leaf anatomy and biomineralization in empetrum rubrum valh ...landscape dominated mainly by...

INTRODUCTION

Genus Empetrum L. originally belonged to the fam-ily Empetraceae, and is in fact still considered as a mem-ber of this family by some authors (Zuloaga et al. 2008).However, according to the circumscription based on mor-phological and molecular evidence (Judd et al. 2002) thisgenus is currently placed within Ericaceae, in the mono-phyletic clade of Empetroideae, which is composed bythree genera: Empetrum (consisting of two circumboreal

and one austral species), Ceratiola Michx. (with a NorthAmerican species), and Corema D. Don (with a NorthAmerican and an European species). These three generashare strong synapomorphies such as expanded anddeeply lobed stigma, small corolla, drupaceous fruit andwind pollination. Empetrum rubrum Valh ex Willd (“mur-tilla”) lives in Patagonia (from Neuquén to Tierra delFuego provinces, Argentina), Chile, the Malvinas/Falk-land and the Tristan da Cunha Islands (Good 1927, Moore1983, León et al. 1988, Correa 1999). This species occurs

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Botanica Complutensis 36: 113-121. 2012 ISSN: 0214-4565http://dx.doi.org/10.5209/rev_BOCM.2012.v36.39449

Leaf anatomy and biomineralization in Empetrum rubrum Valh ex Willd (Ericaceae)

María Gabriela Fernández Pepi1, Mirta Olga Arriaga1 and Alejandro Fabián Zucol2A

Abstract: Fernández Pepi, M. G.; Arriaga, M. O. & Zucol, A. F. 2012. Leaf anatomy and biomineralization in Empetrum rubrum Valh ex Willd(Ericaceae). Bot. Complut. 36: 113-121.

The present paper describes and illustrates features of Empetrum rubrum epidermis and transverse leaf section. It also describes and quan-tifies siliceous and calcium phytoliths.Biomineralization quantification is expressed as the media biomineral content (% from dry weight) withvalues of 1.58% ± 0.51 in silica phytoliths and 0.82% in calcicum phytoliths. E. rubrum is a native species from the austral end of South Ameri-ca. We here compare features of its leaf anatomy and biomineralization quali-quantifications with those from E. nigrum, a native species fromthe northern hemisphere. The comparison between the characters described for this species and those from macroremains and phytoliths recove-red from surface soils provides valuable tools for taxonomical, ecological, paleontological and paleoecological studies. Knowledge of said cha-racters further provides valuable information to infer changes in plant communities in the recent past as well as insight into the environmentaland soil conditions at the time when such changes took place.

Key words: biomineralizations, Empetrum heathlands, Empetrum rubrum, leaf anatomy.

Resumen: Fernández Pepi, M. G.; Arriaga, M. O. & Zucol, A. F. 2012. Anatomía de las hojas y biomineralización en Empetrum rubrum Valh exWilld (Ericaceae). Bot. Complut. 36: 113-121.

En este trabajo se describe e ilustra las características anatómicas de la epidermis y transcorte de hoja de Empetrum rubrum. Se han descri-to y cuantificado a los cuerpos silíceos y cálcicos. La cuantificación de biomineralizaciones se expresa en valores medios del contenido de bio-minerales (% en peso seco) obteniéndose valores de 1,58% ± 0,51 correspondientes a silicofitolitos (biomineralizaciones silíceas) y 0,82% decalciofitolitos (biomineralizaciones cálcicas). La especie E. rubrum es nativa del sur de América del Sur, sus características anatómico foliaresy los caracteres cuali-cuantitativos de sus biomineralizaciones han sido comparados con los de E. nigrum nativa del Hemisferio Norte. Los ca-racteres descriptos para esta especie, al compararlos con macrorestos o silicofitolitos recuperados en suelos recientes, constituyen herramientasde gran valor en estudios de taxonomía, de ecología, paleontología y paleoecología, aportando datos importantes para determinar las modifica-ciones pasadas de estas comunidades vegetales, como así también, las condiciones ambientales y del suelo en que se produjeron dichas modifi-caciones.

