass highly dependent on seed mass and
less on external resource availability (Kitajima 1996). Individuals from different
populations of P. reticulata have similar quantities of seed reserve as there are no
significant differences among populations concerning seed mass (Goulart et al. 2006) or
cotyledons area (present study). Individuals from Atlantic Forest, however, consumed
37
the reserves faster, showing a lower retention time of cotyledons. In spite of the faster
reserve consumption, plants from Atlantic Forest populations did not significantly
outgrew Cerrado ones at early age, during seedling phase. Significantly higher mass
accumulation in plants from Atlantic Forest populations were only observed after 150
days of growth.
Higher biomass accumulation and higher relative growth rate in individuals from
Atlantic Forest core and ecotone could be related to a higher competitive ability in these
populations when compared to Cerrado. Higher growth rates in individuals from
Atlantic Forest may be advantageous by promoting quick occupation of the available
space within the crowded vegetation (Poorter and Garnier 2007). While at Cerrado,
lower growth is related to stress resistant syndrome, typical of plants specialist to low-
resource environment (Chapin et al. 1993).
Besides lower growth rate, lower shoot:root ratio was another characteristic
exhibited by P. reticulata from Cerrado that is frequently related to stress resistant
syndrome (Chapin et al. 1993). Differences in shoot:root ratio has been considered the
most striking difference between Cerrado and Atlantic Forest species (Hoffmann and
Franco 2003). Higher investment in aerial growth in Atlantic Forest plants should be
related to shade avoidance while greater investment in roots in Cerrado plants is
interpreted as a strategy to capture nutrient and water (Hoffmann and Franco 2003).
Ecotypic differentiation
The differences found among populations of P. reticulata from Atlantic Forest,
Cerrado and ecotonal region in individuals growing in our common garden experiment
suggest the existence of genetically based differences among these populations. As
discussed above, these differences occur in an adaptive direction, suggesting that natural
38
selection is an important force promoting genetic divergences between the populations.
This leads us to suggest that there are different ecotypes from Cerrado and Atlantic
Forest in this species. The main differences among ecotypes are related to differences in
the shade avoidance syndrome and the competitive ability, two features that are more
expressed in the forest ecotype with the savanna ecotype being more characterized by
stress resistance traits.
This ecotypic differentiation could lead to niche partitioning among populations
(Schuler 1996). In nursery conditions, the savanna ecotype was able to grow under light
and soil conditions that simulate the real forest habitats. However, at natural conditions,
the savanna ecotype would probably be less successful in Atlantic Forest, since it is a
weaker competitor in a highly competitive environment. In spite of its higher
competitive ability, the forest ecotype would not perform well in Cerrado areas due to
its apparently limited capacity to tolerate stressful conditions. One possible advantage
of the stress resistance syndrome in Cerrado is that by growing slow, plants can
accumulate sugar and nutrients during favorable times, enabling them to grow when
resources are less available (Poorter and Garnier 2007). Indeed, the need to invest in
storage is observed in trees from Cerrado, which commonly invest in root carbohydrate
necessary for resprout after fire (Hoffmann et al. 2003).
Evolutionary trends in savanna-forest boundary
At the boundary between the biomes the populations showed intermediate values
for several studied traits. Considering that the studied boundary is characterized by a
mosaic of adjacent patches of Cerrado and Atlantic Forest physiognomically very
similar to their respective core habitat, it is not likely that the ecotone populations are
ecotypes adapted to intermediate environmental conditions. Alternatively, it is more
39
likely that the ecotone populations are characterized by hybrid individuals between
ecotypes. Hybridization is possible since there is similar flowering period between
populations (Goulart et al. 2005), moreover, evidence of gene flow has being pointed by
Lacerda et al. (2002) based on data from neutral genetic markers.
It is noteworthy that the putative hybrids, in spite of showing intermediate values
for several traits, are more similar to their respective core area ecotype than to the other
habitat ecotype. This pattern was also observed concerning phenological behavior
(Goulart et al. 2005) and fruit and seed morphology and dispersal potential (Goulart et
al. 2006) in this species. These differences can be taken as a suggestion for a strong
habitat-based selection in the patches of Atlantic Forest and Cerrado located in the
boundary between this biomes. In spite of the presence of gene flow among the
populations, natural selection act keeping adaptive differences at each patch.
Ecotypic differentiation is often envisioned as an early stage in the process of
speciation by adaptive radiation, which leads to a latter proliferation of species
accompanied by divergence in the kinds of resources exploited and the morphological
and physiological traits used to exploit the resources (Schuler 1996). Selection pressures
that remove hybrids are important in the speciation process (Schuler 1996) and when
ecotypes are highly dissimilar, hybrids can suffer negative selection due to outbreeding
depression (Hufford and Mazer 2003). Further investigation related to ecotypes survival
and reproduction is necessary to better understand evolutionary trends in this system,
however, the present work show no evidence of selection against possible hybrids
between ecotypes during early developmental phases.
40
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44
CAPÍTULO 2:
To which extent phenotypic plasticity in response to light
is involved in the ecotypic differentiation of a tree species from
savanna and forest habitats?
45
Introduction
Tropical environments include the most diverse plant communities on earth due
to a complexity of biotic and abiotic factors (Givnish 1999, Wright 2002). Among these
factors, spatial and temporal heterogeneity of light has been pointed as an important
mechanism to enable species coexistence, being related to niche partitioning among
plants (Poorter and Arets 2003, Balderrama and Chazdon 2005). Although less studied
and understood with this perspective, phenotypic plasticity in response to light could
also be important for the increase and maintenance of plant diversity (Valladares et al.
2000b). This may seem paradoxical since phenotypic plasticity enables a given
genotype to occupy different environments, so it is usually considered as a retarding
factor of evolutionary change (West-Eberhard 1989). However, plasticity can be a
diversifying factor in evolution since it is in itself a trait subject to natural selection and
can be the result of specialization to a fraction of the environmental heterogeneity
(West-Eberhard 1989, Lortie and Aarssen 1996, Valladares et al. 2000b, Ghalambor et
al. 2007).
Among the most biodiverse ecoregions in the world are the tropical Brazilian
Atlantic Forest and the Brazilian Cerrado, both considered priority hot spots for
conservation (Myers et al. 2000). The Brazilian Atlantic Forest is a typical tropical
forest with dense canopy and comparatively more mesic environmental conditions,
while the Cerrado biome ranges from grassland to woodland, typically being a
xeromorphic savanna named Cerrado sensu stricto. The Cerrado sensu stricto is
characterized by a community of trees and shrubs with a crown cover of 10-60% and a
well developed herbaceous layer (Ratter et al. 1997, Oliveira-Filho and Ratter 2002).
Although occurring in adjacent areas, the Atlantic Forest usually dominates sites of
greater nutrient and water availabilities, while the Cerrado biome is associated to poor,
46
deep and well drained soils (Furley 1992, Haridasan 1992). However, this major pattern
is not always clear and vegetation and soil are so intimately related that it is difficult to
identify cause and effect relationships (Ruggiero et al. 2002, Hoffmann et al. 2005).
From a plant perspective, light is the environmental factor that differ the most
between Atlantic Forest and Cerrado habitats. In the Atlantic Forest the leaf area index
(LAI) is around 5 (Paula and Lemos Filho 2001) and light is the most limiting resource
for plant growth, survival and reproduction (Chazdon et al. 1996). At Cerrado, LAI
values are around 1, with light being highly available in the understory (Miranda et al.
1997). Besides light intensity, Atlantic Forest and Cerrado also differ in light
heterogeneity. Although Cerrado is heterogeneous in light (Moreira 2000), in Atlantic
Forest light varies to a larger extent both spatially and temporally. At forests, light
environments range from deeply shaded understories to full sunlight tree fall gaps and
light variation among microsites may be 50-fold (Niiments 2007). Besides, forest
understories are highly dynamic environments with temporal dynamics spanning from
years (e.g. canopy opening by tree fall) to seconds, since the very low background
understory irradiation is randomly punctuated by short duration but very bright
sunflecks (Pearcy 2007).
Environmental differences in light availability have led to specific and
contrasting adaptations in species from Atlantic Forest and Cerrado (Hoffmann and
Franco 2003). In a previous work, we demonstrated that adaptations to each ecosystem
also occur at a microevolutionary scale, with significant differences among populations
within the same species (Chapter 1). This was studied in the woody species
Plathymenia reticulata (Leguminosae - Mimosoideae), which form distinct ecotypes
adapted to Atlantic Forest and Cerrado, developing traits related to the shade avoidance
47
syndrome and an enhanced competitive ability in the first and traits conferring stress
tolerance in the second (Chapter 1).
