Cranial variation of the greater horseshoe bat Rhinolophus ferrumequinum (Chiroptera: Rhinolophidae) from the central Balkans

Zoologischer Anzeiger 254 (2015) 8–14

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Cranial variation of the greater horseshoe bat Rhinolophus ferrumequinum (Chiroptera: Rhinolophidae) from the central Balkans b ˇ ´ Cabrilo Ivana Budinski a,∗ , Vida Jojic´ a , Vladimir M. Jovanovic´ a , Olivera Bjelic, c a Milan Paunovic´ , Mladen Vujoˇsevic´ a Department of Genetic Research, Institute for Biological Research “Siniˇsa Stankovi´c”, University of Belgrade, Bulevar despota Stefana 142, 11060 Belgrade, Serbia b Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovi´ca 2, 21000 Novi Sad, Serbia c Department of Biological Collections, Natural History Museum, Njegoˇseva 51, 11000 Belgrade, Serbia

a r t i c l e

i n f o

Article history: Received 8 May 2014 Received in revised form 3 September 2014 Accepted 4 September 2014 Available online 16 September 2014 Keywords: Allometry Geographic variation Geometric morphometrics Greater horseshoe bat Sexual dimorphism

a b s t r a c t Cranial size and shape variation of the greater horseshoe bat Rhinolophus ferrumequinum from territories in Serbia and Montenegro was examined using geometric morphometric methods. Statistically significant size and shape differences among specimens from distinct geographic regions (the Carpatho–Balkanides, the Internal and External Dinarides) were observed. Bats from the Carpatho–Balkanides have the smallest crania, while those from the External Dinarides have the largest ones. Compared to specimens from the Carpatho–Balkanides, bats from the other two regions have crania wider in the temporal and elongated in the facial region, while the basicranial region is smaller. Our analysis of sexual size dimorphism revealed no statistically significant differences between males and females. Even though significant sexual shape dimorphism was observed, cranial shape differences among bats from different geographic regions exceeded those between sexes. We also found that size and shape vary with climatic factors. Allometry has statistically significant effect on cranial shape variation and somehow contributes to covariation between cranial shape and environmental variables. Although the examined bats were from a relatively small territory, we have provided new insights into important issues like geographic variation, sexual dimorphism and allometry in this species. © 2014 Elsevier GmbH. All rights reserved.

1. Introduction The greater horseshoe bat, Rhinolophus ferrumequinum (Schreber, 1774) is the largest European horseshoe bat. It has a wide range in the Palearctic, occurring from southern Europe and north-west Africa to China, Korea and Japan, mainly in limestone areas (Dietz et al., 2009). In Europe, this species underwent population decline and range contraction during the last century, and is now rare over most of its distribution (Stebbings and Griffith, 1986). Nevertheless, the greater horseshoe bat is present throughout the territories of Serbia and Montenegro, and the population is stable, as it has the status of a strictly protected wild species (Paunovic´ et al., 2011). The greater horseshoe bat usually inhabits limestone areas, and therefore, its local distribution and dispersal could be constrained by distribution of these geological regions. According to ´ c´ (1996), limestone (karst) in Serbia and Montenegro can be Ciri divided into the Carpatho–Balkanides, the Internal Dinarides and

∗ Corresponding author. Tel.: +381 11 2078332; fax: +381 11 2761433. E-mail address: [email protected] (I. Budinski). http://dx.doi.org/10.1016/j.jcz.2014.09.001 0044-5231/© 2014 Elsevier GmbH. All rights reserved.

the External Dinarides regions in the east, west and south-west, respectively. This species is considered to be sedentary since the distance between summer and winter roosts is usually less than 20–30 km (Mitchell-Jones et al., 1999). Females of R. ferrumequinum show strong natal philopatry to their maternity roosts and some males return to the same mating sites (Rossiter et al., 2000, 2002). Female philopatry, multiple paternity and little inter-colony female exchange suggest that genetic mixing among colonies occurs mainly via extra-colony mating. Strong female philopatry and polygyny might be expected to facilitate genetic drift and divergence among colonies. Thus, genetic differentiation and differences in genetic variability between isolated populations probably reflect the species’ colonization history and also point to reduced gene flow over greater distances (Rossiter et al., 2006). Study of association of phenotype variation with local environmental conditions can provide important insights into the evolutionary history and ecological dynamics of particular species (Marchán-Rivadeneira et al., 2012). The local environmental conditions can induce marked phenotypic differences. In some bat species it has been shown that factors related to temperature and precipitation could be important from the aspect of skull size variation (Burnett, 1983;

