3D dental microwear texture analysis of feeding habits of sympatric ruminants in the Białowieża Primeval Forest, Poland

Forest Ecology and Management 328 (2014) 262–269

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3D dental microwear texture analysis of feeding habits of sympatric _ Primeval Forest, Poland ruminants in the Białowieza Gildas Merceron a,⇑, Emilia Hofman-Kamin´ska b, Rafał Kowalczyk b a b

iPHEP, UMR 7262, CNRS & University of Poitiers, France Mammal Research Institute, Polish Academy of Sciences, Białowiez_ a, Poland

a r t i c l e

i n f o

Article history: Received 18 December 2013 Received in revised form 23 May 2014 Accepted 24 May 2014

Keyword: Bison bonasus Cervus elaphus Capreolus capreolus Alces alces Diet Food niche overlap

a b s t r a c t _ With four species of ruminants, including red deer, roe deer, moose, and European bison, the Białowieza Primeval Forest is unique on the European continent, where only one to three ungulate species are usually found. The present study is the first effort to explore the dietary overlap of a European community of sympatric ruminants using 3D dental microwear texture analysis. Results obtained for ungulates from _ Forest were compared with those of four species with well known differences in diet the Białowieza (semi-wild Heck cattle, African buffaloes, giraffes, and yellow-backed duikers). These ruminants frame the spectrum of expected 3D dental microwear textures among ruminants: C3 and C4 grazers share high anisotropy and low to intermediate complexity while browsers display intermediate to high complexity associated with low anisotropy. No significant differences between browsers (moose and roe deer) and mixed feeders (red deer) were detected by this analysis supporting a significant overlap in diet found _ The results on the fourth ruminant are unequivocal: the European bison is not grazer in Białowieza. but instead is highly engaged in browsing. Furthermore, through 3D dental microwear texture analysis, the high plasticity in feeding behavior of the European bison can be tracked depending on the seasons and on available access to feeding supplements during the winter. Ó 2014 Published by Elsevier B.V.

1. Introduction Evidence of overlap in resource use by sympatric species is essential for the understanding of interspecific competition (Mysterud, 2000). Often, resource use by one species reduces availability of resources for another species or leads to species displacement (Putman, 1996; Ferretti et al., 2008). Competition for resource exploitation is one of the main mechanisms for interaction among large ungulates. Such interaction may occur when ungulates share similar habitats, an overlap in food consumed and when resources are limited (Gordon and Illius, 1989; Mysterud, 2000). Evidence of interactions among ungulates and other groups of species in natural ecosystems is limited (Putman, 1996). _ Primeval Forest, located on the Polish-BelarusThe Białowieza sian border is one of the best preserved forests in Europe (Je˛drzejewska and Je˛drzejewski, 1998). With five species of ungulates (including red deer Cervus elaphus, roe deer Capreolus capreolus, moose Alces alces, European bison Bison bonasus and wild boar Sus scrofa), it is unique on the European continent, largely ⇑ Corresponding author. Tel.: +33 (0)5 49 36 63 05. E-mail address: [email protected] (G. Merceron). http://dx.doi.org/10.1016/j.foreco.2014.05.041 0378-1127/Ó 2014 Published by Elsevier B.V.

inhabited by only one to three ungulate species (Okarma, 1995). Ungulates coexist here with a high diversity of carnivores, including wolves and lynx. These predators hunt roe and red deer almost exclusively (Je˛drzejewska et al., 1997; Je˛drzejewska and Je˛drzejewski, 1998). Deciduous and mixed forests with rich undergrowth and herb layers offer good conditions for ungulates and may limit possible competition among them. In historical times, high densities of ungulates maintained for hunting purposes, together with cattle grazing in the forest (Samojlik and Kuijper, 2013), had a strong impact on tree stands and resulted in species competition. The two largest herbivores, the bison and the moose, were especially shaped by intra- and inter-specific competition for food resources (Je˛drzejewska et al., 1997; Je˛drzejewska and Je˛drzejewski, 1998). Nowadays, populations of ungulates in _ occur in moderate densities and their numbers are Białowieza increasing (Borowik et al., 2013; Kowalczyk et al., 2013). Cervids and European bison – the only bovidae species in the _ Forest – are characterized by different feeding habits Białowieza (Hoffman, 1989). The moose (A. alces) is the largest cervid. Its diet is mainly based on browsing trees and shrubs (birches, willows, rowan etc.) and herbaceous dicots; grasses and sedges being very rare in this diet (Morow, 1976; Franzmann, 1981). The red deer occupies various habitats from forest to moorland. In Europe, its