Palabras claves: biomineralizaciones, murtillar, Empetrum rubrum, anatomía foliar.

1 Laboratorio de Anatomía Vegetal, MACN-CONICET, Ángel Gallardo 470, 2º Piso C1405DJR, Ciudad Autónoma de Buenos Aires, Argen-tina. [email protected], [email protected]

2 Laboratorio de Paleobotánica, CICYTTP- DIAMANTE. CONICET, Materi y España sn, E3105BWA, Diamante, Entre Ríos, [email protected]

Recibido: 26 octubre 2011. Aceptado: 30 marzo 2012.

M. G. Fernández, M. O. Arriaga & A. F. Zucol Leaf anatomy and biomineralization in Empetrum rubrum...

114 Botanica Complutensis 36: 113-121. 2012

as prostrate or cushion subshrub forms, developing theso-called “murtillares”, in plains on the austral slopes ex-tremely exposed to winds, where winter snow can remainfor several months (Roig 1998). It is a heliophilous plantthat lives in acid soils, usually in areas that in the pastused to be covered by “coirón” (Festuca gracillima Hook.f.). When sheeps were introduced into these regions, thescarce availability of forage grass forced animals to over-graze “coirón” causing its slow retreat, with a resultinglandscape dominated mainly by “murtilla” (Correa1999,Anchorena et al. 2001). Recent studies indicate thatsheep-grazing and steady land activity over the last 100years has turned many grass communities into heathlands(Collantes et al. 1989). Given the low value of murtillaas sheep forage, many attemps have been made to substi-tute eroded heath with sown pasture. The recurrent fail-ure of establishing pasture species (Serra 1969) was as-cribed to the alellopathic effect of Empetrum leaf litter.In many places, however, deterioration by overgrazingcontinued and large areas are currently dominated by verypoor and eroded “murtillar”, with scarce soil development(Anchorena et al. 2001).

Phytoliths are a valuable tool for studying ancientvegetation and landscape (Piperno 2006). Most palaeoe-cologically oriented phytolith studies have focused on thereconstruction of grassland composition and tree/grasscover ratios, yielding important general information onthe development and changes of large-scale vegetationcommunities (Alexandre et al. 1997; Barboni et al. 1999,

2007; Scott 2002; Abrantes 2003, Bremond et al. 2005a,b, 2008a, b; Barboni & Bremond 2009). The general an-alytic approach considers all morphotypes and size frac-tions of a phytolith assemblage (Strömberg 2004, Neu-mannn et al. 2009). Although most phytoliths in theassemblages are non-specific and can be used only to re-construct very broad ecological groups, this approach any-way allows the detection of unique morphotypes, givingrise to diagnosis at low taxonomic levels and much moreprecise palaeoecological interpretations (Eichhorn et al.1996). Our work thus contributes to the knowledge of E.rubrum anatomy, especially from the point of view of itsphytolith production, enabling its use as a tool in furtherecological, paleoecological, paleontological and taxonom-ic studies, since leaf epidermis bears relevant identifica-tion features, in addition to its property of resisting di-gestive processes of herbivores given its strongcuticularization and type of cell wall. Phytolith contentscan be identified and quantified when present in soil stra-ta, helping to determine the composition of plant commu-nities on these soils in the recent past.

MATERIALS AND METHODS

To analyze the phytolith production considering the tissu-lar origin of each form, 10 specimens of Empetrum rubrum, col-lected in the Tierra del Fuego ecotone, were sampled (Appen-dix I), sampling sites are affected by different charge ofherbivory and /or environmental conditions (Table 1, Fig. 1).

Fig. 1– Study area. Fueguian ecotone, Tierra del Fuego province (Argentina). A: Ea. San José. B: Ea. Ushuaia. C: Reserva Cora-zón de la Isla. D: Ea. Buenos Aires.