In this work we further investigate local adaptation in P. reticulata populations
hypothesizing that phenotypic plasticity in morphological and physiological traits in
response to light is involved in ecotypic differentiation in this species. We predicted that
Atlantic Forest ecotype should show higher phenotypic plasticity than the Cerrado
ecotype as an evolutionary response to the higher light heterogeneity experienced by
plants in the former ecosystem.
Material and methods
Studied populations
We evaluated four populations of P. reticulata from Minas Gerais state,
southeast Brazil, from which seeds were collected randomly as mixed samples from 10
individuals in September 2004. Two populations are characterized as the Atlantic Forest
semi-deciduous physiognomy being the first located in the biome core area (19°45’S
43°31’W) and the other one in its periphery, in an ecotonal area with the Cerrado
(19°56’S 46°56’W). The two other evaluated populations are in the Cerrado biome,
both characterized as Cerrado sensu stricto. One population is located in the core area of
the biome (18°43’S 45°03’W) and the other one in its periphery, in an ecotonal area
with the Atlantic Forest (19°49’S 43°48’W). Ecotones are environmentally very similar
to their respective core habitat, however, as there are some distinctive traits in
populations of P. reticulata located in ecotones (Lacerda et al. 2002, Goulart et al.
2005, Goulart et al. 2006, Chapter 1), they were here evaluated separately from core
areas. Populations characteristics are more detailed described in Chapter 1.
48
Experimental design
In late December 2004, seeds were submitted to mechanical scarification and
germination was conducted at 28oC in darkness as in Lacerda et al. (2004). After six
days of germination, seedlings were planted in a nursery and were submitted to either
non-destructive or destructive experiment.
In the non-destructive experiment a total of 120 seedlings from each population
were planted in numbered pots (18 cm of diameter and 32 cm of depth) filled with soil
collected in a Atlantic Forest site (pH=5.6; N=1.4 mg/kg; P=2.2 mg/kg; K=128 mg/kg;
Ca2+=1.65 cmolc/kg; Mg2+=0.68 cmolc/kg). Seedlings were equally and randomly
arranged in four growing houses (1 x 8 m), each of them representing a different light
environment: sun and three levels of shade, produced with the use of layers of neutral
shade cloth. The shade cloths were supported by wood frames and stood 1 m above the
tables and covered also its sides. Measures of temperature, relative humidity and
photosynthetic photon flux density were performed in three locations in each house
during an entire sunny summer day at 30-minutes interval. These measures showed that
the houses environment’ differed mainly in irradiance, providing 100%, 53%, 36% and
22% of full sunlight (Table 1). At each house, three blocks were established to test for
homogeneity of irradiance, however they showed no significant effect on the evaluated
traits.
For the destructive experiment, 20 individuals from each population, excluding
the Cerrado in ecotone due to low seed number, were planted in numbered pots (25 cm
of diameter and 40 cm of depth) filled with 3:1 nutrient-enriched peat and sand mixture.
Seedlings were equally and randomly arranged in the growing houses that provided
100% and 22% of full sunlight.
49
Table 1. Environmental conditions in the four growing houses, representing four different light
treatments. Data obtained in sunny summer day for every 30 minutes record, from 7 am to 6 pm. PPFD
stands for photosynthetic photon flux density.
Light treatments (percentage of full sunlight) Environmental variable
100 53 36 22
Daily PPFD (mol.m-2.day-1) 50.3 26.5 18.2 11.0
Maximum PPDF (µmol.m-2.s-1) 2521.0 1249.8 907.6 554.9
Mean PPDF (µmol.m-2.s-1) 1075.7 550.8 375.8 234.0
Minimum PPFD (µmol.m-2.s-1) 42.3 23.3 13.4 7.5
Maximum temperature (oC) 36.1 34.4 35.5 34.5
Mean temperature (oC) 31.5 31.3 31.3 30.3
Minimum temperature (oC) 19.6 19.2 19.2 19.3
Maximum relative humidity (%) 92.7 91.7 93.0 94.1
Mean relative humidity (%) 50.2 50.9 55.3 57.1
Minimum relative humidity (%) 31.5 30.5 36.2 41.3
Morphological and physiological measurements
Individuals in the non-destructive experiment were evaluated after six months of
growth, in June 2005. With the use of a digital paquimeter (0.01 mm precision) and
common ruler, height (cm) and base diameter (mm) were obtained and number of
internodes counted. Mean internode length (cm) was estimated as height / number of
intenodes, considering only individuals with a single bud, and slenderness index
(cm/mm) as height / base diameter.
Half of the individuals in the destructive experiment were harvested after one
month of growth (in January 2005) and the other half after six months of growth (June
50
2005). Plants were separated into leaves, stems and roots, dry mass of each fraction was
weighted after three days at 70oC. Leaf area was measured on a flatbed scanner with
computer software (Easy Quantify), before drying. The following measurements were
calculated for six month seedlings: total dry mass, shoot:root ratio (shoot dry mass / root
dry mass), specific leaf area (SLA = leaf area / leaf dry biomass) and leaf area ratio
(LAR = leaf area / total dry biomass). We also calculated the relative growth rate by the
paring method (Hoffmann and Poorter 2002) where 1212 /)]ln()[ln( ttMMRGR −−=
being M1 and M2 plant dry masses at times t1 (1 month) and t2 (6 months) and net
assimilation rate as )ln)(ln/()( 121212 AAttMMNAR +−−= being M1 and M2 plant
dry masses and A1 and A2 leaf area and at times t1 and t2 .
In vivo chlorophyll fluorescence traits were evaluated in six six-month-old
individuals from each population growing at full sunlight and also at 22% of full
sunlight treatments. Measurements were made with the use of a pulse amplitude
modulated photosynthesis yield analyzer (Mini-PAM, Walz, Germany). Potential
quantum yield of photosystem II was calculated as: mmmv FFFFF /)(/ 0−= where Fm
and F0 are the maximum and the minimum fluorescence respectively, measured in fully
developed leaves after 30 minutes of dark adaptation. Light saturation curves were
obtained using the light curve program of the instrument, and used to determinate
maximum apparent photosynthetic electron transport rate (ETRmax) and saturating
photosynthetically active photon flux density (PPFDsat), following Rascher et al.
(2000). Leaf pigments content were determined in the same individuals by grounding
leaf samples in 80% acetone. Absorbance in the supernatant was measured
spectrophotometrically at 470, 646 and 663 nm and pigment contents were determined
using equations described in Lichtenthaler and Wellburn (1983).
51
Analysis of data
In order to compare light treatments and populations, data were logarithmically
transformed and the assumptions of normality and homoscedasticity were met. Analyses
of variance (ANOVAs) considered as sources of variance light treatment, population
and also population x light treatment interaction. Whenever a factor did not show
significance, it was removed from the model and a new analysis was conducted. Post
hoc Tukey mean comparison tests were performed for population and for light
treatments, for all morphological and physiological traits.
For each population, plasticity was quantified under 22% and 100% of full
sunlight treatments, using the Relative Distance Phenotypic Index (RDPI) described by
Valladares et al. (2006). The relative distances (RD) among trait values for all pairs of
individuals of a given habitat grown in different light environment were determined as
, where j and j’ are individuals belonging to different
light environments i and i’. The RDPI ranges from 0 (no plasticity) to 1 (maximum
plasticity) and is obtained as
)/('' '''' ijjiijjiij xxjidRD +→=→
nxxjidRDPI ijjiij /))/(''( '' +→= ∑ where n is the total
number of RD. Comparisons of RDPIs among populations were made by ANOVAs and
post hoc Tukey test, or by Kruscal-Wallis and post hoc Holm test when non
parametrical distributions were found.
Results
A significant population x light treatment interaction was found for plant height
(Table 2). Height of plants in Cerrado core and in the ecotonal populations did not
significantly respond to the light treatments, while the Atlantic Forest core population
exhibited significant differences among light treatments, with plants in the darkest
shade treatment twice as taller as the ones in the full sunlight treatment (Figure 1A).
52
Plants from Atlantic Forest core and ecotone showed significantly higher height than
Cerrado core and ecotone. Differences were greatest in the darkest shade treatment,
where plants from Atlantic Forest core grew almost three times taller than plants from
the Cerrado core population (Figure 1A).
Table 2. Analyses of variance for morphological and physiological characters recorded in P. reticulata
from four populations and submitted to two or four light treatments. Variance ratios (F values) are
reported with associated level of significance (* = p<0.05; ** = p<0.01; ns = not significant).
