Quaternary International 278 (2012) 81e86
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Diet of Palaeolama major (Camelidae) of Bahia, Brazil, inferred from coprolites Camilla Pires Marcolino a, c, *, Rosy Mary dos Santos Isaias b, Mario Alberto Cozzuol a, Cástor Cartelle c, Mário André Trindade Dantas a a
Laboratório de Paleozoologia, Departamento de Zoologia, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, CEP 31270-010, Belo Horizonte, MG, Brazil Laboratório de Anatomia Vegetal, Departamento de Botânica, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, CEP 31270-010, Belo Horizonte, MG, Brazil c Museu de Ciências Naturais da Pontifícia Universidade Católica de Minas Gerais, R. Dom José Gaspar, 290 e Campus da PUC, CEP 30535-901, Belo Horizonte, MG, Brazil b
a r t i c l e i n f o
a b s t r a c t
Article history: Available online 10 April 2012
In South America, one of the most important themes is the effort to discover the diet of the extinct giant mammals that lived in this continent. This paper presents new data for the species Palaeolama major, acquired by the analysis of plant fragments found in coprolites. This species possibly lived in an open area, feeding on shrubs, in the forest border. Ó 2012 Elsevier Ltd and INQUA. All rights reserved.
1. Introduction The South-American camelids are immigrants of North America, and came to this continent in the Great American Biotic Interchange (GABI; Woodburne, 2010). Some genera originated in North America (Palaeolama, Hemiauchenia) were represented in South America by endemic species, whose fossils are commonly found in the north half of this continent, but can also be found in the north of Argentine Patagonia, and in Peru and the Bolivian plateau (Scherer, 2009). The extant genus (Lama) was endemic to South America, lived in dry and colder environments in the south of the continent, and in the Andean highlands where its representatives were found in high latitudes. It differs from the extinct genus mainly by the smaller size. The diet of current Lama is based almost exclusively on grasses, but herbs and shrubs can also be eaten (Chaves, 2000; Barberena et al., 2009). An analysis of the fecal content may reveal which plants they feed on. Chaves (2000) showed that Festuca pallescens, Poa lanuginosa and Hordeum spp. grass species were the most common, but species of shrubs (Mulinum spinosum), Juncaceae (Juncus sp.), and Cyperaceae (Carex sp.) are also found. Another way of investigating herbivores’ diet is carbon isotope analyses, which show if the animal has fed on C4 or C3 plants. Although the grasses (Poaceae) are commonly C4 plants, the non-Poaceae monocots and dicots are commonly C3 plants (MacFadden, 2005). The diet of the genus Palaeolama and Hemiauchenia in South America was not well-defined yet. One of the methods for
reconstruction of the diet of extinct taxa is the carbon and nitrogen isotopic analyses on tooth enamel. This analysis shows a resume of the animal diet, or at least of a major part of it. However, it does not show in which plants the animal has fed on, showing only if it is a grazer or a browser, and in which environment it lived (Feranec, 2003). When coprolites are available, much more data about the extant animal diet can be recovered. However, the problem with these data is the limitation of samples, and the fact that a coprolite only reflects recent meals. Nevertheless, these data provide substantial information about the vegetation, and the environment where the animal lived, thus establishing at least part of the food chain (Jouy-Avantin, 2003). Angiosperms have been the principal plant group since the Mesozoic, and have macro and micro characters which are used in taxonomic classification (Metcalfe, 1979). Beyond that, the study of cells and tissues of plants can reveal adaptations to specific environments, allowing taxonomical and ecological inferences (Fahn, 1990). These characters can be found in coprolites, and thus, an identification of the plants used in the animal diet as well as the environment where it lived can be determined. The main objective of this paper is to identify coprolites in Palaeolama major, with possible taxonomic precision, to determine the group of plants which this species fed on, and to infer the environment where the animal lived. 2. Materials and methods 2.1. Study area
* Corresponding author. Laboratório de Paleozoologia, Departamento de Zoologia, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, CEP 31270-010, Belo Horizonte, MG, Brazil. E-mail addresses:
[email protected] (C.P. Marcolino), matdantas@ yahoo.com.br (M.A.T. Dantas). 1040-6182/$ e see front matter Ó 2012 Elsevier Ltd and INQUA. All rights reserved. doi:10.1016/j.quaint.2012.04.002
The studied material belongs to P. major, and was collected in the Gruta dos Brejões cave, Morro do Chapéu municipality (Fig. 1), by one of the authors (CC) in 1976. These coprolites are found near a partial skeleton (Fig. 2AeF), and are part of the paleontological
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Fig. 1. Location of the municipality of Morro do Chapéu, Bahia, Brazil.