Two material processing types were implemented, one ofthem to anatomical analysis, where epidermal and leaf cross-sections (10 µm thick) were treated according to traditionalpreparation and mounting techniques for microscopic observa-tion (Arriaga 1983, D’Ambrogio 1986). For phytolith observa-tions the material was washed first with neutral detergent andthen with distilled water, oven-dried and weighed. The phytolithswere obtained following the calcination Labouriau’s technique(Labouriau 1983), summiting each sample to a carbonization at200 °C during 2 h. The carbonized material was chemically di-gested with HCl 5N to remove the remaining organic matter andCaCO3, washed with distilled water and filtered with ash-freefilter paper. The remaining material was calcined at 800 ºC dur-ing 2 h, and the ashes obtained were weighed before mountingfor microscopic observation. In addition, 5 specimens were cal-cined with the Labouriau’s technique, devoided the HCl treat-ment, so as to obtain both the silica and calcium material. Theweights of the samples obtained by both treatments were com-pared to establish a relationship between total biomineraliza-tions weight and that of the silica ones. We also calculated thecontent of phytoliths in relation to the initial dry matter in eachsample. The ashes obtained were mounted in immersion oil formicroscopic observation, and the observed biomineral bodieswere identified and relatively quantified, with a Leica DMLBoptical microscope. For calcium biomineralizations identifica-tion in ashes, epidermis and leaf cross-sections slides we usedthe same optical microscope under polarized light.

For more details on the morphology of the biomineraliza-tions, leaf anatomy observations were made with scanning elec-tron microscopy (SEM) after metallization of the material byevaporation of a thin layer of carbon and gold-palladium, usinga Phillips XL30 SEM of the Scanning Microscopy Service fromthe Museo Argentino de Ciencias Naturales “Bernardino Riva-davia”. Collected specimens, remaining ashes and microscopi-cal slides were deposited in the herbarium and repository of theLaboratory of Plant Anatomy, respectively, of the Museo Ar-gentino de Ciencias Naturales “Bernardino Rivadavia”.

RESULTS

Leaf anatomy. The specimens analyzed have a high-ly revolute leaf with a transverse section that forms an in-dentation in approaching blade edges (Fig. 2F). Leaves arecovered by hairs that intersect (Fig. 2, J), defining an in-terior camera bound by the abaxial side of the lamina(Fig. 2F); where the exposed area of the blade correspondsto the adaxial epidermis (Fig. 2F). The leaf has bifacial or-ganization, with a palisade parenchyma formed by 1-3layers of prismatic cells in adaxial subepidermal positionand a spongy parenchyma towards the abaxial epidermis(Fig. 2F). In the middle area of the transverse section, im-mediately below the central vascular bundle, the intercel-lular spaces are larger, and look like aerenchyma of stel-late cells (Fig. 2D). The central vascular bundle is welldeveloped, with 1 (–2) smaller vascular bundles on eachside (Fig. 2F).

Collateral vascular bundles are surrounded by aparenchyma sheath, unrelated to sclerenchyma (Fig. 2E,H). The transverse section also shows cross connectionsbetween the vascular bundles, formed by conducting tis-sue surrounded by a parenchyma sheath. A layer of isodi-ametric parenchyma cells is observed in abaxial-subepi-dermal position (Fig. 2F, D).

The adaxial epidermis is composed of cells with high-ly thickened walls, strongly cuticularized (Fig. 2B), 19-22µm thick in cross-section, without stomata, formed by iso-diametric cells with smooth or corrugated walls (Fig. 2C),with some lanuginous uniseriate, 1-3 cells and simple-basehairs (Fig. 2K, L), in a row along the curved zone of theblade (Fig. 2G) and over both edge margins (Fig. 2F, J).The abaxial epidermis, on the other hand, consists of un-



Table 1Studied area characteristics. (1) Coronel et al. 2009; (2) Moretto, Pers. Com.

