Importance of Multi-Dimensional Analyses of Resource Partitioning in Highly Mobile Species Assemblages

Popul Ecol (2015) 57:601–611 DOI 10.1007/s10144-015-0508-z

ORIGINAL ARTICLE

Importance of multi-dimensional analyses of resource partitioning in highly mobile species assemblages

1 1 1 Anna Roswag • Nina Inga Becker • Jorge Andre´ Encarnac¸a˜o

Received: 9 December 2014 / Accepted: 25 August 2015 / Published online: 8 September 2015 Ó The Society of Population Ecology and Springer Japan 2015

Abstract Resource partitioning is an essential mecha- assessment of dietary and spatial resource partitioning in nism enabling species coexistence. The resources that are bats. used by an animal are linked to its morphology and ecology. Therefore, similar species should use similar resour- Keywords Bats Á d13C Á d15N Á Molecular diet analysis Á ces. The ecological niche of an individual summarizes all Radio telemetry used resources and is therefore composed of several dimensions. Many methods are established to study different dimensions of an animal’s niche. The aim of this Introduction study was to demonstrate that a combination of suitable methods is needed to study spatial and dietary resource Approximately 8.7 million species coexist worldwide partitioning of sympatric species in detail. We hypothe- (Mora et al. 2011), many of them living in sympatry and sized that, while each individual method might identify sharing resources. Resource partitioning (differing resource differences between species, the combined results of sev- use of sympatric species) is a mechanism that enables the eral methods will lead to a more complete picture of spatial coexistence of species (Schoener 1974). For every species, and dietary resource partitioning. As model organisms we the ecological niche summarizes its living requirements chose the sympatric insectivorous bat species Myotis with regard to resources like habitat, diet, or environmental bechsteinii, M. nattereri, and P. auritus. We examined parameters (Townsend et al. 2008). The multi-dimensional horizontal habitat use by telemetry, vertical habitat use by nature of the ecological niche means that resource parti- measuring d13C, trophic position by measuring d15Nin tioning occurs in several dimensions (Schoener 1974). The wing membrane, and diet composition by molecular fecal most common combination of multi-dimensional resource analysis. Our results show that each method is able to partitioning is diet and habitat as suggested by Schoener provide information about spatial/dietary resource parti- (1974). Spatial segregation of species may result in the use tioning. However, considering further dimensions by of distinct foraging habitats (Pita et al. 2010) or in foraging combining several methods allows a more comprehensive at different heights (Vieira and Monteiro-Filho 2003; Voigt 2010). Dietary resource partitioning includes diet composition but also trophic position within a food web (Siemers et al. 2011; Shiels et al. 2013). Spatial and dietary Electronic supplementary material The online version of this dimensions are not independent of each other as the article (doi:10.1007/s10144-015-0508-z) contains supplementary material, which is available to authorized users. availability of food resources depends on habitat characteristics (Townsend et al. 2008). Several methods are & Anna Roswag suitable to study spatial and dietary resource partitioning of [email protected] species. A direct method to study the habitat use of animals is radio-telemetry (Reynolds 2004; Jhala et al. 2009; Ovi- 1 Mammalian Ecology Group, Department of Animal Ecology and Systematics, Justus-Liebig-University of Giessen, dio et al. 2009; Wikelski et al. 2010). This method has the Giessen, Germany advantage that individual habitat use can be determined in 123 602 Popul Ecol (2015) 57:601–611 detail (Millspaugh et al. 2012). When applying conven- occur sympatrically (e.g., Arlettaz et al. 1997; Hillen and tional radio-telemetry, only one-dimensional habitat use Veith 2013) and, being the only actively flying mammals, can be described (Brown et al. 2009). Thus, vertical seg- occupy the three-dimensional space (Norberg and Rayner regation cannot be determined, although this factor may be 1987). Nursery colonies of European bat species are highly important for species that are able to use different heights philopatric