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diet includes different proportions of browse and grass depending on the season, region and habitat (Gebert and Verheyden-Tixier, 2001). Roe deer - the most widely distributed European ungulate species (Linnell et al., 1998) – is recognized as being a selective feeder (browser) occupying a large set of habitats from open cultivated areas to dense forest (Tixier and Duncan, 1996). The European bison, the largest terrestrial mammal in Europe, restored from captive survivors following its extinction in the wild at the beginning of the 20th century, is found mainly in forest habitats (Krasin´ska and Krasin´ski, 2007; Kerley et al., 2012). It is assumed to be a mixed feeder or grazer (Hoffman, 1989; Krasin´ska and Krasin´ski, 2007; Kowalczyk et al., 2011), however its diet differs strongly from summer to winter due to the supplementary feeding widely used for the management of species conservation (Kerley et al., 2012). In the last two decades, however, agricultural land providing crops of rapeseed and cereals or hay left by farmers in the meadows has been more frequently used in winter and some seasonal migrations can therefore be observed (Kowalczyk et al., 2011; Hofman-Kamin´ska and Kowalczyk, 2012). In the present study, we explore the dietary preferences of the _ Forest through 3D four sympatric ruminants from the Białowieza dental microwear texture analysis (3D-DMTA). The 3D-DMTA is based on the automated quantification of 3D surfaces by using a scale-sensitive fractal analysis. It has proved to be very efficient in discriminating dietary differences between species of primates both extant and extinct (Merceron et al., 2009; Ungar et al., 2010; Scott et al., 2012) as well as ungulates (Ungar et al., 2007; Scott, 2012) and carnivores (DeSantis et al., 2012; Stynder et al., 2012). Because it only provides information for a timescale ranging from a few days to a few weeks (Teaford and Oyen, 1989; Schulz et al., 2013), the dental microwear texture is an appropriate proxy to detect seasonal variations in diet and to pinpoint the exploitation of fallback foods (Merceron et al., 2010), i.e. food items that are consumed when preferred food is not available anymore. Through this 3D dental microwear texture analysis, we will explore the resource partitioning or overlapping among the four sympatric ruminants. We aimed to: (i) establish a first comparative dataset of wild European ungulates for 3D-DMTA, (ii) explore the ecological niche partitioning among a guild of wild and sympatric ungulates including bovids and cervids inhabiting the very same area, and (iii) and then assess the foraging habits of European bison recognized recently as a refugee species in European forests (Kerley et al., 2012). More specifically, we aim to assess the portion of abrasive grasses in the bison’s diet all year round, notably in winter when certain bison herds have access to supplementary hay fodder. Thus, results of this study might have implications for future management strategies for European bison conservation.

2. Material and methods 2.1. Material 2.1.1. The Białowiez_ a forest _ This study was conducted in the Polish part of the Białowieza Primeval Forest (52°350 –52°550 N, 23°300 –24°000 E; 625 km2), one of the best preserved temperate lowland forests in Europe. The dominating habitats found there are mixed coniferous and mixed deciduous forest (pine Pinus silvestris, spruce Picea abies, oak Quercus robur, with admixtures of birch Betula spp., and aspen Populus tremula), which covers 39.3% of the area and rich deciduous forest (oak Q. robur, hornbeam Carpinus betulus, lime Tilia cordata, and maple Acer platanoides) covering 34.9%. Wet alder-ash forest (black alder Alnus glutinosa, ash Fraxinus excelsior) covers 12.6% of the area, coniferous forest (mainly pine P. silvestris, and spruce P. abies)