Ea. San José Ea. Ushuaia Reserva Corazón Ea. Buenos Airesde la Isla

Charge of herbivory Exclusive “guanacos” “Guanacos” and Exclusive “guanacos” “Guanacos”, geese grazing during more domestic livestock grazing during and sheep grazingthan 5 years grazing less than 5 years

Soil features pH= 4.51 (acid) pH = 4.71(acid) pH = 4.71(acid) pH = 4.31(acid) 26.4% organic 17.4% organic 27.6% organic 22-24% organic matter(1) matter(1) matter(1) matter(2)

Environmental Farther north, Low plains, Low plains, Middle altitute, conditions high altitute high humidity high humidity middle humidity

and low humidity



Fig. 2– Empetrum rubrum leaf anatomy. H, L and M, Scanning Electrone Microscopy photomicrographies. A-G and I-K, OpticalMicroscopy photomicrographies. A: Abaxial epidermis overview, stomata can be observed. B: Adaxial epidermis transection,very thick cuticule can be observed. C: Adaxial epidermis overview devoided of stomata. D: Section through palisade andspongy parenchyma. E: Vascular bundle section. F: General leaf transaction. G: Hairs over leaf bending area. H: Vascular bun-dle section. I: Uniseriate multicellular gland hairs. J: Woolen hairs crossing over leaf proximal extremes. K: Uniseriate mul-ticellular wollen hair. L: group of uniseriate multicellular hairs showing wollen aspect. M: Uniseriate multicellular glandularhair. Scale bars: 20 µm.

thickened, scarcely cuticularized cells, 10-14 µm thick incross-section (Fig. 2J). Also observed are abundant stom-ata, most of them anisocytic and some anomocytic(Fig. 2A). Multicellular glandular hairs are also presentwith uniseriate 2-3 cell stalk and a multicellular ellipsio-dal apical head (Fig. 2I, M), where cell divisions take placetransversally to hair length (Fig. 2M).

Biomineral content. The comparative analysis of bio-mineralization percentages taken from samples from E.rubrum specimens is based on different life conditionsfrom the Fuegian ecotone, such as humidity degrees, to-pography, soil, exposure to sun and grazing rates, and wascarried out by comparing the confidence intervals devel-oped with the average values and deviation standards.These intervals overlap do not show significant differenceseither in total or partial biomineralization percentages.Mean values of biomineral contents in relation to the dryweight obtained in both carbonization treatments (i.e. ei-ther with or without HCl) showed higher amounts of totalbiomineralizations than those of silica (Fig. 3). From anaverage of 0.007 g (1.533% dry matter) of biomineral con-tent, the average of silica elements was 0.004 g (0.94%dry matter, the remaining material (0.003 g, or 0.590%)being estimated as biomineralizations of another chemi-cal nature (Fig. 3).

Biomineralization characteristics. Amorphousstructures (silica phytoliths). The main silica biominer-alizations were found in polyhedrical elements, some witha constriction in their middle transversal section, with reg-ular or irregular ends (Fig. 4A-F, N-Q, S, T). Prismatic,elongated, smooth-edged morphotypes were less abundant(Fig. 4G-L) so were globular ones, which range fromspherical to irregular (Fig. 4K, M, O, P, R), and silicified



Fig. 3– Biominerals distribution contents in Empetrum rubrumanalyzed samples, according to with (A) or devoided chlo-ridic (B) treatment. Graphic values are expressed in grams(graphic on the right) and porcentuals (graphic on the left),maximum value (media –/+ standard deviation) minimumvalue on statistical results.

Fig. 4– Biomineralization in leaf of Empetrum rubrum. A-T: Silicophytoliths in ashes from chloridric treatment. U-W and Y: Cal-cium druse in leaf diafanization. V: The same observed in W, under polarizated light. X: Druse in ashes from non chloridic tre-atment. Y: cristals, druses and silicophytoliths in leaf diafanization. Scale bars: 10 µm.

unicellular trichomes. These silica bodies almost com-pletely fill the cell lumina, mostly on the abaxial epider-mis. They are present in the entire cell layer, constitutinga complete siliceous coating.