Sources of variance Traits
Population Light treatment Population x light
Height (cm) 25.00 ** 3.12 * 2.28 *
Slenderness Index (cm.mm-1) 43.96 *** 7.99 *** ns
Internodes length (cm) 16.33 *** ns ns
Dry mass (g) 5.4 * 7.89 * ns
Shoot:root ratio 14.06 *** ns 5.57 *
SLA (cm2.g-1) ns 32.61 *** ns
LAR (cm2.g-1) ns 21.18 ** ns
RGR (g.g.month-1) 10.74 *** ns 4.79 *
NAR (g.cm2.month-1) ns 19.60 ** 4.91 **
Fv/Fm ns ns ns
ETRmax ns ns ns
PPFDsat ns ns ns
Chlorophylls (µm.mg-1) ns 57.88 *** ns
Carotenoids:chlorophylls ratio 8.53 *** 10.42 ** ns
Chlorophyll a:b ratio ns ns ns
53
0
5
10
15
20
25
30
35
40
Hei
ght (
cm)
100% 53% 36% 22%
Cerrado ForestForest in ecotone
Cerrado in ecotone
b ab a a
b ab ab b a ab ab ab c b bc b b a a
A)
0
5
10
15
20
25
30
35
40
Hei
ght (
cm)
100% 53% 36% 22%
Cerrado ForestForest in ecotone
Cerrado in ecotone
b ab a a
b ab ab b a ab ab ab c b bc b b a a
A)
0.00.51.01.52.02.53.03.54.04.5
1 2 3 4 5 6 7 8 9 10
Slen
dern
ess i
ndex
(cm
.mm
-1)
0.0
0.5
1.0
1.5
2.0
2.5
Inte
rnod
e le
nght
(cm
)
Fore
st
Fore
st in
eco
tone
Cer
rado
in e
coto
ne
Cer
rado
22%
36%
53%
100%
c c b a
b b ab a
b b a a
B)
0.00.51.01.52.02.53.03.54.04.5
1 2 3 4 5 6 7 8 9 10
Slen
dern
ess i
ndex
(cm
.mm
-1)
0.0
0.5
1.0
1.5
2.0
2.5
Inte
rnod
e le
nght
(cm
)
Fore
st
Fore
st in
eco
tone
Cer
rado
in e
coto
ne
Cer
rado
22%
36%
53%
100%
c c b a
b b ab a
b b a a
B)
Figure 1. Means ± standard error of morphological non-destructive traits obtained in six months saplings of P. reticulata from four populations (Cerrado, Cerrado in transition, Atlantic Forest in transition and Atlantic Forest) grown under four different light levels (100, 53, 36 and 22% of full sunlight). In A) there is significant population x light treatment interaction, letters inside bars indicate differences among populations within each light treatment while letters above bars indicate differences among light treatment within each population. In B) there is no significant population x light treatment interaction, letters above white bars indicate comparisons among population while letters above black bars indicate comparisons among light treatments. Alphabetical order of letters corresponds with ranking mean value and absence of letters indicates no significant differences considering 95% confidence interval.
54
No significant population x light treatment interaction was found for slenderness
index and internode length (Table 2). Populations significantly differed in slenderness
index and internode length with individuals from Atlantic Forest core showing mean
values almost twice as those from Cerrado core, while ecotonal populations showed
intermediate mean values (Figure 1B). Plants from all populations responded to light
treatments by significantly increasing the slenderness index in the shade, although not
by elongating the internodes since no differences were found among light treatments for
internode length (Figure 1B).
Dry mass, SLA and LAR did not show significant population x light treatment
interaction (Table 2). Populations significantly differed in dry mass but not in SLA and
LAR. Atlantic Forest core and ecotone individuals accumulated more than two times the
dry biomass accumulated by Cerrado ones (Figure 2B). Plants from all populations
showed significantly higher dry mass in full sunlight and significantly higher SLA and
LAR under shade (Figure 2B).
Shoot:root ratio, RGR and NAR showed significant population x light treatment
interaction (Table 2). Among the three evaluated populations, only Atlantic Forest core
showed significant differences in shoot:root ratio among light treatments. In this
population, plants growing in the shade showed mean shoot:root ratio two times bigger
than plants in full sunlight. No significant differences among populations were found in
the full sunlight treatment, while in the shade treatment, the Atlantic Forest core
population showed a significantly higher shoot:root ratio (Figure 2A). Significant
differences between light treatments in RGR were only found for Atlantic Forest
individuals, with higher mean values in full sunlight treatment. Comparisons among
populations showed higher RGR in Atlantic Forest core and ecotone at both light
treatments (Figure 2A). No significant differences among populations were found for
NAR, which significantly differed among light treatments for Cerrado and Atlantic
Forest core populations. In these populations individuals growing in full sunlight
showed higher NAR than those growing in shade (Figure 2A).
55
Figure 2. Means ± standard error of morphological destructive traits obtained in six months saplings of P.
reticulata from three populations (Cerrado, Atlantic Forest in transition and Atlantic Forest) grown under
two different light levels (100 and 22% of full sunlight). In A) there is significant population x light
treatment interaction, letters inside bars indicate differences among population within each light treatment
while asterisk indicate differences between light treatment within each population. In B) there is no
significant population x light treatment, letters above white bars indicate comparisons among populations
while asterisks above black bars indicate comparisons among light treatments. Alphabetical order of
letters corresponds with ranking mean value and absence of letters or asterisks indicates no significant
differences considering 95% confidence interval.
0
2
4
6
8
10
12
1 2 3 4 5 6 7
Dry
wei
ght (
g)
0
50
100
150
200
250
300
350
1 2 3 4 5 6 7SL
A (c
m2 .g
-1)
0
20
40
60
80
100
120
140
160LA
R (c
m2 .g
-1)
Fore
st
Fore
st in
eco
tone
Cer
rado
22%
100%
b a a
*
*
*
0123
hoot
:r
4567
Soo
t rat
io
0.600.70
0.800.901.001.10
1.201.30
1 2 3
RGR
(g.g
.mon
th -1
)
0.000
0.003
0.006
0.009
0.012
0.015
NA
R (g
.cm
2 .mon
th -1
)100% 22%
ForestForest in ecotone
Cerrado
*
*
*
*
b b a
b a abb b a
B)A)
Dry
mas
s(g)
0
2
4
6
8
10
12
1 2 3 4 5 6 7
Dry
wei
ght (
g)
0
50
100
150
200
250
300
350
1 2 3 4 5 6 7SL
A (c
m2 .g
-1)
0
20
40
60
80
100
120
140
160LA
R (c
m2 .g
-1)
Fore
st
Fore
st in
eco
tone
Cer
rado
22%
100%
b a a
*
*
*
01
4567
Soo
t rat
io
23
hoot
:r
0.600.70
0.800.901.001.10
1.201.30
1 2 3
RGR
(g.g
.mon
th -1
)
0.000
0.003
0.006
0.009
0.012
0.015
NA
R (g
.cm
2 .mon
th -1
)100% 22%
ForestForest in ecotone
Cerrado
*
*
*
*
b b a
b a abb b a
01
4567
Soo
t rat
io
23
hoot
:r
0.600.70
0.800.901.001.10
1.201.30
1 2 3
RGR
(g.g
.mon
th -1
)
0.000
0.003
0.006
0.009
0.012
0.015
NA
R (g
.cm
2 .mon
th -1
)100% 22%
ForestForest in ecotone
Cerrado
*
*
*
*
b b a
b a abb b a
B)A)
Dry
mas
s(g)
56
No significant population x light treatment interaction was found for
photosynthetic traits and leaf pigment content (Table 2). For photosynthetic traits
(Fv/Fm, ETRmax, PPFDsat), differences among populations and differences among
light treatments were not significant (Table 2 and Figure 3A). For leaf pigment content,
only carotenoids:chlorophyll ratio showed significant differences among populations,
with higher mean values in Cerrado core and ecotone. Comparison between light
treatments showed significant differences for chlorophyll content and
carotenoids:chlorophyll ratio, but not to chlorophyll a:b ratio. Individuals growing in the
shade showed significantly higher chlorophyll and lower carotenoids:chlorophyll ratio
than those growing in full sunlight (Table 2 and Figure 3B).
Phenotypic plasticity in response to light was comparatively higher for dry mass,
NAR, SLA, LAR and chlorophyll content (final mean RDPI ranging from 0.28 to 0.41).