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covers 6.2%, and open habitats within the forest cover 7% (Sokołowski, 2004). Most of habitats are covered with rich undergrowth dominated by hornbeam (37%) and Norway spruce (22%) (Podgórski et al., 2008; Kowalczyk et al., 2011). The Forest is surrounded by agricultural areas with meadows, wastelands and arable land interspersed with small woodlands. The study area _ National Park (17%) and the exploited included both Białowieza forests (83%, of which 34% is protected). The latter are exploited by clear cutting small areas for natural regeneration or replanting. _ Primeval Forest is transitional, The climate of Białowieza between the Atlantic and continental types, with clearly marked cold and warm seasons. The average annual temperature is 7 °C. The coldest month is January (average daily temperature is 4.8 °C), and the warmest is July (18.4 °C). Snow cover persists for 60–96 days per year with a maximum recorded depth of 95 cm (Je˛drzejewska and Je˛drzejewski, 1998). The densities of the four species of ruminants are as follows: European bison 0.7 individual per km2, red deer (C. elaphus) 6.0 individual per km2, roe deer (C. capreolus) 2.0 individual per km2 and moose (A. alces) 0.08 individual per km2. As well as this, the density of wild boar (S. scrofa) reaches 5.4 individual per km2 (Borowik et al., 2013). 2.1.2. The Białowiez_ a ruminants 3D dental microwear texture analysis is applied on 86 speci_ Forest, including 19 roe mens of ruminants from the Białowieza deer, 14 red deer, 8 moose, and 45 European bison. All specimens were living free before being culled or found dead in the _ Forest and their skulls are stored in the mammal collecBiałowieza _ (Poland). For tion of the Mammal Research Institute, Białowieza most of them, date of death and gender are known. All moose and roe deer were culled in winter. The red deer sample is more heterogeneous including individuals shot in winter and spring, and 6 individuals over 14 red deer for which date of death is unknown. However, they were shot during the hunting period in fall and winter (Appendix 1). Among the 45 individuals of European bison, 39 can be clustered in four groups defined by period of death and access to supplementary hay fodder in winter (Table 1). The first group of bison (Bison #1; N = 18) is composed of individuals sampled from spring to summer (from April to September). During that span of time, the _ offers understory vegetation in the primary forest of Białowieza abundant resources for bison (Falin´ski, 1986; Je˛drzejewska et al., 1997). The groups Bison #2 (N = 8), Bison #3 (N = 7), and Bison #4 (N = 6) consist of individuals sampled in late autumn and winter (from November to early March; Table 1). Bison #2 group is composed of individuals from populations intensively fed with hay (3–5 times a week); Bison #3 with individuals less intensively fed with hay (usually once a week). Non-fed individuals using mainly agricultural areas in winter compose the fourth group (Bison #4; Table 1; see also Kowalczyk et al., 2011). The rest of the bison sample (6 individuals) were sampled in October. We made the choice to exclude them from the intra-population analysis in order to keep samples with sharp differences in vegetal resources access (see Appendix 1). 2.1.3. A comparative dataset Our analysis includes 58 individuals representing four species of ruminants with clearly defined and well known differences in diet to serve as reference points and frame the spectrum of expected 3D dental microwear textures among ruminants (Table 1). The African buffaloes (Syncerus caffer; N = 8) from open landscapes in Central and Eastern Africa and the semi-wild, non-fed Heck cattle (Bos taurus; N = 8) from Oostvaardersplassen (Netherlands) are grazers; the former mostly foraging on C4 and the latter on C3 monocotyledonous (Sinclair, 1977; Estes, 1991;

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Table 1 Descriptive statistics (mean m, median med, and standard deviation sd) of dental microwear textural parametersa for extant species (A) used as reference for comparisons with _ (B) and groups of Bison bonasus depending of the season of death and their access to the supplementary fodder (C). Bison #1 is mostly composed of the wild game from Białowieza specimens sampled in summer and marginally in late spring. Bison #2, #3, #4 were sampled in late autumn and winter. Bison #2 were intensively fed, Bison #3 less intensively fed, and Bison #4 non-fed (see text for details). Samples

a

n

epLsar3.6 lm (103)

Asfc

Hasfc81cells

Tfv

m

med

sd

m

med

sd

m

med

sd

m

med

sd

(A) Bos taurus Syncerus caffer Giraffa camelopardalis Cephalophus sylvicultor

8 8 17 25

0.694 2.142 2.744 5.296

0.653 1.961 2.680 5.204

0.195 0.699 1.464 2.054

7.30 6.53 1.99 3.31

6.92 7.09 1.02 2.80

2.24 1.87 1.59 2.02

0.857 0.630 0.829 0.642

0.807 0.540 0.773 0.467

0.239 0.354 0.393 0.464

20789.5 33781.9 27461.8 34605.2

24405.6 35254.5 31202.7 34791.9

11425.6 6527.5 14853.2 6707.7

(B) Alces alces Capreolus capreolus Cervus elaphus Bison bonasus

8 19 14 45

3.006 4.475 4.096 4.836

2.024 2.432 3.673 4.247

2.385 4.666 3.000 2.478

3.88 4.43 3.86 3.63

4.71 4.12 3.32 3.59

2.38 2.36 2.14 1.91

0.711 1.025 0.887 0.420

0.669 0.746 0.703 0.329

0.427 1.096 0.497 0.239

26319.7 27800.0 35395.8 32725.2

27103.3 33596.1 35263.6 34893.9

18630.9 12864.8 9076.2 9784.8

(C) Bison Bison Bison Bison

18 8 7 6

5.364 3.469 6.190 4.767

4.916 3.048 5.087 3.924

2.351 1.435 3.503 2.689

2.93 4.83 3.26 3.92

2.99 4.50 1.93 4.43

1.36 1.31 2.75 1.47

0.371 0.565 0.333 0.338

0.310 0.474 0.329 0.308

0.158 0.352 0.182 0.177

34192.0 35666.2 28659.8 28000.4

36982.5 37400.0 34538.5 31482.6

10044.6 6050.3 10939.9 11273.7

#1 #2 #3 #4

n: numbers of individuals per sample; Asfc: complexity; epLsar: anisotropy; Hasfc: heterogeneity of complexity, Tfv: textural fill volume.