Crystal structures (calcium phytoliths). As regardscrystals, we observed the presence of simple, square andprismatic crystals 0.50-1 µm long (Fig. 4Y) distributed incells of both parenchyma. We also observed larger druseswith a diameter of 1-2.5 µm, formed by the concrescenceof simple crystals located in the spongy parenchyma (Fig.4U-Y). All crystal structures show refringence when ob-served under polarized light (Fig. 4, V)

DISCUSSION AND CONCLUSIONS

Previous studies of Empetrum rubrum (Rossow, inCorrea 1999) described their leaves as narrow and longi-tudinally ridged on their abaxial surface. The presentanalysis shows that these leaves are strongly revolute andthat their groove edges are simply leaf margins facing eachother, with hair intercrossing between both margins. Ac-cording to Gibelli (1876) and Gruber (1882) (sensu Sol-ereder 1908) leaf folding is due to the fact that the lowerepidermal cells, located in both lateral portions of the cen-tral vascular bundle, grow faster than the rest of the cellsof both epidermis. In cross-section, the leaf is seen as ahollow structure, with the lower epidermis located towardsthe inner part, presenting abundant glandular hairs. Theadaxial epidermis that forms the periphery of the cross-section lacks stomata and has a very thick cuticle. Unise-riate multicellular hairs are common in young leaves, es-pecially along leaf margins.

Although Empetrum is represented by only threespecies worldwide, and has medicinal and food value (Al-tan & Özdemir 2004), only the anatomical structure of E.nigrum L. has been described so far (Solereder 1908, Met-calfe & Chalk 1950). We are thus here contributing to theknowledge of the anatomical structure of the australspecies used as forage by native and domestic herbivores,especially in seasons when trophic overlapping is presentin Patagonian grasslands (Fernández Pepi et al. 2009a, Al-varenga et al. 2009).

The present observations on E. rubrum leaf anatomyagree with the type, tissue position, presence and locationof simple or compound crystals of E. nigrum described bySolereder (1908) and Metcafe & Chalk (1950). Observa-tions on Patagonian Gaultheria L., a genus from the samefamily placed within another group, namely within sub-family Vaccinioideae (sensu Kron 1997, Kron et al. in Juddet al. 2002), also showed druses placed in special idioblasts

or in lumen of chlorenchymatic cells. In G. phyllyreifolia(Pers.) Sleumer, druses were also observed on the hypo-dermis (Hermann & Pérez Cuadra 2011).

Concerning the biomineral content in Ericaceae, ina phytolith analysis of alpine tundra flora Carnelli et al.(2001) reported the presence of 0.16% silica in relationto leaf dry matter of E. nigrum. Hodson et al. (2005) re-ported silica content in several species of Ericaceae,where that of E. nigrum was between the mean values ofthose mentioned for this family, and equivalent to 0.39%of dry mass.

The data obtained from our analysis show 0.94% ofsilica biomineralizations and 0.59% of other types of bio-mineralizations, higher than the ones described above. Al-though these differences in biomineralization percentagescan be attributed to the characteristics of each species (E.rubrum vs E. nigrum) they can also derive from their dif-ferent environmental conditions or phenological stage(Bertoldi de Pomar 1975, Zucol 2001). Indeed, Carnelli etal. (2001) studied subalpine-alpine species in a transition-al zone of heathland, stunde trees and meadows above thetimberline of the central Swiss Alps at 2000-2300 m a.s.l.Our studies, on the other hand, were carried out regardingthe Fuegian ecotone, between the steppe and the southernforest area covering the central part of the island wherethe relief is more pronounced than that of the steppe andis characterized by a mosaic of small patches of open for-est of Nothofagus antarctica alternating with bunch ofgrasses in the higher parts and herbaceous communitiesin the depressions (Cassola et al. 1975). The marked dif-ferent environmental conditions of these two regions couldaccount for the differences in biomineralization percent-ages mentioned above. Indeed, according to Piperno(1988) there are environmental factors such as climate,soil, moisture and plant age, which affect the developmentof silica bodies, regulating the concentration of dissolvedsilica available in plants.