Intermediate plasticity was found for height, slenderness index, root:shoot ratio,
ETRmax and PPFDsat (final mean RDPI between 0.17 and 0.24). Traits as internode
length, RGR, Fv/Fm, carotenoids:chlorophyll ratio and chlorophyll a:b ratio showed to
be comparatively less plastic (final mean RPDI between 0.03 and 0.14). Comparisons of
phenotypic plasticity among populations rendered several significant differences. For
morphological traits (height, slenderness index, LAR and NAR) plasticity was higher in
Atlantic Forest core population, while for leaf pigments contents (chlorophyll,
carotenoids:chlorophyll ratio, chlorophyll a:b ratio), higher plasticity was found either
in Atlantic Forest core or both ecotonal populations. Traits such as internode length, dry
mass, shoot:root ratio, SLA, RGR and the ones related to photosynthetic response did
not show differences in plasticity among populations (Table 3).
57
Figure 3. Means ± standard error of physiological traits obtained in six months saplings of P. reticulata
from four populations (Cerrado, Cerrado in transition, Atlantic Forest in transition and Atlantic Forest)
grown under two different light levels (100 and 22% of full sunlight). In A) traits related to
photosynthetic performance are present and in B) leaf pigments contents are presented. Letters above
white bars indicate comparisons among populations while asterisks above black bars indicate
comparisons among light treatments. Alphabetical order of letters corresponds with ranking mean value
and absence of letters or asterisks indicates no significant differences considering 95% confidence
interval.
0.5
6
7
8
9
1 2 3 4 5 6 7 8
Fv/F
m
0.
0.
0.
0.
0
1000
2000
3000
4000
Chlo
roph
ylls
(µg.
mg -1
)
0.0
0.1
0.2
0.3
1 2 3 4 5 6 7 8
Caro
teno
ids:c
hlor
ophy
lls ra
tio
0102030405060708090
0
1 2 3 4 5 6 7 8
ETR
max
10
0
0
0
0
0
0
0
0
0
PPFD
sat
10
20
30
40
50
60
70
80
0
1
2
3
4
Chlo
roph
yll a
:b ra
tio
Fore
st
Fore
st in
eco
tone
Cer
rado
in e
coto
ne
Cer
rado
22%
100%
a ab c bc
*
*
Fore
st in
eco
tone
Cer
rado
in e
coto
ne
Cer
rado
22%
100%
Fore
st
0.5
6
7
8
9
1 2 3 4 5 6 7 8
Fv/F
m
0.
0.
0.
0.
0
1000
2000
3000
4000
Chlo
roph
ylls
(µg.
mg -1
)
0.0
0.1
0.2
0.3
1 2 3 4 5 6 7 8
Caro
teno
ids:c
hlor
ophy
lls ra
tio
0102030405060708090
0
1 2 3 4 5 6 7 8
ETR
max
10
0
0
0
0
0
0
0
0
0
PPFD
sat
80
70
60
50
40
30
20
10
0
1
2
3
4
Chlo
roph
yll a
:b ra
tio
Fore
st
Fore
st in
eco
tone
Cer
rado
in e
coto
ne
Cer
rado
22%
100%
a ab c bc
*
*
Fore
st in
eco
tone
Cer
rado
in e
coto
ne
Cer
rado
22%
100%
Fore
st
A) B)
58
Table 2. Plasticity indexes (RDPI) of morphological and physiological traits obtained in of P. reticulata
from four populations. Characters marked with “np” are non parametric, medians are presented and
compared by Kruskal Wallis (Chi-square value) and post hoc Holm test, for the others, means are
presented and compared by ANOVA (F values) and post hoc Tukey test. Levels of significance are * =
p<0.05; ** = p<0.01; *** = p<0.001; ns = not significant and alphabetical order of letters correspond with
ranking mean or median value.
Traits Cerrado Cerrado in
ecotone
Forest in
ecotone Forest F Ch-sq
Height np 0.17 b 0.16 b 0.15 b 0.29 a - 86.69 ***
Slenderness index np 0.11 c 0.20 b 0.22 a 0.21 ab - 56.62 ***
Internode length np 0.14 0.14 0.10 0.13 - ns
Dry mass 0.45 0.32 0.45 ns -
Shoot:root 0.21 0.21 0.29 ns -
SLA 0.33 0.21 0.31 ns -
LAR 0.23 b 0.22 b 0.42 a 9.62 *** -
RGR np 0.10 0.06 0.10 - ns
NAR 0.27 b 0.24 b 0.44 a 6.26 ** -
Fv/Fm np 0.03 0.03 0.02 0.02 - ns
ETRmax np 0.13 0.18 0.21 0.14 - ns
PPFDsat 0.18 0.17 0.20 0.19 ns -
Chlorophyll 0.26 b 0.35 b 0.52 a 0.30 b 10.49*** -
Carotenoids:chlorophyll np 0.10 b 0.17 a 0.17 a 0.11 b - 15.70 **
Chlorophyll a:b 0.08 b 0.13 a 0.16 a 0.12 a - 9.67 *
59
Discussion
Phenotypic plasticity in response to light
Comparative studies that evaluate phenotypic plasticity among functional plant
groups are important in determining the implications of plasticity for the distribution,
spread and persistence of populations, and also for understanding patterns of
evolutionary diversification (Sultan 2000). In the past few years, the accumulated
knowledge about plastic responses of plants to the light environment suggested the
existence of the general trend of higher plasticity in sun-adapted versus shade-tolerant
plant species (Valladares et al. 2000a, Grime and Mackey 2002, Valladares et al. 2005).
The trend may depend, however, on the traits analyzed since sun plants seems to be
more plastic for physiological features while plasticity for morphological and
architectural traits seems to be greater in shade plants (Valladares et al. 2002, Niinemets
and Valladares 2004, Valladares et al. 2007).
Ecotypic differentiation towards lower plasticity in populations from more
homogeneous light environment was reported by Balaguer et al. (2001) in a comparison
of Mediterranean oak populations from habitats with different levels of light
heterogeneity. The results of the present work point to a similar pattern for P. reticulata
populations, with lower values for plasticity indexes in Cerrado core compared to the
other populations. Significantly higher plasticity in morphological traits (height,
slenderness index, LAR and NAR) was found in Atlantic Forest core, while for leaf
pigments contents, significantly higher plasticity was found in Atlantic Forest core and
both ecotonal populations.
These results can be interpreted as habitat based selection for plasticity. Higher
morphological plasticity in response to light in Atlantic Forest saplings suggests higher
efficiency in exploiting this limiting resource than plants from Cerrado. Morphological
60
plasticity has been linked to an enhanced capacity to survive and grow in the understory
(Valladares et al. 2000b) as a particular mechanism to optimize resource acquisition in
plants (West-Eberhard 1989). The evolutionary advantage and the benefits of plasticity
are better understood than its disadvantages, limits and costs (DeWitt et al. 1998), but a
reduced plasticity can be advantageous under stressful conditions where a conservative
resource use is crucial (Valladares et al. 2007). In this context, lower plasticity in
response to light in Cerrado saplings may be one more feature related to the stress
resistance syndrome (Chapin et al. 1993). The lower plasticity could prevent Cerrado
plants under a temporally favorable circumstance to grow too large or to produce
structures that are too expensive to be sustained once conditions deteriorate, as has been
discussed for evergreen woody plants in both tropical and Mediterranean ecosystems
(Valladares et al. 2000a, b).
Hoffmann and Franco’s (2003) explored macroevolutionary processes across
Cerrado and Forests by comparing congeneric pairs of savanna and forest plant species.
These authors concluded that plasticity was higher in Cerrado species, although
exceptions were reported. Considering that many Cerrado species occur in several
microhabitats within the Cerrado, from grasslands to woodlands, higher plasticity in
some Cerrado species is likely to be explained at least in part by the fact that these
species occur over a wider range of environmental conditions and not because savanna
in Cerrado is more heterogeneous with regards to light than forests as argued by
Hoffmann and Franco (2003). In fact, evidences that plants occurring over a wider
range of light environments show higher plasticity in response to light than the ones that
occur over a narrower range has already being reported by Popma et al. (1992). Thus,
the apparent contradictory results found here can be reconciled with those of Hoffmann
and Franco (2003) by considering that their results show rather similar levels of
61
plasticity in Cerrado vs Forests species and by realizing the different level of analysis,
micro vs. macroevolutionary, i.e. populations vs species. Our study suggests that the
ecotypic differentiation of the populations of P. reticulata involves differentiation not
only in certain functional traits but also in the plasticity of these traits to gradients of
light availability leading to an overall more conservative resource use strategy in
Cerrado than in Atlantic Forest populations.