Vulink and Drost, 1991; Vulink et al., 2000). The giraffe (Giraffa camelopardalis; N = 17) is a leaf browser (Leuthold, 1978; Estes, 1991) whereas the yellow-backed duiker (Cephalophus sylvicultor; N = 25) includes up to 80% of fruits along with dicot foliages in its diet (Lumpkin and Kranz, 1984; Wilson, 2005). 2.2. Methods 2.2.1. Teeth and dental facets This study is focused on the paracone lingual enamel facets and the protoconid buccal enamel facets of the second upper and lower molars respectively (Fig. 1). These dental facets share a similar dental microwear pattern because they occlude during the chewing–shearing phase. Accordingly, data from lower and upper molars are combined in a single sample (Schulz et al., 2010). The molds were made with a polyvinylsiloxane material, polymerized by an addition reaction (Regular Body Microsystem, Coltene President), and were prepared in a formwork (Putty soft, Coltene President). The mold reproduces only the shearing facets in which we are interested. Casts were made using a transparent polyurethane resin (Ebalta MG709/20) according to the procedures described in Merceron et al. (2012). 2.2.2. Scanning and pre-processing procedures for surface data A 180  180 lm area was scanned at the center of the dental facet using a TalySurf interferometry-based profilometer fitted with a 50 objective (Fig. 1). The lateral sampling interval is 0.36 lm and the vertical sampling interval is lower than the nanometer scale. First, scans were leveled using TalyMap software using the rotation option. Missing data were replaced by an estimation calculated using the elevation of the surrounding points. The third step was erasing any abnormal relief due to exogenous particles on the enamel surfaces. A final surface leveling was performed. Resulting data were analyzed with Toothfrax and SFrax software using a scale-sensitive fractal analysis (Surfract, http://www.surfract.com; Brown and Siegmann, 2001; Scott et al., 2006). 2.2.3. Variables Scale Sensitive Fractal Analysis (SSFA) is applied to length profiles (length-scale analysis) and to three-dimensional surfaces (area-scale and volume-filling scale analyses; Scott et al., 2006). Four parameters are used here to distinguish the different dental

microwear textures: area-scale fractal complexity (complexity or Asfc hereafter), exact proportion of length scale anisotropy of relief (anisotropy or epLsar hereafter), textural fill volume (Tfv), and heterogeneity of area-scale fractal complexity (heterogeneity of complexity with 81 cells or Hasfc hereafter; Table 1; Fig. 2). For a detailed description of the variables, including the calculation procedures, see Scott et al. (2006). Because we did not use the same magnification as Scott et al. (2006), we had to slightly update the protocol according to our own settings. The differences in settings concern the scale at which the anisotropy is calculated (3.6 lm in the present study meaning about 10 times the pixel size). 2.2.4. Statistics The first analysis aims to explore differences between rumi_ and the taxa used as refnants including species from Białowieza erence points. The null hypothesis of this set of analyses of variances (ANOVAs) is that there is no significant differences between species (Table 2). The second set of ANOVAs is focused on the European bison, to explore the variations in diet over the year depending on the availability of food in the forest and the access to supplementary fodder in winter. The null hypothesis is that there is no significant differences between the four groups of European bison. When the null hypothesis of the ANOVAs are rejected (Table 2), post hoc comparisons are then employed to determine the sources of significant variations (Table 3). The conservative Tukey’s Honest Significant Differences (HSD) test is combined with Fisher’s Least Significant Differences (LSD) test (Table 2) to balance risks of Type I and Type II errors (Sokal and Rohlf, 1969; Cook and Farewell, 1996). All data are first rank-transformed before ANOVAs to mitigate the violation of assumptions inherent to parametric analyses of variance (Conover and Iman, 1981). 3. Results A first set of univariate statistical analyses indicates significant differences in 3D dental microwear texture parameters between species (Tables 1A and B and 2A). Differences concern complexity (Asfc), anisotropy (epLsar), heterogeneity of complexity (Hasfc), and textural filling volume (Tfv). Results of the pairwise comparisons test are reported in Table 3A. Because complexity (Ascf) and anisotropy (epLsar) discriminate at best species from each other,