collection of the “Museu de Ciências Naturais da Pontifícia Universidade Católica de Minas Gerais”, with the number MCL 002. This cave is part of a giant complex of caves in the north region of the Bahia state, Chapada Diamantina, where currently the Caatinga vegetation is predominant. Two types of coprolites were found: the first one is small, oval, and was excreted from the digestive tract (Fig. 3A), as currently seen in extant camelids. The second one is bigger, with no definite format, interpreted as intra-intestinal material (Fig. 3B). This material probably belongs to the same specimen. The first type was ejected by the animal before its death, while the second type was exposed by the carcass decomposition. 2.2. Plant anatomical analyses Three samples of 1.61 g of P. major coprolites were used for microscopic analyses. The material was rehydrated in cool 20% glycerin, for 1 min in a microwave, at high potency. Later, the samples were maintained in a hotplate at 45 C for 10 min. The analyses of the plant fragments found in the cropolite were done in a Petri dish under a stereomicroscope. Plant fragments were fixed in FAA50 (37e40% formaldehyde, glacial acetic acid and 50% ethanol, 1:1:18, v/v) and preserved in 70% ethanol (Johansen, 1940). The description of the anatomical aspects was made with semipermanent and permanent sections made with the plant fragments. For the semipermanent preparations, the samples were clarified with 50% sodium hypochlorite, stained with 1% astrablue and safranin O (9:1, v/v). The sections were mounted in Kaiser’s jelly glycerin (Kraus and Arduin, 1997). For the permanent preparations, the samples were dehydrated in ethyl series and embedded in Historesin LeicaÒ, according to the fabricant specifications. The material was sectioned (6 mm) in a rotary microtome (Jung Biocut mod 2035), stained in Toluidine blue (0.05%, v/v) buffered in 20 mM sodium benzoate (pH 4.4) (O’Brien and McCully, 1981). The results were studied using light microscopy (Olympus BH2-BHS).
Fig. 2. Palaeolama major bones associated MCL 003 with the coprolites. (AeB) cranial fragment; (C) mandibular fragment; (D) maxilar fragment; (E) hair; (F) hull.
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(Fig. 4AeB). The guard cells are similar in size to the basic ones. The non-glandular trichomes are unicellular, isolated or grouped (Fig. 4A). The glandular trichomes were distinguished by their unicellular head (Fig. 4B). Their basal cells were not evident, and they were less frequent in the samples. In the transverse sections, two morphotypes were observed. Morphotype I was more abundant, and had unisseriate thick-walled epidermal cells. The leaf lamina was thin in the internervural regions, and the minor veins protruded and form a small rib either to the adaxial or abaxial surface. The major veins had more prominent ribs (Fig. 4CeD). Prismatic crystals occurred in the endodermis (Fig. 4D). The vascular tissues were well developed and involved by pericyclic fibers, 3e4 layered in the major veins and 1e3 layered in the minor veins (Fig. 4C). Morphotype II had bisseriate epidermis with thin walled cells, and isobilateral mesophyll. The palisade cells were one-layered on both surfaces, and 3 times higher than wide. The median region of leaf lamina had a reduced paravenal parenchyma. This morphotype had thin walled clorophyllous parenchyma cells, and reduced vascular bundles with parenchymatic sheath immersed in the paravenal parenchyma. Druses occurred in the endodermis, in the paravenal parenchyma, and in the palisade (Fig. 4E). The veins of morphotype I ramify in a closed angle (35e45 ), with a parallel arrangement observed in transverse section (Fig. 4F). 3.3. Crystals The cells of the bundle sheathes (¼ endodermis) had prismatic, relatively large crystals. The crystalliferous cells were isodiametric and isolated, with one crystal per cell. The prismatic crystals and druses (Fig. 4G) occurred in the parenchyma cells of the stem pith. Fig. 3. coprolites MCL 002 of Palaeolama major. (A) ejecteds coprolites; (B) intra intestinals coprolites.