Functional heterogeneity of the light environment
The light environment tends to be more homogeneous in open habitats while
forest understories are highly variable regarding light availability both in time and in
space (Örgren and Sundin 1996). The observed environmental heterogeneity, however,
does not always match the heterogeneity really experienced by organisms, which are
named structural and functional heterogeneity, respectively (Li and Reynolds 1995,
Gómez et al. 2004). In order to test the hypothesis that phenotypic plasticity is higher in
habitat with more heterogeneous light environment, we assumed that functional light
heterogeneity experienced by P. reticulata saplings in Atlantic Forest was higher than
that in Cerrado. Our assumption, however, disagree with that of Hoffmann and Franco
(2003) since they assumed a greater light heterogeneity in savannas from Cerrado than
in Forests. Without a specific determination of the functional light heterogeneity
experienced by seedlings and saplings in each of these habitats, the hypotheses of both
the present work and those by Hoffmann and Franco (2003) can be proposed. However,
in support to our assumption, it is unlikely that saplings from Cerrado experience a
greater gradient in light resource than those in the Forests, since the very low levels of
light availability observed in the Forests are never found in the Cerrado while both
ecosystems have zones of similarly high levels of irradiance. Thus, further investigation
62
is needed to better understand light as ecological and evolutionary factor in these
ecosystems and to better interpret micro and macroevolutionary processes in the
Cerrado-Atlantic Forest boundary. It is important to bear in mind that plastic responses
to light of species and populations are not only explained by the heterogeneity of the
light environment experienced in each case but also by a complexity of co-occurring
biotic and abiotic factors, which can set important limits to the expression of plasticity
(Valladares et al. 2007).
Functional traits and ecotypic differentiation
Besides showing ecotypic differentiation in phenotypic plasticity, the results of
this work also indicates ecotypic differentiation in functional traits, reinforcing the
findings of Chapter 1 and bringing new evidences. Concerning morphological traits,
more evident shade avoidance syndrome and competitive ability in Atlantic Forest and
stress resistance traits in Cerrado was reported.
For leaf pigment content, the results reinforce the notion that irradiance directly
affects composition and concentration of pigments (Rosevear et al. 2001). Individuals
submitted to shade improved light interception by showing higher chlorophyll
concentration (Johnson et al. 1997) while the ones submitted to sun maximized
photoprotection by showing higher carotenoids:chlorophyll ratio (Demmig-Adams and
Adams 1992). The results also show a genetic determination on pigment features since
the savanna ecotype showed higher carotenoids:chlorophyll ratio than the forest
ecotype.
Concerning photosynthesis traits, we expected that Atlantic Forest individuals to
show higher levels of photoinhibition as shade plants have a decreased capacity to
dissipate excess light energy (Reich et al. 2003). We also expected the Cerrado
63
individuals to show higher light saturation levels for photosynthesis, according to
common features of sun-adapted plants (Bazzaz 1979). In this work, however, no
significant differences between light treatments nor between populations in
photosynthetic traits were observed. This agrees with Nicotra et al. (1997) who reported
that physiological traits related to photosynthetic performance are under strong
stabilizing pressure and may show little or no variation among genotypes.
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Valladares F, Wright JS, Lasso E, Kitajima K, Pearcy RW. 2000b. Plastic phenotypic response
to light of 16 congeneric shrubs from a Panamanian rainforest. Ecology 81(7): 1925-1936.
Valladares F, Chico JM, Aranda I, Balaguer L, Dizengremel P, Manrique E, Dreyer E. 2002.
Greater high light seedling tolerance of Quercus robur over Fagus sylvatica is linked to a
greater physiological plasticity. Trees, Structure and Function 16: 395-403.
Valladares F, Arrieta S, Aranda I, Lorenzo D, Sánchez-Gómez D, Tena D, Suaréz F, Pardos JA.
2005. Shade tolerance, photoinhibition sensitivity and phenotypic plasticity of Ilex
aquifolium in continental Mediterranean sites. Tree Physiology 25: 1041-1052.
Valladares F, Sanchez-Gomez D, Zavala MA. 2006. Quantitative estimation of phenotypic
plasticity: bridging the gap between the evolutionary concept and its ecological
applications. Journal of Ecology 94: 1103-1116.
Valladares F, Gianoli E, Gómez JM. 2007. Ecological limits to plant phenotypic plasticity. New
Phytologist 176: 749-763.
West-Eberhard MJ. 1989. Phenotypic plasticity and the origins of diversity. Annual Review of
Ecology and Systematic 20: 249-278.
Wright JS. 2002. Plant diversity in tropical forests: a review of mechanisms of species
coexistence. Oecologia 130: 1-14.
68
CAPÍTULO 3:
How important is soil fertility in driving ecotypic differentiation of a
tropical tree species from savanna and forest habitats?
69
Introduction
All over the world, plant communities’ diversity, structure and biomass are
severely affected by soil characteristics such as hydrological status, texture, nutrient
availability and also biotic elements as root feeders, symbionts and decomposer
organisms (as reviewed by Lawrence 2003, Baltzer and Thomas 2005, Wijesinghe et al.
2005). Although such edaphic characteristics may vary considerably at local and
temporal scales, especially in tropical environments (Sultan and Bazzaz 1993, Baraloto
et al. 2006), there is much evidence of plant specialization to soil properties (Baltzer
and Thomas 2005). So, in a general way, soil characteristics are thought to be important
selective agents in plant evolution (Baraloto et al. 2006, Macel et al. 2007).
In the tropics, soil plays an important role in forest and savanna distributions,
with the first one usually correlating with higher water and nutrients availability (Furley
1992, Haridasan 1992). However, this major pattern is not always clear and vegetation
and soil are so intimately related that it is difficult to identify cause-and-effect
relationships (Ruggiero et al. 2002, Hoffmann et al. 2005). In Brazil, the Atlantic Forest
and the Cerrado provide a good model of study of plant specialization to soil conditions
as these are adjacent forest and savanna habitats with distinct major soil characteristics.
The Atlantic Forest originally covered more than 1 million km2, being a vast and
diversified biome with different physiognomies found along rainfall, temperature,
altitude, longitude and also soil gradients (Oliveira-Filho and Fontes 2000, Oliveira-
Fillho and Ratter 2002, Resende et al. 2002). Although soil characteristics strongly vary
along the Atlantic Forest biome (Resende et al. 2002), in general nutrient is much less
limiting than in Cerrado. The Cerrado biome once covered about 2 million km2 of
central Brazil, being constituted by xeromorphic vegetation that varies from grassland
to woodlands. The most typical Cerrado physiognomy is a savanna vegetation named
70
Cerrado sensu stricto characterized by a community of trees and shrubs with crown
cover of 10-60% and a well developed grassy ground layer (Ratter et al. 1997, Oliveira-
Filho and Ratter 2002). Cerrado sensu stricto is generally found in poor and deep soils,
usually also characterized as acid, well-drained and with high levels of exchangeable
aluminum (Oliveira-Filho et al. 1989, Haridasan 1992, Ruggiero et al. 2002).
Plants from Atlantic Forest and Cerrado are considered different functional
groups as they show different physiological and life history attributes (Hoffmann et al.
2005). There are evidences of habitat specialization in the earlier phases of growth
between them, mainly related to growth and survival in response to vegetation cover
(Hoffmann and Franco 2003, Hoffmann et al. 2004) and to fire incidence (Hoffmann
and Moreira 2002). Concerning specialization to soil properties, little is known about
differences in the nutrients requirements between them (Hoffmann et al. 2005), but as
nutrients are more limited resource at Cerrado than at Atlantic Forest, some level of soil
specialization in plants is expected.
The leguminous tree species Plathymenia reticulata (Mimosoideae) provides a
good model to evaluate soil specialization as it can be found both at Atlantic Forest and
Cerrado habitats. A previous study suggested that the wide distribution of this species is
related to the existence of ecotypes adapted to different light and soil environments
(Chapter 1). In the present study, we further investigate populations’ responses to soil
and explore the extent of this environmental factor in driving ecotypic differentiation in
P. reticulata. Specifically, we tested the hypothesis that individuals are locally adapted,
showing morphological and physiological traits that enhance performance in their home
habitat soil condition. Moreover, we hypothesized that the locally adapted
characteristics should be shown even by individuals growing in a different soil
condition from their home habitat. Our prediction for these hypotheses is that Atlantic
71
Forest individuals should show traits adapted to a more fertile and competitive habitat,
while Cerrado individuals should show traits adapted to less fertile and more stressful
habitat. Further, we hypothesized that ecotypes should differ in levels of phenotypic
plasticity in response to soil. We expected to find higher plasticity in Atlantic Forest
individuals as it enhances competitive ability (Grime 2002) and lower plasticity in
Cerrado individuals, as it confers stress tolerance (Chapin et al. 1993).