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Fig. 1. Occlusal views of right upper toothrow of a modern bison (MRI PAS 169-573). The 3D-DMTA is run on the lingual shearing facets of the paracone on second molars _ moose (A ; #AC 325), roe deer (highlighted facets in red). A-D show four 3D surface simulations (180 lm  180 lm) of enamel shearing facets of ruminants from Białowieza: (B ; #B-PRV 5), red Deer (C ; #551), and the European bison (D ; #MRI PAS 169-613). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2. Schematic microwear surfaces showing low and high values for the four investigated textural parameters: anisotropy, complexity, heterogeneity of complexity, and texture fill volume (modified after Scott et al., 2006). For the latter, the texture fill volume corresponds to the difference between the volumes modeled with smaller columns and with larger columns (see Scott et al., 2006 for details).

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Table 2 Analyses of variance on species used as reference for comparisons and on the wild _ (A), and on the four groups of Bison bonasus from Białowieza _ game from Białowieza depending on season of death and their access to supplementary fodder (B). Variables (A) Asfc epLsar Hasfc Tfv (B) Asfc epLsar Hasfc Tfv a

Effect Residual Effect Residual Effect Residual Effect Residual Effect Residual Effect Residual Effect Residual Effect Residual

SSa

MSa

F

p

83213.4 165606.6 62772.4 186047.1 70605.5 178214.5 24182.3 224637.2

11887.6 1217.7 8967.5 1368.0 10086.5 1310.4 3454.6 1651.7

9.7624

<0.001

6.5552

<0.001

7.6973

<0.001

2.0915

<0.05

458.58 4481.42 1001.78 3937.72 420.93 4519.07 591.36 4348.64

152.86 128.04 333.93 112.51 140.31 129.12 197.12 124.25

1.1938 2.9681

0.326 <0.05

1.0867

0.367

1.5865

0.210

SS: sum of squares; MS: mean of squares.

biplot graphs show species dispersion with these two variables (Figs. 3 and 4). Among the four species used as reference (Table 1A), there is no significant difference between African buffaloes and Heck cattle with the exception of the textural filling volume (Tfv; Tables 1A and 3A; Fig. 3). These two grazing bovines differ from yellow-backed duikers in having lower complexities of enamel surfaces (Asfc) and higher anisotropies to a lesser extent (epLsar; Tables 1A and 3A; Fig. 3). Moreover, the Heck cattle have a higher heterogeneity of complexity than duikers (Hasfc; Tables 1A and 3A). Heck cattle and African buffaloes also differ from giraffes in having higher anisotropy (epLsar; Tables 1A and 3A; Fig. 3). Giraffes have higher complexity than cattle but significantly lower than duikers (Asfc; Tables 1A and 3A; Fig. 3). Finally, giraffes have higher heterogeneity of complexity than duikers (Hasfc; Tables 1 and 3A). _ Forest have lower anisotThe three cervids from the Białowieza ropy than the grazing Heck cattle and African buffaloes, but higher values than the leaf-browsing giraffes (epLsar; Tables 1 and 3A; Fig. 3). These cervids also differ from Heck cattle in having higher complexity but lower values compared to the fruit-browsing

yellow-backed duikers (Asfc; Tables 1 and 3A; Fig. 3). It is worth mentioning that there is no significant difference between cervids, _ meaning that their dietary habits partly overlap in the Białowieza Forest. The 45-individual sample of European bison significantly differ from the two grazing bovines, African buffaloes and Heck cattle, in having higher complexity and lower anisotropy (Asfc & epLsar; Tables 1 and 3A; Fig. 3). The only difference between bison and duikers is in the heterogeneity of complexity, yellow-backed duikers having higher values (Hasfc; Tables 1 and 3A). Bison differ from giraffes in having higher complexity (Asfc) and anisotropy (epLsar) as well as lower heterogeneity of complexity (Hasfc; Tables 1 and 3A; Fig. 3). Such dental microwear textures clearly exclude grazing habits and instead support a high component of browse in the diet of the European bison. However, the differences in comparison to the leaf-browsing giraffe strongly support intakes of tough as well as hard and brittle material such as seeds, woody material or shoots. Also, bison differ from the three cervids in having lower values in heterogeneity of complexity (Hasfc) and marginally higher complexity than moose and roe deer (Ascf, Table 1 and 3A; Fig. 3). This also supports the consumption of harder and tougher items compared to moose and roe deer. The second set of univariate statistical analyses indicates significant differences in anisotropy between the four groups of bison (epLsar; Tables 1C and 3B; Fig. 4). Bison #2 (including the specimens intensively fed with hay in winter) have significantly higher anisotropy (epLsar) than Bison #1 and Bison #3; specimens from the group Bison #1 being sampled in summer while vegetation is abundant in the forest, whereas Bison #3 is composed of specimens less intensively fed (Tables 1C and 3B). There is no significant difference between Bison #2 and Bison #4 (Fig. 4). 4. Discussion 4.1. Dental microwear textures of reference species The dental microwear textures of the four ruminants used as reference (African buffaloes, Heck cattle, giraffes and yellow backed duikers) closely reflect their differences in feeding preferences. The combination of low to intermediate complexity (Asfc) with high values in anisotropy (epLsar) found for African buffaloes and Heck cattle fits with what is expected for grazing bovids (Ungar et al., 2007; Scott, 2012; see also Schulz et al., 2010). Grasses as well as sedges and rushes are monocotyledonous angiosperms. These plants contain, in most cases, a higher