and usually occupy the same habitats through- or depths (Wilson 2010). Overall habitat use can also be out an individual’s life (e.g., Entwistle et al. 2000; Rivers estimated indirectly by analyzing stable isotope ratios, the et al. 2006). European bats are almost exclusively insec- ratio of the heavy to the light isotope of an element (dX, tivorous (Dietz et al. 2009) feeding on several arthropod where X is the heavy isotope of the respective element). orders (Vaughan 1997). Therefore, every bat species Different isotopes may be used to test different assump- should occupy its own niche that at least slightly differs in tions. For example on a large geographical scale, hydrogen the dietary and/or spatial dimensions from that of other isotope ratios (d2H) provide insights into animal migration species. because d2H is correlated to latitude (Hobson 1999). Car- We chose the European forest-dwelling bats Myotis bon isotope ratios (d13C) are suitable to estimate foraging bechsteinii (Kuhl), Myotis nattereri (Kuhl), and Plecotus grounds (Sullivan et al. 2006) or foraging height (Voigt auritus (Linnaeus) that are very similar in their morphol- 13 2010) of animals. d C differs between C3/C4 plants and ogy and ecology (Dietz et al. 2009; Becker et al. 2013). marine/terrestrial systems (i.e., foraging grounds) and it Their nursery colonies can coexist on a very small spatial was shown for tropical forests that leaves’ d13C is corre- scale (Otto et al. 2013). All three species forage mainly lated to vegetation height (DeNiro and Epstein 1976; within deciduous forest (Entwistle et al. 1996; Kerth et al. Medina and Minchin 1980). Nitrogen stable isotopes ratios 2001; Smith and Racey 2008) and have relatively short and (d15N), are suitable to indirectly study the diet of species, broad wings with a low aspect ratio and wing loading because 15N accumulates in a characteristic way within a (Norberg and Rayner 1987). This leads to a high maneu- food web and therefore, provides information about the verability and enables them to forage within highly clut- trophic level of individuals (DeNiro and Epstein 1981). A tered space (Aldridge and Rautenbach 1987). In addition to single sample can simultaneously provide information on catching prey in flight, the three species forage by gleaning several dimensions e.g., on habitat use (d13C) and diet i.e., they collect their prey from vegetation surfaces (d15N). By choosing different tissues for analysis of stable (Neuweiler 1989; Siemers and Swift 2006). Species- isotope ratios, short (e.g., liver), intermediate (e.g., mus- specific differences in digestive efficiency (Becker et al. cle), or long (hair) time periods can be studied (MacAvoy 2012), retention times (Roswag et al. 2012), energetics et al. 2006). Stable isotope ratios in tissues always reflect a (Becker et al. 2013), and thermoregulation (Otto et al. mixed signal of all used resources (Hobson 1999) and thus 2013) are strong indicators for resource partitioning. do not allow detailed conclusions regarding individual prey This study assessed if it is possible to determine species. Morphological or molecular fecal analyses are resource partitioning in highly similar species by combin- suitable tools for studying an animal’s diet in more detail. ing stable isotope analysis, telemetry, and molecular Both techniques provide individual short-term information methods. We hypothesize that while each method might be about ingested prey items. Morphological fecal analysis able to detect differences between similar species, the can determine prey organisms only to the order or family combination of different methods will lead to a more level. While rare or highly digestible prey is often under- comprehensive picture of resource partitioning. represented, volume and proportion of the diet can be examined accurately (Whitaker et al. 2009). Molecular fecal analysis has the advantage of being able to detect Methods even rare prey organisms at the species-level (Casper et al. 2007; Razgour et al. 2011). However, no information about Study area and data collection the volume of consumed prey items is provided by this method. By a combination of several methods, strengths of The study was conducted in a small-sized (60 ha) decidu- one method can compensate the weakness of another. ous forest in Central Germany (50°270N, 8°490E). This Combining molecular fecal