Material and methods
Studied populations
We evaluated four populations of P. reticulata from Minas Gerais state,
southeast Brazil, from which seeds were collected randomly as mixed samples from 10
individuals in September 2004. Two populations are characterized as the Atlantic Forest
semi-deciduous physiognomy being the first located in the biome core area (19°45’S
43°31’W) and the other one is located in its periphery, in an ecotonal area with the
Cerrado (19°56’S 46°56’W). The two other evaluated populations are in the Cerrado
biome, both characterized as Cerrado sensu stricto. One population is located in the core
area of the biome (18°43’S 45°03’W) and the other one in its periphery, in an ecotonal
area with the Atlantic Forest (19°49’S 43°48’W). Populations’ characteristics are more
detailed described in Chapter 1.
Soil chemical properties were evaluated in all populations by randomly
collecting 10 soil samples at 0 to 20 cm of depth. Soil samples were analyzed at the
“Laboratório de Química Agropecuária” from the “Instituto Mineiro de Agropecuária”
and their properties are shown in Table 1.
72
Table 1. Chemical analysis of soil from samples provided from the four studied populations of P.
reticulata (Cerrado, Cerrado in ecotone, Atlantic Forest in ecotone and Atlantic Forest) from the two
different soil treatments used in a nursery experiment (Cerrado and Atlantic Forest). V% stands for base
saturation and m% for aluminum saturation.
Populations Soil treatments Chemical parameters
Cerrado Cerrado in ecotone Forest in ecotone Forest Cerrado Forest
pH in water 5.0 4.5 5.2 4.15 5.2 5.6
Organic matter (g/Kg) 24.8 24.8 26.0 51.2 18.0 28.4
N (g/Kg) 1.2 1.2 1.3 2.4 1.0 1.4
C (g/Kg) 1.2 1.2 2.4 1.3 1.0 1.4
P (mg/Kg) < 1 < 1 < 1 5.3 < 1 2.2
K (mg/Kg) 93 26 44 46.5 48 128
Al 3+ (cmolc/Kg) 2.44 1.35 0.66 4.15 0.86 0.27
Ca 2+ (cmolc/Kg) 0.08 0.35 0.86 0.24 0.36 1.65
Mg 2+ (cmolc/Kg) 0.17 0.08 0.11 0.14 0.13 0.68
V% 4.72 8.13 20.74 3.11 17.01 44.01
m% 83.37 72.95 37.99 89.34 58.39 9.21
Nursery experiment and data collection
In late December 2004, seeds were submitted to mechanical scarification and
germination was conducted at 28oC in darkness as in Lacerda et al. (2004). After six
days of germination, 30 seedlings from each population were planted in pots (18 cm of
diameter and 32 cm of depth). Half of the pots were filled with soil collected at a
Cerrado site and the other half with soil from an Atlantic Forest site. Chemical analyses
of soils are shown in Table 1.
During six months (from January to June 2005), seedlings grew in a nursery that
provided about 11.0 mol.m-2.day-1, representing around 22% of full sunlight. Under this
73
irradiance level, greater differences among populations of P. reticulata were reported in
a previous study (Chapter 1). After this period, morphological and physiological traits
were evaluated.
Morphological data were taken with the use of a digital paquimeter (0.01 mm
precision) and common ruler, height (cm), maximum diameter of the crown (cm) and
base diameter (mm) were obtained and number of internodes counted. Mean internode
length (cm) was estimated as height / number of intenodes, considering only individuals
with a single bud, and slenderness index as height (cm) / base diameter (mm).
In vivo chlorophyll fluorescence traits were evaluated in six individuals, with six
months, from each population and submitted to each soil treatmetn. Measurements were
made with the use of a pulse amplitude modulated photosynthesis yield analyser (Mini-
PAM, Walz, Germany). Potential quantum yield of photosystem II was calculated as:
where Fmmmv FFFFF /)(/ 0−= m and F0 are the maximun and the minimum
fluorescence respectively, measured in fully developed leaves after 30 minutes of dark
adaptation. Light saturation curves were obtained using the light curve program of the
instrument, and used to determinate maximum apparent photosynthetic electron
transport rate (ETRmax) and saturating photosynthetically active photon flux density
(PPFDsat), following Rascher et al. (2000). Leaf pigments content were determined in
the same individuals by grounding leaf samples in 80% acetone. Absorbance in the
supernatant was measured spectrophotometrically at 470, 646 and 663 nm and pigment
contents were determined using equations described in Lichtenthaler and Wellburn
(1983).
74
Field experiment and survival censuses
After one year of growth in the nursery, individuals were transplanted into a
field experiment part of an experimental vegetation recovery project of Atlantic Forest.
After several years of land cleaning, the area is now characterized by an open field, the
soil is acid (pH=4.5), rich in organic matter (41.4 g/Kg), with low base saturation
(V%=4.09) and high aluminum saturation (m%=81.01).
In January 2006, twenty individuals from each four populations from each soil
treatment were transplanted, totaling 160 individuals. The individuals were randomly
positioned in the area, distant 3 meters from each other. They were transplanted to holes
of about 25 cm of diameter and 40 cm of depth. Individuals were numbered, and the
population provenance and nursery soil treatment were registered. Mortality censuses
were made after 6 months (July 2006) and after 20 months (September 2007).
Analysis of data
Comparisons among soil treatments and populations in the nursery experiment
were made after logarithm transformation of data. First, Analyses of variance
(ANOVA) were conducted with the sources of variance: soil treatment, population and
also the population x soil treatment interaction. However, as the interaction was not
significant for all evaluated traits, one-way ANOVAs were conducted for soil treatment
and population separately. Comparisons among populations were also made using post
hoc Tukey test.
For each population, plasticity was quantified using the Relative Distance
Phenotypic Index (RDPI) described by Valladares et al. (2006). The relative distances
(RD) among trait values for all pairs of individuals of a given population grown in
different soil treatment were determined as )/('' '''' ijjiijjiij xxjidRD +→=→ , where j and
75
j’ are individuals belonging to different light environments i and i’. The RDPI ranges
from 0 (no plasticity) to 1 (maximum plasticity) and is obtained as
where n is the total number of RD. RDPIs showed
non-parametric distributions even after transformations, so comparisons among
populations were made then by Kruscal-Wallis and post hoc Holm test.
nxxjidRDPI ijjiij /))/(''( '' +→=∑
In the field experiment, differences in the survival among population and among
individuals from different soil treatments were estimated by the Kaplan-Meier product-
limit method. The chi-square test was used to test significant survival differences.
Results
Six months saplings growing in the nursery showed significant differences in
morphological traits among populations (Table 2). Independently of soil treatment,
Atlantic Forest core population had the highest values from all morphological traits.
Atlantic Forest core showed almost three times the mean height and crown diameter,
and approximately twice the slenderness index and internode length found in the
Cerrado core population. Populations from the ecotonal area showed a tendency towards
intermediate values for morphological traits. Concerning comparisons between soil
treatments, significant differences were found for height and crown diameter with the
higher mean values in the individuals from the forest soil treatment (Table 2 and Figure
1).
For photosynthesis traits, P. reticulata individuals showed mean Fv/Fm of 0.79
(± 0.01), mean ETRmax of 80.34 (± 2.78) and mean PPFDsat of 586.70 (± 55.03), no
significant differences were found among populations nor between soil treatments
(Table 2 and Figure 2). Measures of pigment content showed significant differences
among populations but not between soil treatments. Concerning comparisons among
76
populations, a tendency towards higher chlorophyll content was found in Atlantic Forest
core and both ecotonal populations while compared to Cerrado core population. Higher
mean values for carotenoids:chlorophyll ratio was found in Cerrado core and Cerrado
ecotone, while for chlorophyll a:b, higher ratio was found Cerrado core, followed by
both ecotonal population and lower values in Atlantic Forest core (Figure 2).
Table 2. Analysis of variance of morphological and physiological traits recorded on P. reticulata from
four different populations and two soil treatments. Variance ratios (F values) are reported with associated
level of significance (* = p<0.05; ** = p<0.01; ***=p<0.001; ns = not significant).