I. Budinski et al. / Zoologischer Anzeiger 254 (2015) 8–14

Marchán-Rivadeneira et al., 2012). Santana and Lofgren (2013) observed that cranial shape in rhinolophids follows two major divisions that could reflect adaptations to dietary and environmental differences in African versus South Asian distributions. Morphological variation of greater horseshoe bat has been studied using traditional morphometric techniques. Previous results indicated the presence of sexual dimorphism in body weight and external measurements (Caubère et al., 1968; De Paz, 1995a; Dietz, 2007; Dietz et al., 2006), as well as in wing measurements (De Paz, 1995a; Dietz, 2007; Dietz et al., 2006), with females usually being larger than males. Kryˇstufek (1993) analyzed the geographic variation of R. ferrumequinum in south-eastern Europe through twelve skull measurements and found clinal variation of size with a gradual increase from the west to the east. Based on 42 external, dental and skull measurements, De Paz (1995b) described a similar pattern of morphological variation of R. ferrumequinum in the west Palearctic, where it increases in size from west to east. On the contrary, Dietz et al. (2006) did not find significant regional differences after analyzing the wing morphology of the greater horseshoe bat. Within mammals, geometric morphometrics has been applied mostly in rodents, primates and carnivores, but has also been used to describe cranial size and shape variation in bats, e.g. Myotis auriculus and Myotis evotis (Gannon and Rácz, 2006), Myotis nigricans (Bornholdt et al., 2008), Myotis myotis, Myotis blythii and Myotis punicus (Evin et al., 2008), Monophyllus redmani (Mancina and Balseiro, 2010) and Artibeus lituratus (MarchánRivadeneira et al., 2012). Recently, Santana and Lofgren (2013) analyzed cranial modularity in 22 species of rhinolophid bats, as well as skull shape variation in relation to habitat across rhinolophids. Besides crania and mandibles, bat wings also represent morphological structures suitable for the application of geometric morphometric techniques. Thus, Birch (1995) compared wing shape among four species of bats, while De Camargo and de Oliveira (2012) studied sexual dimorphism in Sturnira lilium wings. In this study we used geometric morphometric methods to analyze cranial size and shape variation of bat populations inhabiting different areas (the Carpatho–Balkanides, the Internal and External Dinarides). Regarding environmental factors, studied regions are characterized by similar topographic relief (limestone), but the External Dinarides region is different from the Internal Dinarides and the Carpatho–Balkanides in respect to the climate type (Mediterranean/sub-Mediterranean versus continental/mountainous) and vegetation (Mediterranean/subMediterranean deciduous woodlands and shrubs versus continental deciduous and coniferous woodlands). Climatic data of the three geographic regions are given in Table 1. As flying animals, bats are characterized by a high potential for gene exchange between populations. However, considering the biology of the greater horseshoe bat (sedentary and philopatric), we can assume that gene flow may be greater among nearby populations and therefore, genetic exchange is more likely to occur among populations from one geographic region, than among those from different regions. Data collected from bat banding from the Centre for Animal Marking in Belgrade showed that movements of R. ferrumequinum were within, but not among geographic regions (Hutterer et al., 2005). Accordingly, we expect to find size and shape differences between bats from three geographic regions. As already mentioned, previous studies reported sexual dimorphism in body weight, external (Caubère et al., 1968; De Paz, 1995a; Dietz, 2007; Dietz et al., 2006) and wing measurements (De Paz, 1995a; Dietz, 2007; Dietz et al., 2006). Therefore, we examined the existence of morphological differences between the sexes. Finally, we tested whether the effect of size on shape (allometry) contribute to overall shape variation.

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Fig. 1. Geographic distribution of analyzed localities of the greater horseshoe bat (Rhinolophus ferrumequinum) from Serbia and Montenegro (see Appendix).