Table 3 Pairwise differences for microwear texture parameters between species (A) and between groups of Bison bonasus (B). (see Table 1 captions and text for details). Significance at p < 0.05 is indicated in normal font for Fisher’s LSD tests and in bold font for both Tukey’s HSD and Fisher’s LSD tests. Bison #1 is mostly composed of specimens sampled in summer and marginally in late spring. Bison #2, #3, #4 were sampled in late autumn and winter. Bison #2 were intensively fed, Bison #3 less intensively fed, and Bison #4 nonfed (see text for details). Species

Bos taurus

Syncerus caffer

(A) Syncerus caffer Cephalophus sylvicultor Giraffa camelopardalis Alces alces Capreolus capreolus Cervus elaphus Bison bonasus

Tfv Asfc epLsar Hasfc Tfv Asfc epLsar Asfc epLsar Asfc epLsar Asfc epLsar Tfv Asfc epLsar Hasfc Tfv

(B) Samples Bison #2 Bison #3 Bison #4

Bison #1 epLsar

Cephalophus sylvicultor

Giraffa camelopardalis

Asfc epLsar epLsar epLsar epLsar Asfc epLsar Asfc epLsar Hasfc

Asfc epLsar Hasfc Asfc Asfc Asfc Hasfc

epLsar epLsar epLsar Asfc epLsar Hasfc

Bison #2

Bison #3

epLsar

Alces alces

Capreolus capreolus

Cervus elaphus

Asfc Hasfc

Asfc Hasfc

Hascf

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microwear textures. This is a new piece of evidence to consider, the food items themselves rather than the exogenous particles being the main factor for controlling dental wear. Alternative hypotheses suggest dental wear could be driven by the abundance of exogenous grit in diet (Sanson et al., 2007; Lucas et al., 2013). These authors state that grit and dust (actually quartz particles) are harder than enamel and phytoliths as well. They then conclude that enamel is not scratched by silica phytoliths but by exogenous grit. A closer look at the present results emphasizing similarities between Heck cattle from European wetlands and African buffaloes from African open savannah do not support this alternative hypothesis but instead evidences the role of food in the dental microwear genesis. High complexity associated with low anisotropy found for fruit-browsers such as the yellow-back duiker, and intermediate complexity with low anisotropy found for leafbrowsers such as the giraffe fit with the textures expected for fruit- and leaf-browsing ruminants respectively (Ungar et al., 2007; Scott, 2012). Differences in heterogeneity of complexity and texture fill volume are more difficult to interpret. 4.2. Dental microwear textures and food resources overlapping among cervids

Fig. 3. Distribution (mean ± confidence interval of the mean at 95%) of reference_ depending on the complexity (Asfc) and species and ruminants from Białowieza anistropy (epLsar) on the enamel shearing facets.

Fig. 4. Distribution (mean ± confidence interval of the mean at 95%) of reference_ depending on the complexity (Asfc) species and samples of Bison from Białowieza and anistropy (epLsar) on the enamel shearing facets. Bison #1 is mostly composed of specimens sampled in summer and marginally in late spring. Bison #2, #3, #4 were sampled in late autumn and winter. Bison #2 were intensively fed, Bison #3 less intensively fed, and Bison #4 non-fed (see text for details).

concentration of silica phytoliths compared to dicotyledonous herbaceous plants, bushes and trees (Hodson et al., 2005 and citations therein) which results in pronounced scratches and low complexity and high anisotropy. Although the Heck cattle graze on C3 monocotyledonous in wetlands and the African buffaloes graze on C4 monocotyledonous, there is little difference in their dental