Traits Comparisons among
populations
Comparisons between
soil treatments
Height (cm) 24.20 *** 14.53 **
Slenderness index (cm.mm-1) 53.70 *** ns
Diameter of crown (cm) 18.93 *** 9.04 **
Internode lenght (cm) 17.86 *** ns
Fv/Fm ns ns
ETRmax ns ns
PPFDsat ns ns
Chlorophylls (µm.mg-1) 4.11 * ns
Carotenoids: chlorophylls ratio 15.87 *** ns
Chlorophyll a:b ratio 4.09 * ns
77
0
1
2
3
4
5
6
Slen
dern
ess i
ndex
(cm
.mm
-1)
0
5
10
15
20
25
30
35
1 2 3 4 5 6 7 8
Crow
n di
amte
r (cm
)
0.0
0.5
1.0
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2.0
2.5
Inte
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ngth
(mm
)
Fore
st
Fore
st in
eco
tone
Cer
rado
in e
coto
ne
Cer
rado
Fore
st so
il
Cer
rado
soil
Fore
st
Fore
st in
eco
tone
Cer
rado
in e
coto
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Cer
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Fore
st so
il
Cer
rado
soil
0
5
10
15
20
25
30
Hei
ght (
cm)
d c b a *
c c b a
c b a a *
b b a a
0
1
2
3
4
5
6
Slen
dern
ess i
ndex
(cm
.mm
-1)
0
5
10
15
20
25
30
35
1 2 3 4 5 6 7 8
Crow
n di
amte
r (cm
)
0.0
0.5
1.0
1.5
2.0
2.5
Inte
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ngth
(mm
)
Fore
st
Fore
st in
eco
tone
Cer
rado
in e
coto
ne
Cer
rado
Fore
st so
il
Cer
rado
soil
Fore
st
Fore
st in
eco
tone
Cer
rado
in e
coto
ne
Cer
rado
Fore
st so
il
Cer
rado
soil
0
5
10
15
20
25
30
Hei
ght (
cm)
d c b a *
c c b a
c b a a *
b b a a
Figure 1. Means ± standard error of morphological traits obtained in six months saplings of P. reticulata
from four different populations (Cerrado, Cerrado in ecotone, Atlantic Forest in ecotone and Atlantic
Forest) submitted to two soil treatments (Cerrado soil and Atlantic Forest soil). Letters above white bars
indicate comparisons among populations while asterisks above black bars indicate comparisons among
soil treatments. Alphabetical order of letters corresponds with ranking mean value. Absence of asterisks
indicates no significant differences considering 95% confidence interval.
78
20
80
100
120
ETR
max
60
Figure 2. Means ± standard error of photosynthesis traits (A) and leaf pigment content (B) obtained in six
months saplings of P. reticulata from four different populations (Cerrado, Cerrado in ecotone, Atlantic
Forest in ecotone and Atlantic Forest) submitted to two soil treatments (Cerrado soil and Atlantic Forest
soil). Letters above white bars indicate comparisons among populations, alphabetical order corresponds
with ranking mean value and absence of letters indicates no significant differences considering 95%
confidence interval. No significant differences between soil treatments were detected.
0
40
0.6
0.7
0.8
Fv/F
m
0
100
200
300
400
500
600
700
800
PPFD
sat
Fore
st
Fore
st in
eco
tone
Cer
rado
in e
coto
ne
Cer
rado
Fore
st so
il
Cer
rado
soil
2000
3000
4000
5000
Chlo
roph
ylls
(µg.
mg -1
)
b ab a ab
0.00
0.05
0.10
0.15
0.20
0.25
Caro
teno
ids:c
hlor
ophy
lls ra
tio
a a b b
15
20
25
30
35
Chlo
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ylls
a:b
ratio
a ab ab b
Fore
st
Fore
st in
eco
tone
Cer
rado
in e
coto
ne
Cer
rado
Fore
st so
il
Cer
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soil
A) B)
20
80
100
120
ETR
max
60
40
0
0.6
0.7
0.8
Fv/F
m
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100
200
300
400
500
600
700
800
PPFD
sat
Fore
st
Fore
st in
eco
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Cer
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in e
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Cer
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st so
il
Cer
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soil
2000
3000
4000
5000
Chlo
roph
ylls
(µg.
mg -1
)
b ab a ab
0.00
0.05
0.10
0.15
0.20
0.25
Caro
teno
ids:c
hlor
ophy
lls ra
tio
a a b b
15
20
25
30
35
Chlo
roph
ylls
a:b
ratio
a ab ab b
15
20
25
30
35
Chlo
roph
ylls
a:b
ratio
a ab ab b
Fore
st
Fore
st in
eco
tone
Cer
rado
in e
coto
ne
Cer
rado
Fore
st so
il
Cer
rado
soil
A) B)
79
Among the evaluated traits, comparatively higher plasticity in response to soil
was found for height and crown diameter (mean RDPI of 0.22 and 0.20 respectively).
Intermediate plasticity was found for slenderness index, internode length, ETRmax,
PPFDsat, chlorophyll content and chlorophyll a:b ratio (mean RDPI between 0.12 and
0.17). Comparatively lower plasticity was found for the traits Fv/Fm and
carotenoids:chlorophylls ratio (mean RDPI lower than 0.10). Comparisons of
phenotypic plasticity among populations showed significant differences in six over ten
evaluated traits. For height, higher plasticity was found in Atlantic Forest core
population. The traits: slenderness index, internode length, Fv/Fm and chlorophyll a:b
ratio, showed a major tendency of higher plasticity in ecotonal populations while
compared to core ones. For PPFDsat significantly higher plasticity was found in
Atlantic Forest in ecotone, followed by the core populations and lower values in
Cerrado in ecotone. No significant differences in plasticity among populations were
found for diameter of crown, ETRmax, chlorophyll content and carotenoids:chlorophyll
ratio (Table 3).
Concerning individuals transplanted to field conditions, the mortality censuses
showed that 36,48% of the plants died after 20 months in the field. There was no
significant difference in survival in individuals from different populations (Chi-
square=1.36; p=0.714) (Figure 3). Significant differences in survival were found in
individuals from different soil treatments (Z=-2.19; p=0.028) with higher mortality rate
in those from the Cerrado soil treatment (Figure 3).
80
Table 3. Plasticity indexes (RDPI) of morphological and physiological traits obtained in of P. reticulata
from four populations. Medians are presented and compared by Kruskal Wallis (Chi-square value) and
post hoc Holm test. Levels of significance are * = p<0.05; ** = p<0.01; *** = p<0.001; ns = not
significant. Alphabetical order of letters corresponds with ranking median value.
Populations Traits
Cerrado Cerrado in ecotone Forest in ecotone Forest
Chi-square
Height 0.21 b 0.21 b 0.19 b 0.27 a 17.18 **
Slenderness index 0.13 b 0.16 a 0.16 a 0.12 b 17.32 **
Diameter of crown 0.26 0.20 0.17 0.18 ns
Internode length 0.12 b 0.17 a 0.16 ab 0.14 b 9.81 *
Fv/Fm 0.013 b 0.025 a 0.013 ab 0.021 ab 8.52 *
ETRmax 0.17 0.13 0.19 0.20 ns
PPFDsat 0.13 b 0.05 c 0.29 a 0.17 b 12.34 **
Chlorophylls 0.10 0.13 0.12 0.13 ns
Carotenoids: chlorophylls ratio 0.08 0.09 0.09 0.10 ns
Chlorophyll a:b ratio 0.06 c 0.25 a 0.14 b 0.14 b 9.65 *
.
81
cer mat16 18 20 22 24 26 28 30 32 34
0.5
0.6
0.7
0.8
0.9
1.0
c ct mmt16 18 20 22 24 26 28 30 32 34
0.5
0.6
0.7
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1.0
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0.7
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Cum
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Time (months)
A)
B)
CerradoCerrado in ecotoneForest in ecotoneForest
Cerrado soilForest soil
cer mat16 18 20 22 24 26 28 30 32 34
0.5
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Time (months)
A)
B)
CerradoCerrado in ecotoneForest in ecotoneForest
CerradoCerrado in ecotoneForest in ecotoneForest
Cerrado soilForest soilCerrado soilForest soil
Figure 3. Cumulative survival over time in individuals planted in field. In A) individuals submitted to two
different soil treatments during the nursery experiment, with the ones in from the Atlantic Forest soil
treatment surviving significantly less than the ones submitted to Cerrado soil. In B) individuals from four
populations (Cerrado, Cerrado in ecotone, Atlantic Forest in ecotone and Atlantic Forest) with no
significant differences in survival among them. Analyses were performed with the Kaplan–Meier product
limit, considering 95% confidence interval.