In our study, no significant differences were found inbiomineralization percentages (total and partial) of sam-ples from different Fuegian ecotonal sites which differedin humidity, topography, soil, exposition and grazing rates.Collantes et al. (1989) reported that the presence of “mur-tillares” of Empetrum in the steppe of Tierra del Fuego isprobably associated with anthropogenic causes, particu-larly sheep overgrazing. The scarcity of other plant com-munities associated with Empetrum sp. could be due ei-ther to the fact that Empetrum has a very low organicmatter degradation rate, thus inhibiting the growth of oth-er species (Monteith 1970), or to soil acidification result-ing from the blockade of N mineralization, low levels of



Ca++ and high content of Al+++, which shows an alterationin soil evolution (Collantes et al. 1989). Although envi-ronmental conditions and herbivore load are different fromthose in the steppe, in our ecotone there has been an in-crease of “murtillar” in overgrazed places. The differentsampling areas where organic matter and charge were stud-ied did not seem to affect the rates or types of biominer-alization observed. Their respective environmental param-eters did not influence leaf biomineralization of the speciesstudied. While environmental factors are considered to in-fluence biomineralization rates, ecotone conditions do notaffect the incorporation of minerals for phytolith forma-tion. Thus, no significant differences were found amongbiomineralization values. Soil acidity was the most con-stant of all factors in our ecotone sampling sites, allowingthe availability of the same quantities of silice. FernándezHonaine et al. (2011) found no significantly different sil-ical contents in plants from different sampling sites, un-like the case of young and scenescent individuals, whichshowed higher biomineralization percentages. The mate-rial we studied was all at fruit stage, so maturity differ-ences do not apply.

Because biomineralizations last for so long withoutany degradation (Rovner 1983, Piperno 1988), furtherknowledge of this process may allow its future applicationin paleontological and paleoecological studies to infer landcover development in the recent past in a particular area,and to look into the kind of soil and the chemical condi-tions that affected it at a given time. The description of thetypes of silica phytoliths and of leaf anatomy will thus con-tribute to the identification of diet macroremains in the

region where this species develops and to soil researchesthereon. Paleoecological studies on peatlands of Tierra delFuego (a different type of plant community) used phy-toliths as a tool for paleoenvironmental reconstruction(Benvenuto et al. 2008, 2009), concluding that certain lev-els reflect a stream pattern decrease or decrease of envi-ronmental moisture which resulted in a thicker plant cov-er, where they found some dicotyledons such as Asteraceaeand Ranunculaceae. Previous phytolith descriptive stud-ies on this ecotone were carried out on Gunnera magel-lanica Lam. (Acosta Ricci et al. 2011) and on dominantgrass species Poa pratensis L., Festuca magellanica Lam.(Fernández Pepi et al. 2009b, 2010) and Phleum alpina L.(Fernández Pepi et al. 2011a), and constitute reference ma-terial for studies on changes in these communities in therecent past as a result of overgrazing. The first profiles an-alyzed show high abundance of grasses in a land very sel-dom used for grazing (Fernández Pepi et al. 2011b). Webelieve that the data we are here providing on Empetrumwill definitely add to the study of ecotone changes in com-munities of Tierra del Fuego.

ACKNOWLEDGEMENTS

We are indebted to Dr. Alicia Moretto, Lic. Julio Escobar and Lic.Eugenia Alvarenga for their f ield trip collaborations. To Mrs. MaríaDolores Montero for assistance with histological samples and hand careof herbarium material. We also thank to the owners of the field estab-lishments Ushuaia, San José and Buenos Aires and to the Ranger fromthe Park “Reserva Corazón de la Isla”, their kind and hospitality dur-ing field trips. Special thanks to reviewers who generously improve thepaper with their comments.



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Appendix ISpecimens examined

ARGENTINA. TIERRA DEL FUEGO. Dpto. Ushuaia: Ea. BuenosAires, March 2008, Fernández Pepi et al., BA 92194; Ibidem, Feb. 2009,BA 92195; Ea. San José, Nov. 2007, Fernández Pepi et al., BA 92197& BA 92190; Ibidem, March 2008 Fernández Pepi et al., BA 92198;

Ibidem, Ea. Ushuaia, Nov. 2007, Fernández Pepi et al., BA 92197, BA92192, BA 92193; Reserva Corazón de la Isla, Nov. 2007, FernándezPepi et al., BA 92191; Ibidem, March 2008, Fernández Pepi et al., BA92199.

leaf anatomy and biomineralization in empetrum rubrum valh ...landscape dominated mainly by...

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