2. Materials and methods 2.1. Sample composition and data collection A total of 181 crania of the greater horseshoe bat (R. ferrumequinum) from Serbia and Montenegro were analyzed (Fig. 1). All specimens were from the collection of the Natural History Museum (Belgrade, Serbia). Overall, we examined 60 (25 males and 35 females) crania from the Carpato–Balkanides, 99 (42 males and 57 females) from the Internal Dinarides and 22 (12 males and 10 females) from the External Dinarides. Detailed list of localities and sample sizes is given in Appendix. Most bat species finish their growth in age of 6–10 weeks, and R. ferrumequinum has been shown to grow full skeletal size in about 60 days (Dietz et al., 2007; Hoying and Kunz, 1998; Ransome, 1989). Full cranial suture closure and eruption of all adult dentition were criteria for discrimination of juveniles from adults. Only adult specimens were analyzed. Digital images (4000 × 3000 pixels resolution) of crania in the ventral view were obtained using Canon PowerShot SX20 IS. Crania were supported by modeling clay with the palate positioned parallel to the photographic plane. Eighteen two-dimensional landmarks, selected to describe the overall shape of the analyzed morphological structure, were digitized using TpsDig software (Rohlf, 2010) (Fig. 2). Landmarks (definitions see Table 2) were collected only on the right side of the cranium to avoid redundant information in symmetric structures. Landmarks were of three different types (type I – points defined by the juxtaposition of different

Fig. 2. Landmarks recorded on the cranium in ventral view of the greater horseshoe bat (Rhinolophus ferrumequinum). See Table 2 for landmark definitions.

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I. Budinski et al. / Zoologischer Anzeiger 254 (2015) 8–14

Table 1 Mean values of 19 climatic factors extracted from WorldClim (Hijmans et al., 2005) for three geographic regions. C-B – the Carpatho–Balkanides; ID – the Internal Dinarides; ED – the External Dinarides.

Annual mean temperature [◦ C] Mean monthly temperature range [◦ C] Isothermality Temperature seasonality Max temperature of warmest month [◦ C] Min temperature of coldest month [◦ C] Temperature annual range [◦ C] Mean temperature of wettest quarter [◦ C] Mean temperature of driest quarter [◦ C] Mean temperature of warmest quarter [◦ C] Mean temperature of coldest quarter [◦ C] Annual precipitation [mm] Precipitation of wettest month [mm] Precipitation of driest month [mm] Precipitation seasonality Precipitation of wettest quarter [mm] Precipitation of driest quarter [mm] Precipitation of warmest quarter [mm] Precipitation of coldest quarter [mm]

C-B

ID

ED

9.47 9.22 31.08 770.49 25.37 −4.29 29.66 16.94 1.19 18.61 -0.28 694.00 89.44 43.61 25.47 238.94 22.06 6.81 145.61

10.38 9.47 32.00 748.50 25.68 −3.90 29.58 17.55 5.87 19.25 0.80 787.63 92.17 49.08 21.29 257.08 20.66 31.50 177.62

12.00 8.85 31.83 698.33 26.82 −1.00 27.82 6.63 20.50 20.50 3.43 1274.50 163.50 50.50 31.55 449.00 139.82 31.03 402.75

tissues, type II – points of maximum curvature and the innermost points of concavities and type III – anteriormost or posteriormost points of a structure) after Bookstein (1991). 2.2. Geometric morphometrics and statistical analyses The landmark coordinates were superimposed using Generalized Procrustes Analysis (GPA) (Dryden and Mardia, 1998; Rohlf, 1999; Rohlf and Slice, 1990) to eliminate differences due to the scale, position, and orientation, and to extract size variables (centroid size – CS) and shape variables (Procrustes coordinates) from landmark data. Variation in size between the sexes and the geographic regions was explored by analysis of variance (ANOVA) of centroid size. Multiple regression models were used to explore the effects of climatic factors on the cranial size variation. Environmental data were extracted from 19 WorldClim climatic layers (Hijmans et al., 2005). Starting from the most extensive regression model, that incorporates all 19 climatic factors as explanatory variables, backward selection was performed toward the narrowest, and the best fitting model, that includes only the significant factors. To test for Table 2 Definitions of landmarks digitized on the cranium in ventral view of the greater horseshoe bat (Rhinolophus ferrumequinum). 1 2 3 4 5 6 7 8 9 10 11

12 13 14 15 16 17 18

Anteriormost point of the maxilla Anteriormost point of the first molar Premaxillary incisura Anteriormost point of the second molar Mandibular arcade incisura Palatal incisura Anteriormost point of the zygomatic arch Anteriormost point of the foramen ovale Lateralmost point of zygomatic arch Proximal point of articular fossa Lateral margin of articular fossa where it intersects outline of the temporal bone in photographic plane Posteriormost intersection between tympanic bulla and cochlea Proximal point of the cochlea Skull lateral extremity in the level of auditory region Anterior limit of foramen magnum Tip of paraoccipital process Posterior limit of foramen magnum Posteriormost point of cranium