82
Discussion
Responses to soil
The results of the nursery experiment showed that, concerning morphological
traits, plants responded to the higher levels of nutrients and organic matter in the
Atlantic Forest soil treatment by showing significantly higher height and crown
diameter than plants submitted to Cerrado soil. For leaf pigment, we expected
chlorophyll content to be higher in plants submitted to Atlantic Forest soil treatment as
higher availability of nutrients, especially nitrogen and magnesium, is known to
correlate with an increase in chlorophyll content (Johnson et al. 1997, Dale and Causton
1992). Leaf pigments, however, were not influenced by soil treatments, in this species
these traits are more influenced by environmental light and by genes, as discussed on
the next topic. Also, photosynthesis performance was not significantly influenced by the
soil treatments, contraring the expectation that improvement on soil nitrogen levels
would have a positive effect on photosysten II efficiency and a reduction on
photoinhibition (higher Fv/Fm) (Field 1983, Johnson et al. 1997). Photosynthesis traits
are probably very stable in this species, showing low responsiveness to soil and light
environment (Chapter 2) and also being very conserved among populations. Our results
for populations of P. reticulata agree with Nicotra et al. (1997) reports that
photosynthesis traits usually show little or no variation among genotypes.
Habitat specialization
Common garden experiments are frequently used to test genotypic differences
between provenances, however it may be hard to distinguish between divergence caused
by drift or by natural selection (Hufford and Mazer 2003). In the present work, we
considered as evidence of natural selection and local adaptation the fact that Atlantic
83
Forest individuals showed higher mean values for morphological traits while compared
to Cerrado ones and the differences in pigment content found among populations, as
those differences occurred in an adaptive direction.
Concerning morphological traits, higher mean values of height, crown diameter,
slenderness index and internode length in the Atlantic Forest core population when
compared to Cerrado, show higher competitive ability in the first. The competitive
ability should be a response to a crowded habitat, highly limited in light resource (see
Chapter 1) but it also can be considered a response to higher nutrient levels, as more
productive habitats show higher competition intensity (Grime 2002). On the contrary,
lower mean values of morphological traits in Cerrado individuals, specially the reduced
stature, are probably an adaptation for survival on infertile soils, as demonstrated for
several other species (Grime 2002). The results reinforce that P. reticulata from
Cerrado, independently of soil conditions, show more evident stress resistance traits
than Atlantic Forest’s. These traits should enable plants to accumulate more reserves of
nutrient elements in the infertile soil habitat (Chapin et al. 1993, Grime 2002),
increasing nutrient use efficiency (Reich et al. 1992).
Both Cerrado and Atlantic Forest habitats may show seasonal climate, although
the length and the severity of the dry season are more marked at Cerrado (Oliveira-
Fillho and Fontes 2000, Oliveira-Filho and Ratter 2002, Goulart et al. 2005). P.
reticulata individuals are adapted to the seasonal water deficit by showing deciduous
habit, losing leaves during drier months and avoiding transpiration water loss (Goulart
et al. 2005). Cerrado populations may show more evident adaptation to this seasonality
by showing reduced morphological traits, including stature and leaf area (Chapter 1),
which may enhance water use efficiency (Dudley 1996, Gonzalez-Astorga et al. 2003,
Silvera et al. 2003). The deciduous habit, however, is not usually found in species from
84
nutrient poor habitat, on the contrary, evergreeness represent a lower tissue turnover
strategy that enhance nutrient economy in infertile soils (Chapin et al. 1993, Grime
2002). P. reticulata individuals probably overcome the nutrient deficit caused by leaf
loss by showing association with nitrogen fixing microorganism (Faria and Lima 1998).
The results of this study bring new evidences that reinforce the genetic
determination on pigment content in P. reticulata populations. Under similar
environmental conditions, Cerrado core and ecotonal populations showed higher
carotenoids:chlorophyll ratio when compared to Atlantic Forest, which may be
interpreted as a mechanism to maximize photoprotection (Demmig-Adams and Adams
1992). On the contrary, Atlantic Forest core and ecotonal populations showed
mechanisms to improve light interception, as higher chlorophyll concentration and
lower cholorophyll a:b ratio (Johnson et al. 1997, Dale and Causton 1992). As
discussed, differences among populations in leaf pigment are more likely to be
explained by local adaptation to light environment than to soil properties. These
differences in pigment composition among ecotypes, though, did not promote
differences in the evaluated photosynthesis traits.
Phenotypic plasticity
The results of this study suggest that soil properties are important in driving
morphological local adaptation of P. reticulata to either more competitive or more
stressful habitat, suggesting ecotypic differentiation between Atlantic Forest and
Cerrado populations, respectively. The ecotypic differentiation, however, was not
supported by pattern of variation in plasticity levels among populations. We expected to
find lower phenotypic plasticity in response to soil in Cerrado populations as it is
related to the stress resistance syndrome, being advantageous by preventing individuals
85
from infertile habitat to grow too large under a temporally favorable circumstance that
may soon deteriorate (Chapin et al. 1993, Valladares et al. 2000a, b). On the other hand,
we expected to find higher plasticity in Atlantic Forest as higher competitors show
flexibility to respond rapidly to changes in the distribution of resource within the habitat
(Grime 2002). For P. reticulata, however, the evaluated traits showed a general pattern
of higher plasticity in populations from ecotonal area when compared to Atlantic Forest
and Cerrado core. Higher plasticity in ecotonal populations in response to soil could be
explained as a strategy of plants located in the boundary between biomes to adjust to
different soil environments, considering that plasticity is important in the process of
colonizing new ecological space (West-Eberhard 1989).
While comparing plastic responses to soil and to light (data reported in Chapter
2), we found that for chlorophyll and carotenoids:chlorophyll ratio plasticity in response
to light is significantly higher that in response to soil (T tests, respectively t=4.120,
p=0.0062 and t=2.460, p=0.0491). This result is in accordance to the fact that leaf
pigments content are more strongly influenced by light than soil. All the other evaluated
traits showed to be equally plastic in response to light and soil (p>0.05, data not shown),
showing that, for most of the traits, populations of P. reticulata are equally plastic in
response to these two different environmental factors. However, responses to light and
soil drive processes in different directions: responses to light show a major pattern of
higher plasticity in Atlantic Forest than in Cerrado populations, reinforcing ecotypic
differentiation (Chapter 2); this ecotypic difference was not corroborated by responses
to soil, for which higher plasticity was found in ecotonal populations.
86
Survival in field conditions
The data showed that after 20 months in the field, individuals’ survival rate was
still dependent on the soil type used in the initial growth condition. The ones submitted
to Cerrado soil treatment during the nursery experiment showed higher mortality rate
than the ones submitted to Atlantic Forest soil treatment, independently of the
population provenance. This result is consistent with the fact that P. reticulata shows
association with nitrogen fixing microorganisms (Faria and Lima 1998) and during the
nursery experiment, seedlings submitted to forest soil probably had higher initial
mycorrhizal infection. In fact, a higher mycorrhizal infection in seedlings is known to
improve individuals’ success (Dickie et al. 2007).
No differences were observed in mortality rates among populations during the
studied period. It is early to conclude, though, that ecotypes do not differ in survival
during sapling phase and further censuses are needed. Plant success can be strongly
affected by extremes events and perturbations (Osmond et al. 1987) so the study period
may not have reflected yet the characteristic environmental conditions of the field sites.
Concluding remarks
The previous studies (Chapter 1 and 2) clearly suggested ecotypic differentiation
in P. reticulata populations in response to differences in light environment between
Atlantic Forest and Cerrado. The results of the present study also point to the existence
of ecotypes specialized to different soil conditions, although the evidences were not as
clear as for light. It is important to consider, though, that responses to soil are harder to
characterize than to light. Whereas light is characterized solely of a particular range of
photon flux densities and spectral distribution, soil properties are many and interact in a
complex manner (Dale and Causton 1992). Morphological differences found among
87
populations were interpreted as adaptation to a more fertile and competitive habitat
(forest ecotype) or to a less fertile and stressful habitat (savanna ecotype). However, the
differences found among populations in leaf pigment are more likely to be explained by
local adaptation to light environment than to soil properties and forest and savanna
ecotypes could not be differentiated concerning phenotypic plasticity in response to soil
nor concerning survival in the field.
Hoffmann and Franco (2003) evaluated macroevolutionary responses across
savanna and forests by comparing congeneric pairs containing one Cerrado and one
Forest species and reported no difference in response to soil nutrient level between these
functional groups. The results of the present study add to this knowledge showing that
soil nutrient level may play a role in microevolution of Cerrado and Atlantic Forest
plants, although light environment is probably more important in driving the
evolutionary process.
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