2.3. Histochemical tests Histochemical tests were made in the historesin included material to verify the presence of lignins and suberin. For the lignin test, Wiesner reagent was applied (Lin and Dance, 1992). The reaction was monitored by 1 h, every 10 min. To the suberin test, Sudan IV in 70% ethanol was used directly in the samples, for 20 min. The slides were mounted in the reagents, and observed using light microscopy (Johansen, 1940). 3. Results 3.1. General aspects The analyses of plant material collected in the coprolites allow the identification of leaf and stem fragments, present together with little fragments of quartzite. The leaves are membranaceous and the stem branches are slender and woody, indicating they belong to an herb or low to medium size shrub. Neither the leaf nor the stem fragments reacted for lignin and suberin detection. 3.2. Leaf fragments Two kinds of leaf fragments could be distinguished. The first one was epidermis dissociated by the action of digestive fluids. The second was cut in small pieces probably by the teeth of the animal, and had to be clarified for the observation of venation pattern or included in historesin for transverse sections. The basic cells of the epidermis have slightly sinuous walls, anomocytic stomata, and non-glandular and glandular trichomes
3.4. Stem fragments Distinct anatomical features were observed in the stem fragments. More slender branches had papilose epidermis, with thickwalled cells especially in the outer periclinal surface. The cortex can be divided in two portions. The external one was a 2e3 layered periderm, and the internal one had remnants of the endodermis with prismatic crystals and 3e5 layered thick-walled pericyclic fibers. The phloem was not evident due to the rupture of the sections in the original positions. The xylem portions were wellpreserved, with vessel elements in linear arrangement (Fig. 5A). Fragments in more advanced secondary growth had only the xylem portion and the pith preserved. The xylem portion had large vessel elements, 2e4 grouped, rarely isolated (Fig. 5B). Prismatic crystals occurred in the pith. Fibers were abundant and the medullary rays distinct (Fig. 5B). Some fragments had large isolated vessels in linear arrangement interspersed with abundant fibers. Larger fragments had two growth rings, the internal one with isolated vessels in linear arrangement, and the external layers with no vessels and thick-walled fibers. The external ring is marked by large, 2-grouped vessels, associated with abundant fibers. The parenchymatic rays were evident (Fig. 5C). In some fragments, the medium lamella and the fibers were distinctly stained by Toluidine blue. The medium lamella stained light purple and the fibers blue. The dissociated material was constituted of vessel elements with simple perforation plates (Fig. 5D) and terminal tails of variable dimensions (Fig. 5E). The most abundant were those with short tails. T-shaped sclereids (Fig. 5F) and perforated ray cells were commonly found. The parenchyma cells had variable shapes and dimensions, from isodiametric with truncated terminal walls, to elongated with cuneate terminal walls. The fibers were frequent, short and thin, commonly associated with parenchymatic cells with crystals.
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Fig. 4. Epidermal fragments: (AeB) trichomes. (A) unicellular non-glandular trichomes. (B) glandular trichome; (CeE) Leaf fragments in transversal sections of two different morphotypes: morphotype I (CeD) general aspect of the leaf fragment. Note the vein forming a prominent rib on both leaf surfaces. The outer periclinal epidermal cell walls of the adaxial surface are thicker than those of the abaxial surface. (D) Leaf fragment in polarized light (PL) showing crystal distribution in endoderm and scattered in mesophyll cells. (E) General aspects of morphotype II. Note isobilateral mesophyll with vascular bundles immersed. Vascular tissue in detail. (F) Clarified leaf fragment observed in polarized light. Note crystals associated with bundles. (G) Isolated or grouped crystals in pith cells of a stem branch (scale bars ¼ 100 mm in A, C, D, E, F; 50 mm in B; 25 mm in G).