None of the differences in dental microwear textures between _ Forest is significant (Fig. 3). This is cervids from the Białowieza not unexpected because their feeding preferences tend to overlap. Indeed, these three cervids are highly engaged in browsing. The significant differences between the cervids and giraffes on one hand and with yellow-backed duikers on the other hand exclude a leaf-dominated diet or high frugivory, respectively. Although differences are not significant, the lower values in complexity (Asfc) for A. alces compared to the two deer could effectively reflect the predominance of leaf-dominated browsing in the moose diet compared to the wider feeding spectrum of the two species of deer. Indeed, leaf-browsing species such as the giraffe have lower complexity than mixed feeders and browsers including fruits and seeds in their diet such as the yellow backed duiker (Scott, 2012). The red deer is a mixed feeder. Herbaceous vegetation compose up to 70% of its diet. Moreover, grasses may overdominate over forbs. The roe deer is a browser and a selective feeder. Its diet includes leaves, shoots, seeds, fruits; monocots representing less than 5% of the diet. However, based on stomach content analysis of sympatric roe and red deer, Storms et al. (2008) conclude that although the red deer is more plastic in its feeding preferences, its dietary composition overlaps, especially in winter, with that of the roe deer. Still through stomach analysis conducted on the two species of _ Forest, Ge˛bczyn´ska (1980) found differences deer in the Białowieza between the two species in fall and winter. However, these differences are moderate and concern items consumed in large quantities: woody material. Indeed, the red deer includes more woody material consumption (64–71%) than the roe deer (49–58%) (calculated from Tables 2 and 4 in Ge˛bczyn´ska, 1980 and expressed as grams of the total dry weight of identified items in the stomach content). 4.3. Dental microwear textures and the feeding ecology of the European bison at Białowiez_ a When considered as whole (without taking into account subsamples), the European bison shows similarities with the sample of C. elaphus clearly indicating that the European bison from the _ Forest is more of a mixed feeder than a grazer, includBiałowieza ing a wide spectrum of feeding items in its diet. Field observations clearly support the abundance of dicots in the European bison’s diet. Borowski et al. (1967) proposed the first compilation of published data regarding the dietary composition of the European

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bison in the wild. More than 200 species are eaten and among those 5–25 plants are preferred. Almost 29% of the plants eaten are monocotyledonous (grasses, sedges). Forbs (herbaceous dicots) and trees/bushes represent 31% and 39% of the eaten species, respectively. In terms of dry matter weight in stomach contents, forbs together with grasses represent 50% of the food bolus, the rest being composed of tree and bush foliages, shoots and fine bark (Borowski et al., 1967 and citations therein). Later, Borowski and Kossak (1972) concluded that monocotyledonous and dicotyledonous herbs (forbs) collectively represent 67% of the dietary bolus. All these ecological data are in keeping with what the 3D-DMTA extracts from the enamel surface analysis. Grasses and grass-like plants such as rushes and sedges do not constitute a major element _ is but a secondary food resource. The European bison in Białowieza not a grazer (not even a variable grazer sensu Gagnon and Chew, 2000) but a mixed feeder. This compilation does not take into account the seasonal changes in diet. A closer look at the variations among sub-samples will help to draw a more detailed picture. The only significant difference between the four groups of the European bison concerns anisotropy (EpLsar), a variable positively correlated to the amount of abrasive monocotyledonous (Ungar et al., 2007; Schulz et al., 2010; Scott, 2012). Bison #1 composed of specimens which died in the summer differs from the intensively-fed bison (Bison #2). The latter tend more to be grazers due to their access to intensive supplementary feed and hay. Due to the higher amount of silica in grasses, hay is more abrasive than browse and is responsible of the higher anisotropy. Bison #1 group has access to huge amounts of vegetation in the forest during the summer: fresh grasses but also foliage, twigs, shoots, buds and fruits. The less intensively fed group, Bison #3 does not differ from Bison #1 but does differ from Bison #2 suggesting a higher amount of browse compared to Bison #2. This result was expected, the assumption being that restricted access to supplementary fodder encourages bison to forage for other foods available in the forest during winter: tree shoots, bark and dry vegetation available in more productive habitats and forest gaps. These elements are not as abrasive as monocotyledonous but some of them like bark are tougher than the softer foliage available in summer and spring and thus require more processes to be broken down before ingestion. Bison #4 is composed of specimens foraging mostly outside the forest during the winter. These individuals forage mainly on hay stored by farmers and meadows maintained by EU subsidies (Hofman-Kamin´ska and Kowalczyk, 2012). Occasionally they also forage on winter crops of rapeseed and cereals accessible under the snow cover and shrubby vegetation available on forest edges (Kowalczyk et al., 2011). Such a diet, mixing monocotyledonous, herbaceous dicots and woody vegetation, explains why this group has an intermediate value in anisotropy as well as in complexity between Bison #1 and Bison #2.