analysis with measurement of forest is very homogenous in its characteristics, e.g., tree d15N will, for example, provide information on prey age and species composition, and consists mainly of oak spectrum at high resolution and on the importance of dif- and beech trees. It is surrounded by orchards, agricultural ferent prey items independent of their digestibility. land, and further deciduous forests. To reduce data varia- A prerequisite for the study of resource partitioning is a tion as a result of seasonally changing environmental and stable assemblage of coexisting species. Bats (Chiroptera) physiological conditions data acquisition was limited to are suitable model organisms since several species can early pregnancy season (approximately two weeks at the 123 Popul Ecol (2015) 57:601–611 603 beginning of May). This reproduction period proceeds increase of the home range size over the number of fixes. similar in the three species (Dietz et al. 2009; Otto et al. Based on this we were able to determine the minimum 2015). Research followed the guidelines of the American amount of points reflecting the maximal home range of Society of Mammalogists (American Society of Mam- every species (M. bechsteinii n = 605, M. nattereri malogists 1998). The study was ethically and methodically n = 562, P. auritus n = 406). To identify core foraging approved by the Nature Conservation Authority and the areas for the three species we calculated 50 % kernels Animal Care Authority of the administrative district of using the software ArcGis (version 10.1, ESRI, California, Giessen, federal state of Hesse. USA) with the Kernel Density Estimation and Isopleths We chose female bats to ensure a stable coexistence of functions of the Geospatial Modelling Environment Tool species. Bats were captured by mist-netting. Species, sex, (Beyer 2012). and reproductive status of each individual were visually determined. We categorized reproductive status as preg- Dimension 2 and 3—vertical habitat use (d13C) nant or non-pregnant by abdominal palpation (Racey and trophic position (d15N) 2009). The study was limited to females in early pregnancy to ensure comparability of stable isotope ratios (Roswag Wing membrane samples were washed three times with a et al. 2014). Captured individuals were individually held in 1:1 chloroform/methanol mixture. Wing membrane and sterile cloth bags for 30 min to collect individual fecal leaf samples were dried in an oven at 60 °C until constant samples (n = 5 per species). We took wing membrane mass was reached. Leaf samples were ground in a mixer samples only from individuals without wing membrane mill (MM 400 Retsch, Germany). A minimum of 0.1 mg of injuries (e.g., fissures or holes) using a biopsy punch (ø wing membrane or 2.0 mg of leaf samples was required for 3.5 mm; plagiopatagium; M. bechsteinii: n = 4, M. nat- isotope mass spectrometer analyses (Delta V, Thermo tereri n = 5 and P. auritus n = 5). Samples were stored in Scientific, Germany). The precision of measurements was 98 % ethanol at -20° C until further analysis. Radio- 0.09 % and 0.12 % (n = 15) for d13C and d15N, respec- transmitters (LB-2 N, Holohil Ltd., Ontario, Canada, tively. Based on Eq. 1 we calculated d15N and d13C in per 0.35 g) were attached with skin bond adhesive (50.01, mille: Sauer, Lobbach, Germany) to the dorsal fur of individuals Rsample with an adequate body mass of [7.0 g (Aldridge and dX ¼ À 1 Â 1000 ð1Þ Rreference Brigham 1988; Willis and Brigham 2003)(M. bechsteinii n = 4, M. nattereri n = 4, and P. auritus n = 3). There- where Rsample/Rreference represents the ratio between the after, bats were released at the capture site. The safety and heavy (15N, 13C) and the light (14N, 12C) isotope of element animal-health compatibility of these methods has been X of the sample (wing membrane or leaf) and the inter- confirmed by several recaptures of healthy, reproducing national standard (air-N2 or Pee Dee Belemnite (PDB)), 13 individuals over the years. To quantify the increase of d C respectively. We used acetanilide and L-phenylalanine as depending on the forest height, we took beech leaves at 2, laboratory standards that were calibrated to air-N2 and 10, and 20 m height (minimum of 20 leaves per sample) PDB. from three different trees. Leaf samples were stored at -20 °C until further analysis. Dimension 4—diet (molecular fecal analysis)