4. Discussion Anatomical characters are efficient in the classification of the high taxonomic vascular groups of plants. In archaeological and palaeobotanical studies, only plant fragments are usually found. These materials can be identified only by comparative studies with extant flora (Metcalfe, 1979). The vascular system, especially the xylem, presents the most important anatomical character of the sporophyte. It can be useful to distinguish organs of the same species in ferns, gymnosperms and angiosperms. It is commonly well preserved in the fossil record (Gifford and Foster, 1974), and is a diagnostic character for angiosperms. The material found in the coprolites of P. major did not present tracheids, which excluded gymnosperms and ferns. The mesophyll
structure of the morphotypes I and II, the distribution of the crystals in the perivascular region, the morphology of the stem fragments which seemed to belong to the morphotype I, and the vessel elements and fibers of the secondary xylem diagnosed their angiosperm origin (Fahn, 1990; Evert, 2006). The use of angiosperms in extant guanaco (Lama guanicoe) diet was reported by some authors (cf. Chaves, 2000; Barberena et al., 2009). Nevertheless, they assumed that these animals fed basically on grasses, Juncaceae and sedges (¼ Poales) according to the Angiosperm Phylogeny Group (APG, 2009). The samples of the coprolites of P. major did not belong to any of these groups of plants. The leaves of grasses have unmistakable structures, such as vascular bundles of different sizes, regularly alternate and connected by anastomoses (Esau, 1976). The epidermal cells are typically buliform, suberose and siliceous, and were not observed in the
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Fig. 5. Fragments of stem branches. (AeC) Stem branches in cross sections. (A) Periderm like dermal system, gelatinous fibers in the outer cortex, and secondary xylem with isolated vessel elements with ample pores. The rupture of the material occurred in the region of phloem. (B) Secondary xylem with isolated and grouped vessel elements, numerous fibers, and evident parenchymatic rays. (C) Fragment with two growth rings marked by numerous fibers. (DeF) Dissociated xylem cells. (D) Vessel element with simple perforation plate and pit wall thickening. (E) Vessel element with simple perforation plate and elongated tail. (F) T-shaped sclereid. (Scale bars ¼ 100 mm in AeC; 50 mm in DeF).
studied material. In the stems of Juncaceae and Cyperaceae, the vascular bundles are amphixylematic (Esau, 1976), differently from the collateral bundles observed in the coprolites of P. major. In a general view, the monocot leaf varies in form and structure, and in some cases may be similar to a eudicot leaf (Esau, 1976). However, the vascular tissues in the material studied excluded monocots. According to the APG III (Angiosperm Phylogeny Group, 2009), the Angiosperms are divided in nine groups: monocots, Amborellales, Nymphaeales, Austrobaileyales, Chlorantales, Magnoliidae, Comelinides, Ceratophyllum, and Eudicotyledoneae. The accurate and comparative analysis of the synapomorphies of these nine groups with the anatomical features of the samples obtained from the coprolites of P. major led to the elimination of eight of these groups, the monocots, Amborellales, Nymphaeales, Austrobaileyales, Chlorantales, Magnoliidae, Comelinides, and Ceratophyllum. Amborellales were excluded because they present homogeneous mesophyll, subepidermic layers similar to a hypoderm, and bundles of different sizes with sclereids with hypocrepiform thickenings (¼ horseshoe-shaped) (Carlquist and Schneider, 2001). These features were not observed in the samples. The Nymphaealles are typically found in aquatic environments, showing hydromorphic structures, such as large air chambers in leaves and stems, forming flotation structures, and star-shaped parenchyma cells between the chambers (Cutler et al., 2007). In the interpretation of Carpenter (2006), the hydropots are synapormophies of the Nymphaealles, and cells secreting volatile oils are synapormophies of Austrobaileyales. The stem and leaf fragments found in the coprolites did not have such structures. The serrate margin observed in morphotype I leaf fragments could be related to the Chlorantales. Nevertheless, the plants from