5. Conclusion This study is the first attempt to explore the ecological niche partitioning between member of a unique guild of sympatric ruminants in Europe through 3D dental microwear textural analysis. Being an observer-free approach, the 3D-DMTA allows the objective exploration of differences in the feeding habits of a given species or population. The present 3D-DMTA supports previous pioneering studies on ungulates (Ungar et al., 2007; Schulz et al., 2010; Scott, 2012). Indeed, the results on the four species used as reference points to frame the model are clearly defined. Grazers significantly differ from browsers mostly in having higher anisotropy and lower complexity. Among browsers, fruit browsers have higher complexity _ than leaf browsers. The 3D-DMTA on ruminants from Białowieza

clearly supports the hypothesis that cervids overlap in their feeding preferences, which is not unexpected for browsers (roe deer and moose) and mixed feeders (red deer) exploiting the same habitat. The high rate of overlap in food habits may indicate an absence of competition and sharing of common resources (Gordon and Illius, 1989; Putman, 1996; Mysterud, 2000). Longterm data analysis by Je˛drzejewska et al. (1997) showed that the _ Forest were shaped feeding habits of ungulates in the Białowieza by intra- and interspecific competition for food at high densities, especially at the beginning of 20th century, which was an effect of extensive supplementary feeding and the extermination of large predators. Much lower densities in latter decades allowed the _ Forest withshared use of the rich food resources of the Białowieza out competitive interactions. This probably resulted in a higher overlap of the diet and microwear patterns. _ bovid are The results of the 3D-DMTA on the Białowieza unequivocal: the European bison is highly involved in browsing. Tree leaves and shoots during spring and summer are more easily digestible than grass, which may explain mixed feeding foraging by bison. The 3D-DMTA on the different sub-samples also track the high plasticity in feeding behavior for the European bison. Indeed, specimens sampled during periods where dense understory vegetation was available within the forest differ in dental microwear texture from those sampled in winter at the close vicinity of the supplementary hay fodders. When the access to supplementary hay fodder is reduced or even removed, the European bison either completes its diet with browse in the forest or broadens its home range out of the forest to exploit the hay left by farmers and other farm crops (Hofman-Kamin´ska and Kowalczyk, 2012). Acknowledgments This study was financed by the Polish National Science Centre (Grant No N N304 301940) and the Project ANR TRIDENT (ANR13-JSV7-0008-01, PI: G. Merceron). We are also grateful to the sup_ and port of the Mammal Research Institute PAS in Białowieza iPHEP (UMR 7262, CNRS & University of Poitiers). We are grateful to the former director of the Mammal Research Institute for the access to mammalian collection and to N. Brunetière from Pprime Institut at the University of Poitiers for the access to the Taylor Hobson 3D non contact surface Profilometer. We also would like to thank Dr. L. Sönnichsen and T. Borowik for the access to addi_ region. tional mandibles of red deer and roe deer from Białowieza Finally, we thank Seth Ramdarshan (http://srtranslations.wordpress.com/) for improving the English. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foreco.2014. 05.041. References Borowik, T., Cornulier, T., Je˛drzejewska, B., 2013. Environmental factors shaping ungulate abundances in Poland. Acta Theriol. 58, 403–413. Borowski, S., Kossak, S., 1972. The natural food preferences of the European bison in seasons free of snow cover. Acta Theriol. 17, 151–169. Borowski, S., Krasin´ski, Z.A., Miłkowski, L., 1967. Food and role of the European bison in forest ecosystems. Acta Theriol. 12, 367–376. Brown, C.A., Siegmann, S., 2001. Fundamental scales of adhesion and area scale fractal analysis. Int. J. Mach. Tools Manu. 41, 1927–1933. Conover, W.J., Iman, R.L., 1981. Rank transformations as a bridge between parametric and nonparametric statistics. Am. Stat. 35, 124–129. Cook, R.J., Farewell, V.T., 1996. Multiplicity considerations in the design and analysis of clinical trials. J. Roy. Stat. Soc. 159, 93–110. DeSantis, L.R.G., Schubert, B.W., Scott, J.R., Ungar, P.S., 2012. Implications of diet for the extinction of Saber-toothed cats and American lions. PLoS ONE 7, e52453.

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