Dimension 1—horizontal habitat use (radio- For identification of prey species, the DNA of prey telemetry) organisms in the feces were isolated using the QIAampÒ DNA Stool Mini Kit (Qiagen, Hilden, Germany) with Spatial positions of individuals were logged every 15 min modifications according to Zeale et al. (2011). Amplifica- during the entire nocturnal activity period by the ‘homing- tion, library preparation and amplicon-sequencing were in’ method (White and Garrot 1990; Encarnaçaõ 2012) conducted by Seqlab (Sequence Laboratories Go¨ttingen using a VR500-receiver and a HB9CV-hand-held antenna GmbH, Go¨ttingen, Germany). A 157 bp long fragment of with amplifier (Wagener Telemetrieanlagen, Ko¨ln, Ger- the mitochondrial cyotochrome c oxidase I (COI) gene was many). The exact location was determined based on signal amplified by PCR with the purified primers ZBJ-ArtF1c strength using a calibration curve (R2 = 0.71). Roosts were and ZBJ-ArtR2c (Zeale et al. 2011), which are particularly located during the day, and the exact position of each roost suitable to amplify a wide spectrum of arthropods com- was recorded by GPS. monly found in the feces of insectivorous bats (Razgour For every species we calculated the minimum convex et al. 2011). The forward primer were modified to 50-bar- polygon as a measure for the home range using ArcGis code-marked ‘fusion primer’ prior to PCR allowing iden- (version 10.1, ESRI, California, USA) and plotted the tification of individual samples. After sample cleaning all 123 604 Popul Ecol (2015) 57:601–611 P samples were pooled equimolar and sequenced using the n p p qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffii¼1 ij ik Ion Torrent sequencer. Primer and barcodes were trimmed Ojk ¼ P P ð4Þ n p2 n p2 off and all sequences\100 bp and[180 bp were removed i¼1 ij i¼1 ik (BioEdit, version 7.2.5; Hall 2013). For each sample, where pij is the proportion that resource i is of the total sequences were clustered to Molecular Operational Taxo- resources used by species j; pik is the proportion that nomic Units (MOTUs) (jMOTU, version 1.0.6; Ghoorah resource i is of the total resources used by species k, and et al. 2010; Jones et al. 2011). We tested a series of n is the total number of resources states. thresholds and, based on the inflection point, estimated a threshold of 2 % to be suitable for MOTU assignment. Similar thresholds were chosen for MOTU assignment in Statistics other studies (e.g., Bohmann et al. 2011; Clare et al. 2011; Razgour et al. 2011). We excluded singletons (i.e., MOTUs To test for a phylogenetic signal in data of wing membrane containing only one sequence) from further analyses d13C, d15N, and H0 we constructed a phylogenetic tree of because these sequences are most likely sequencing our study species. We used sequences of the subunit 1 of induced errors (Valverde and Mellado 2013). Representa- the mitochondrial protein NADH dehydrogenase (ND1) tive sequences of remaining MOTUs were compared to published by Mayer and von Helversen (2001). Sequences reference sequences from BOLD (Barcode of Life Data of P. auritus, M. bechsteinii, and M. nattereri were System) using the identification engine. Matches with a downloaded from GenBankÒ (NIH genetic sequence sequence similarity [98.0 % were used for further analy- database; accession numbers: AF401368–AF401374, sis. We used criteria developed by Razgour et al. (2011)to AF401459–AF401462, AF401439, AY033978, verify matches on species-, genus- and family-level. We AY033984). Sequences were aligned using the ClustalW calculated prey accumulation curves on the family level for algorithm. The neighbor joining algorithm based on the every species (EstimateS, version 9.1.0; Colwell 2013). Kimura (1980) assumptions was used for phylogenetic To compare the dietary niche of the three species, we reconstruction. Substitution rates and patterns were calculated the proportion (frequency of occurrence/total assumed to be homogeneous. Sequence analyses were occurrences) of prey arthropods on family- and order-level, conducted using the software MEGA (version 6.0; Tamura the standardized Levins’ measure of niche breadth (Eq. 2) et al. 2013). On this basis, data of wing membrane d13C, and Shannon’s diversity index (Eq. 3). d15N, and H0 were tested for phylogenetic independence (test for serial independence (TFSI); Phylogenetic Inde- P 1 B ¼ n 2 ð2Þ pendence) (Reeve and Abouheif 2003). i 1 pi ¼ Values of d13C and d15N were checked for normal distribution using the Shapiro–Wilk-test. Spearman’s rank B À 1 correlation was used to test for a statistical dependence Standardized as: B ¼ 13 A n À 1 between leaves’ d C and their height in the vegetation. We compared d13C and d15N of bat tissues using an ANOVA where B is Levins’ measure, p is the proportion of fecal i followed by an honestly significant difference-test (HSD). samples in which prey i was found and n is the number of All statistical analyses were performed using the software possible preys in the diet. package Statistica 10.0 for Windows (StatSoft Inc., Tulsa, Xn 0 OK, USA). Results are given as mean ± SD. H ¼À pi log pi ð3Þ i¼1 where pi is the proportion of fecal samples in which prey i was found. Results We calculated niche overlap between species using Pianka’s measure (Eq. 4, Pianka 1973). We used the null Dimension 1—horizontal habitat use (radio- model analysis software EcoSim (Entsminger 2012) to test telemetry) if niche overlap is significantly different from that expected by chance. The Randomization Algorithm 3 was used to The accumulation curves of the home range of every spe- generate 10,000 matrices of random prey spectra and cies reached a plateau [Fig. S1 in Electronic Supplemen- generated niche overlap was compared to observed niche tary Material (ESM)]. Core foraging areas were exclusively overlap. Resulting P values were controlled for Type-I and within the forest, with the exception of M. nattereri that Type-II errors using false-discovery-rate-control (FDR- also used nearby orchards as foraging grounds (Fig. 1). The control; Verhoeven et al. 2005). core foraging areas differed in size, with values decreasing 123 Popul Ecol (2015) 57:601–611 605