significant variation in shape, we performed a multivariate analysis of variance (MANOVA) with Procrustes coordinates as the dependent variables, and sex and geographic region as the independent variables. Subsequently, we performed MANOVA using reduced number of variables, i.e. the first 20 principal components (PCs) that explained 90.9% of total variance, to examine the potential sensitivity of parametric test to the number of shape variables (Hair et al., 1998; Seetah et al., 2012). Since the results of this MANOVA were very similar to those of previous one, all further analyses were done on Procrustes coordinates. To decide whether to pool sexes or not, we constructed unweighted pair-group method using arithmetic averages (UPGMA) diagram from the matrix of Procrustes distances calculated between mean male and female cranial shape within each geographic region. Procrustes distance is an absolute measure of the magnitude of shape difference between two configurations (Klingenberg et al., 2003). In addition, we performed multivariate regression of shape variables onto the sex coded as dummy variable (Cardini and Elton, 2008) within each geographic region. The significance of these regressions was assessed by using a permutation test with 10,000 iterations against the null hypothesis of independence between sex and shape (Good, 1994). To assess shape variation among the analyzed geographic regions, we used Canonical Variate Analysis (CVA). All shape variables were regressed onto the first two CV axes and a scatter plot was displayed. Shape changes along CV1 axis were illustrated by thin plate spline (TPS) deformation grids. Additionally, Procrustes distances were obtained by pairwise comparisons of the mean shapes of specimens from different geographic regions. The statistical significance of the observed Procrustes distances for all pairwise comparisons was estimated with a permutation test against the null hypothesis of no mean difference between groups (Good, 1994). Subsequently, Bonferroni correction was made. Allometric effect on shape variation was examined by multivariate regression of Procrustes coordinates onto log-transformed CS (log CS) using a permutation test with 10,000 iterations against the null hypothesis of independence between size and shape (Good, 1994). The amount of allometric shape variation was quantified as a percentage of the total shape variation. Shape differences caused by allometry were visualized with TPS deformation grids. To test the homogeneity of regression slopes among bats from the analyzed geographic regions, multivariate analysis of covariance (MANCOVA) was performed with shape variables as dependent variables, geographic region as the categorical factor and log CS as the covariate.

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To examine covariation between shape and environmental factors we used a two-block partial least squares (2B-PLS) method with shape variables as the first block and Z-transformed climatic data as the second block of variables. As shape variables, for overall shape variation we used Procrustes coordinates, while for non-allometric component of shape variation we used residuals from the multivariate regression of Procrustes coordinates on logtransformed CS. RV coefficient, a multivariate analog of the squared correlation (Escoufier, 1973), represents an overall measure of association between the blocks. The significance level for the correlation between the blocks was evaluated using a permutation test with 10,000 iterations. Statistica v. 5.1 (StatSoft Inc, 1997) was used for ANOVA, MANOVA, MANCOVA and multiple regression models, whereas all other analyses were carried out using MorphoJ v. 1.06a software (Klingenberg, 2011). 3. Results 3.1. Size variation Statistically significant differences in cranial centroid size among the bats from analyzed geographic regions were observed, while the effects of sex and sex × geographic region interaction were not statistically significant (Table 3). Crania of bats from the External Dinarides were the largest, followed by those from the Internal Dinarides, while the smallest were found in bats from the Carpatho–Balkanides populations (Fig. 3). The post hoc Tukey honest significant difference test for unequal sample sizes (Tukey-uss test) revealed that all region pairs showed statistically significant (P < 0.05) size differences for the examined morphological structure, except the Internal versus the External Dinarides pair. The most pronounced differences in size were between bats from the Carpatho–Balkanides and the External Dinarides (P = 0.0006). Table 3 Analysis of variance (ANOVA) for centroid size. Effect

SS

df

F

P

Sex Geographic region Sex × geographic region Error

0.0497 7.6819 0.5262 67.6821

1 2 2 175

0.13 9.93 0.68

0.7205 0.0001 0.5078

Fig. 3. Plot of centroid size means, standard deviations and standard errors for bats from three geographic regions (ID – the Internal Dinarides; C-B – the Carpatho–Balkanides; ED – the External Dinarides). Significance levels at *P < 0.05 and **P < 0.001 after Tukey-uss test; ID vs. ED not significant.