this clade have oil cells and the absence of calcium crystals. Its vessel elements have oblique terminal walls with scalariform perforation plates (Watson and Dallwitz, 1992). These anatomical characteristics were not observed in the studied material. Even though the analyzed samples were crystal rich, such as the comeliniid, in this clade the crystal are raphids (Watson and Dallwitz, 1992), a crystal type not observed in the coprolites of P. major. As well as in the Nymphaealles, in the clade Ceratophyllum aquatic species predominate, which eliminates the possibility of this identification for the samples. The Magnoliidae is constituted of four orders and 20 families, and are as diverse as the eudicotyledons, which constitute threequarters of all the angiosperms (Soltis and Soltis, 2004). The xylem of the magnoliids has basal features, namely, tracheids as the tracheary elements (Herendeen et al., 1999). The xylem was the best preserved plant tissue in the samples of the coprolites. Nevertheless, tracheids were not observed in the material, due to the passage through the digestive tract of P. major. Instead, fibers and vessel elements were abundant. When present in the magnoliids, the perforation plate of the vessel elements are reticulate or scalariform (Herendeen et al., 1999), differing from the simple perforation plates observed in the samples. These features reduce the probability that the plants used for the diet of the Palaeolama belong to this clade. The anatomical evidence observed in the samples indicated the probability that these plant fragments belonged to a representative of the clade of the eudicots. However the great diversity in number of taxa, and morphological structures of this group of Angiosperms, do not allow a detailed taxonomic identification. For such a purpose, a floristic inventory of the extant flora together with anatomical studies of the species, and palynological data of the local flora in the Pleistocene is necessary.
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In the material studied, morphotype I was the most frequent, identified by the presence of endoderm and pericyclic fibers with prismatic crystals, very similar in the vascular bundles of leaves or branches. Leaf characteristics, such as thin-walled cells and poorly lignified mesophyll, are regarded as belonging to mesophytic plants not exposed to stressful levels of light or water. The differences observed in staining of the secondary xylem walls in the stem fragments are characteristic of wood tension (Beiguelman, 1962; Mendes and Paviani, 1997), influencing the flexibility of the branch, and commonly observed in current cerrado plants. However, there may be degradation of the walls by digestion or by time. 5. Final remarks The morphological analyses of plant fragments indicated that P. major found in Bahia, Brazil had an eudicot-based diet. No grass fragments were found in the samples. These results are similar to those reported for the Palaeolama found in North America, in which isotopic analyses assigned a diet based on C3 plants (MacFadden, 1997; Kohn et al., 2005), and a tendency of feeding in forest environments (Kohn et al., 2005). The species in the genus Palaeolama are usually considered to have a diet composed of grasses and shrubs, and, thus, are interpreted as indicators that the animals preferred open environments (Janzen and Martin, 1982). However, as this data and the previous isotopic analyses show, this is not applicable to the Palaeolama. The C3 plant diet suggested for the Palaeolama is consistent with what is expected for the fauna which crossed from North to South America, in the Great American Biotic Interchange (GABI). For instance, the Equidae have an opportunistic diet, varying from grasses to shrubs, and the Bovidae have a diet based exclusively on grasses. The Bovidae were not present in South America fauna. However, the Palaeolama which lived in Bahia, Brazil, probably lived in forest border areas, inferred by the presence of crystals and sediments in the coprolites. The vegetation of the site where this species lived should have a type of Seasonal Dry Forest, perhaps a Cerrado, which were present in this region in the Pleistocene (De Oliveira et al., 2005). Thus, the Palaeolama maintained a diet of C3 plants, even though grasses were present in this region in the Pleistocene (Suguio, 1999; De Oliveira et al., 2005). The data corroborate the suggestion of Chaves (2000) that the Palaeolama did not feed on grasses. References Angiosperm Phylogeny Group, 2009. An update of Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG III. Botanical Journal of the Linnean Society 161 (2), 105e121. Barberena, R., Zangrando, A.F., Gil, A.F., Martínez, G.A., Politis, G.G., Borrero, L.A., Neme, G.A., 2009. Guanaco (Lama guanicoe) isotopic ecology in southern South
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