Fig. 1 Core foraging areas (50 % kernel) and day roosts and of M. bechsteinii (fixes: n = 605, 50 % kernel: grey- dotted line, roosts: triangle), M. nattereri (fixes: n = 562, 50 % kernel: grey line, roosts: square), and P. auritus (fixes: n = 406, 50 % kernel: black- dotted line, roosts: circles). Upper left corner: location of study site in Germany

from M. nattereri (42.31 ha) over M. bechsteinii (22.64 ha) (-24.23 ± 0.33 %) displayed signiﬁcantly lower d13C to P. auritus (13.80 ha). Overlap of core foraging areas was than M. bechsteinii (-23.49 ± 0.24 %, HSD, P = 0.005) high for P. auritus (95 % overlap with M. bechsteinii and and M. nattereri (-23.44 ± 0.16 %, HSD, P = 0.001). 63 % overlap with M. nattereri), intermediate for M. Myotis nattereri and M. bechsteinii did not differ signiﬁ- bechsteinii (58 % overlap with P. auritus and 48 % overlap cantly from each other (HSD, P = 0.959). with M. nattereri), and low for M. nattereri (26 % overlap with M. bechsteinii and 20 % overlap with P. auritus). Dimension 3—trophic position (d15N)

Dimension 2—vertical habitat use (d13C) Tissue d15N contained no phylogenetic signal (TFSI, P = 0.2) and differed signiﬁcantly among species

There was a significant positive correlation between (ANOVA, F2,11 = 25.53, P = 0.0001, observed leaves’ d13C and their height in the vegetation (Spearman’s power = 1, Fig. 3). Myotis bechsteinii had significantly rank correlation, R = 0.74, P = 0.02, Fig. S2 in ESM). lower d15N (2.88 ± 0.50 %) compared to M. nattereri Tissue d13C values were phylogentically independent (7.08 ± 1.33 %; HSD, P = 0.0003) and P. auritus (TFSI, P = 0.3) and species differed significantly in their (5.18 ± 0.41 %; HSD, P = 0.009). Plecotus auritus had 13 15 tissue d C values (ANOVA, F2,11 = 14.8, P = 0.0008, significantly lower d N than M. nattereri (HSD, observed power = 0.99, Fig. 2). Plecotus auritus P = 0.014).

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We identified 29 arthropods belonging to 7 different orders. Eighteen arthropods (62 %) were identified at species-level, 10 (34 %) at genus-level, and 1 (3 %) at family-level (Table 1). To analyze the prey spectrum, we excluded one insect that was categorized as non-prey item: plant lice (Anoecia corni). Plecotus auritus fed on 22 arthropod species, M. bechsteinii on 13, and M. nattereri on 15. Main prey orders for P. auritus (each 31 %, Fig. 4) were Diptera and Coleoptera. Major families in the diet of P. auritus were Limoniidae (17 %), Elateridae (17 %), Carabidae (13 %), and Tipulidae (13 %). For M. bechsteinii Diptera (50 %) and Coleoptera (30 %) were main prey orders (Fig. 4). The families Limoniidae (28 %), Tipulidae (22 %), and Carabidae (17 %) were common in the diet of M. bechsteinii. Myotis nattereri fed mainly on the prey orders Diptera (36 %) and Coleoptera Fig. 2 Wing membrane’s d13C(%)ofM. bechsteinii, M. nattereri, (36 %, Fig. 4). For M. nattereri Limoniidae (21 %), and P. auritus. Significant differences are indicated by different Tipulidae (17 %), Bibionidae (17 %), and Curculionidae letters. Shown are the mean ± standard error (box) and the standard (17 %) were major prey families. Prey accumulation deviation (whiskers) curves did not reach a plateau (Fig. S3 in ESM). The diet of P. auritus showed the highest diversity (H0 = 2.89) followed by M. nattereri (H0 = 2.51) and M. bechsteinii (H0 = 2.38). Values of H0 were not significantly correlated with species’ phylogeny (TFSI, P = 0.2). Niche breath was relatively high and very similar for all species

(M. nattereri BA = 0.67, M. bechsteinii BA = 0.68, and P. auritus BA = 0.67). Dietary niche overlap was relatively high for all species pairs. Overlap was greatest between

M. bechsteinii and M. nattereri (Ojk = 0.75, P = 0.001; FDR signiﬁcance-level = 0.017), intermediate between

M. bechsteinii and P. auritus (Ojk = 0.72, P = 0.006; FDR signiﬁcance-level = 0.03), and lowest between M.

nattereri and P. auritus (Ojk = 0.60, P [ 0.05; FDR signiﬁcance-level = 0.05).

Discussion

15 Fig. 3 Wing membrane’s d N(%)ofM. bechsteinii, M. nattereri, The present study demonstrated that a combination of and P. auritus. Significant differences are indicated by different letters. Shown are the mean ± standard error (box) and the standard different methods enables a better assessment of spatial and deviation (whiskers) dietary resource partitioning than each method individually. This is especially true for studies of resource partitioning in similar species. Seasonality has to be considered Dimension 4—diet (molecular fecal analysis) when investigating resource partitioning (Thalmann 2001). To exclude data variation caused by seasonal effects, our All fecal samples produced PCR amplicons. The study confined to the early pregnancy season with the sequencing run generated 521,603 sequences. After fil- drawback of a limited sample size. This drawback tering, 1417 sequences representing MOTUs containing notwithstanding, our results showed that, although the more than one sequence remained. These were compared study species used similar foraging habitats and diets, to references in BOLD. Of these sequences, 595 matched resource partitioning was demonstrable on all studied to an arthropod with a sequence similarity of [98.0 %. dimensions.

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Table 1 Identified arthropods Order Family Species P. auritus M. bechsteinii M. nattereri in the fecal samples of Myotis bechsteinii, M. nattereri, and Araneae Linyphiidae Tenuiphantes flavipes 00 1 Plecotus auritus (n = 5 per species) Tenuiphantes sp. 0 0 1 Blattodea Blattellidae Ectobius sylvestris 10 2 Ectobius sp. 2 1 0 Coleoptera Carabidae Carabus problematicus 43 1 Carabus sp. 1 0 0 Curculionidae Curculio glandium 11 4 Curculio sp. 0 0 1 Elateridae Dalopius marginatus 51 1 Dalopius sp. 3 0 1 Dermaptera Forficulidae Chelidurella guentheri 20 0 Chelidurella sp. 1 0 0 Diptera Bibionidae Bibio johannis 11 4 Bibio sp. 0 2 1 Limoniidae Limonia nubeculosa 55 4 Limonia sp. 0 2 2 Sphaeroceridae Coproica hirtula 10 0 Tipulidae Tipula fendleri 10 0 Tipula hortorum 11 0 Tipula nubeculosa 23 1 Tipula submarmorata 30 0 Tipula sp. 44 4 Hemiptera Aphididae Anoecia corni*10 0 Lepidoptera Noctuidae Apamea scolopacina 20 0 Apamea sp. 1 0 0 Nolidae Pseudoips prasinana 01 0 Unknown 11 1 Tortricidae Crocidosema plebejana 10 0 Tortrix viridana 10 0 Numbers indicate how many fecal samples contained the respective insect. Insects marked with an asterisk were excluded from prey spectrum analysis. Insects given in bold were consumed by all three bat species

Dimension 1—horizontal habitat use

The accumulation curves of the home ranges reached asymptotes for every species indicating that the obtained habitat use is representative for these colonies during our study period. As in other studies, P. auritus and M. bechsteinii both foraged exclusively within the forest (Entwistle et al. 1996; Kerth et al. 2001) resulting in nearly identical core foraging areas, while M. nattereri also used other habitats (Smith and Racey 2008). Considering solely our results concerning the horizontal habitat use would suggest that only M. nattereri showed a clear horizontal segregation because overlap with the two other species was low (Fig. 1). These data would lead to the interpretation, that spatial resource partitioning occurs between M. nattereri Fig. 4 Proportions (%) of prey insect orders in the diet of M. bechsteinii, M. nattereri, and P. auritus and the two other species, but not between M. bechsteinii and P. auritus.

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Dimension 2—vertical habitat use determination: Razgour et al. (2011) and Hope et al. (2014)]. However, the consumed proportion differs in It was shown that d13C is suitable to study the vertical every study. This is most probably caused by a local and habitat use of species. The main foraging height of tropical seasonal shift in prey availability (Shiel et al. 1991). In frugivorous bats can be estimated using the ‘Canopy general, the three study species had a similar diet and prey Effect’ (Voigt 2010). This effect is based on the signifi- diversity. Nonetheless, M. bechsteinii fed mainly on cantly lower d13C values of leaves from the lower com- arthropods that are known to be primary consumers like pared to the upper canopy in tropical forests (Medina and Limoniidae or Tipulidae (Gillot 1980) while P. auritus and Minchin 1980). The current study described the ‘Canopy M. nattereri fed in addition on arthropods that are descri- Effect’ also for temperate forests. As temperate bats subsist bed as secondary and tertiary consumers like Forficulidae on insects that in turn feed on leaves, differences in d13Cof or Carabidae (Gillot 1980; Foelix 1982). Additionally, the leaves might indicate differences in the foraging height of diet of M. nattereri contained more secondary and tertiary temperate bat species. Tissues of insects feeding on leaves consumers (e.g., Araneae) compared to P. auritus. The of the upper vegetation should have higher d13C compared niche breadth and niche overlap suggest that M. bechsteinii to insects feeding on the lower vegetation. As the carbon had the lowest dietary resource partitioning with P. auritus isotopes flow through the food web, bats feeding on insects and M. nattereri. Plecotus auritus and M. nattereri, how- of the upper vegetation should also have higher d13C ever, showed significant resource partitioning. compared to bats foraging on insects of the lower vegetation. Tissues of P. auritus displayed significantly lower Considering all dimensions d13C values than the other two species, suggesting that P. auritus foraged at lower heights than the Myotis species. Horizontal habitat use (dimension 1) revealed resource This indicates that resource partitioning occurred between partitioning between M. nattereri and the other two spe- P. auritus and M. bechsteinii/M. nattereri but could not be cies, but not between M. bechsteinii and P. auritus. observed between the Myotis species. Incorporating vertical habitat use (dimension 2), resource partitioning between M. bechsteinii and P. auritus became Dimension 3—trophic position apparent. This study therefore revealed a clear spatial resource partitioning of all three species in the three We measured d15N in wing membranes that can be used as dimensional space. There was a trophic resource parti- a ‘trophometer’ because d15N increases in a characteristic tioning of the three species (dimension 3) but this result is way with trophic level (2–4 %, Fry 2006). Wing mem- difficult to interpret without known prey spectra. Including branes differed significantly in their d15N values in the the molecular fecal analysis (dimension 4), different decreasing order M. nattereri, P. auritus, and M. bech- trophic positions within in the food web became inter- steinii. This shows that the species fed on different trophic pretable because the species’ diets covered primary, sec- levels, indicating dietary resource partitioning among ondary, and tertiary consumers to different amounts. In them. However, different values could be caused by feed- turn, interpreting the results of molecular fecal analysis ing on different arthropods or by feeding on the same alone would not lead to the conclusion that every bat arthropods to different proportions. species fed on distinct trophic levels because important prey arthropods (e.g., Carabus problematicus or Limonia Dimension 4—diet nubeculosa) were consumed by all three species. These different results might be caused by the different time We examined the prey spectrum as a measure of resource spans covered by the two analyses. Fecal samples provide partitioning on the dietary dimension. The diet of the three only a snapshot of a species’ diet because the ingested prey species has been described on order-level in the literature is usually excreted within a few hours (Roswag et al. (for review see Vaughan 1997). As expected due to the 2012). In contrast, stable isotope ratios in wing membranes deliberately limited sampling period and the resulting reflect a mixed signal of the diet over a few weeks (Voigt limited sample size, prey accumulation curves were not et al. 2009). Therefore, fecal samples might be more suit- saturated. The aim of the study was not to describe the able for detecting short-term variation in prey items, for complete prey spectrum of the study species but to deter- example temperature introduced changes, while stable mine detailed differences in consumed prey items without isotopes in wing membranes cover a longer period; how- the influence of prey seasonality. Despite the small sample ever at a lower resolution. Combining the two methods size, the main prey orders found in this study were also provided a clear picture of prey composition. The molec- described as important prey in the literature [morphological ular fecal analysis revealed the greatest dietary niche prey determination: Vaughan (1997); molecular prey overlap between M. bechsteinii and P. auritus/M. nattereri. 123 Popul Ecol (2015) 57:601–611 609

This can be explained by considering horizontal and ver- Becker NI, Encarnaçaõ JA, Tschapka M, Kalko EKV (2013) tical habitat use. The habitat use of M. bechsteinii showed Energetics and life-history of bats in comparison to small mammals. Ecol Res 28:249–258 more overlap with M. nattereri (vertical habitat use) and P. Beyer HL (2012) Geospatial Modelling Environment. version 0.7.2.1. auritus (horizontal habitat use) than that of M. nattereri Available at: http://www.spatialecology.com/gme. Accessed 01 and P. auritus. Therefore, species using more similar for- Jul 2014 aging grounds might have more similar dietary niches than Bohmann K, Monadjem A, Lehmkuhl Noer C, Rasmussen M, Zeale MRK, Clare EL, Jones G, Willerslev E, Gilbert MTP (2011) species using less similar foraging grounds. Molecular diet analysis of two African free-tailed bats (Molos- In summary, we showed that a combination of suitable sidae) using high throughput sequencing. 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