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The best fitting multiple regression model incorporated four climatic factors as statistically significant: mean monthly temperature range (P = 0.0012), isothermality, i.e. mean monthly temperature range/annual temperature range (P = 0.0026), minimal temperature of the coldest month (P = 0.0023), and maximal temperature of the warmest month (P = 0.0014). The centroid size enlarges with the increase of maximal temperature of the warmest month and isothermality, as well as with the decrease of minimal temperature of coldest month and the narrowing of the mean monthly temperature range. 3.2. Shape variation Multivariate analysis of cranial shape variation unveiled statistically significant differences among the analyzed geographic regions (Table 4). This analysis also showed significant sexual dimorphism. However, the UPGMA diagram (not shown), constructed from the matrix of Procrustes distances calculated between mean male and female cranial shape within each geographic region, revealed that the sexes of each geographic region first grouped together. Thus, cranial shape differences among bats from different geographic regions exceeded those between sexes. Additionally, after performing multivariate regressions of shape variables onto the sex coded as dummy variable we found absence of sexual shape dimorphism (P < 0.05) within each geographic region. Accordingly, the sexes were pooled throughout all following analyses. Subsequently performed multivariate analyses also disclosed shape variation among the analyzed geographic regions. The External Dinarides and the Carpatho–Balkanides tend to segregate along the first CV axis (Fig. 4). When compared with bats from the Carpatho–Balkanides, specimens from the External Dinarides had skulls elongated in the facial region, enlarged in the Table 4 Multivariate analysis of variance (MANOVA) for the shape variables. Effect

Wilks

df1, df2

F

P

Sex Geographic region Sex × geographic region

0.7341 0.4797 0.6291

32, 144 64, 288 64, 288

1.63 2.00 1.17

0.0280 0.0001 0.1916

Fig. 4. Canonical variate analyses (CVA) scatter plot. ID – the Internal Dinarides; C-B – the Carpatho–Balkanides; ED – the External Dinarides. Shape changes are presented in the form of TPS deformation grids along CV1 axis. For better perception of differences in form, shape changes are magnified three times.

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Fig. 5. Allometric shape variation. Shape changes are presented in the form of TPS deformation grids amplified by a factor of 3. ID – the Internal Dinarides; C-B – the Carpatho–Balkanides; ED – the External Dinarides.

temporal and reduced in the basicranial region. Additionally, pairwise comparisons of the analyzed geographic regions for cranium revealed statistically significant Procrustes distances between the mean shapes of specimens from the Carpatho–Balkanides and the External Dinarides (Pd = 0.0100; P = 0.0024), as well as between those from the Carpatho–Balkanides and the Internal Dinarides (Pd = 0.0072; P = 0.0003). Multivariate regression of shape variables on log-transformed CS revealed that shape changes were significantly (P < 0.0001) correlated with changes in size. Allometry explained 2.4% of the total shape variation. With increasing cranial size facial and temporal regions became elongated, whereas, due to antero-posterior and medio-lateral narrowing, the basicranial region was reduced (Fig. 5). The results of MANCOVA showed that interaction of geographic region and log-transformed CS was not significant for cranial shape (Wilks = 0.6872, F64,288 = 0.93, P = 0.6313), indicating that allometric slopes were homogenous among the analyzed geographic regions (Fig. 5). For overall shape variation, 2B-PLS analysis showed that the association between cranial shape and climate variables was statistically significant (RV = 0.06; P = 0.0152). Subsequently performed 2B-PLS analysis using residuals from the multivariate regression of shape variables on log-transformed CS revealed that the association between non-allometric component of shape variation and climate variables was not statistically significant (RV = 0.05; P = 0.1321). 4. Discussion To the best of our knowledge, this study is the first that used geometric morphometric methods to describe size and shape variation of the cranium in R. ferrumequinum. Although examining bats from a relatively small territory, it has provided new insights into important issues like geographic variation, sexual dimorphism and allometry in this species. Analysis of variance revealed statistically significant differences in cranial centroid size among specimens of the greater horseshoe bat from distinct geographic regions. Previous results indicated a clinal size increase in the greater horseshoe bat from west to

east (Caubère et al., 1968; De Paz, 1995a, 1995b; Kryˇstufek, 1993; Palmeirim, 1990). A similar pattern of clinal size variation appears in other bats, e.g. M. myotis, M. blythii, Myotis nattereri (Benda and Horaˇcek, 1995), Rhinolophus euryale and Rhinolophus blasii (Popov and Ivanova, 2002). On the other hand, we observed that bats from the External Dinarides (specimens from the south-western parts) have the largest, while those from the Carpatho–Balkanides (specimens from the north-eastern parts) have the smallest crania. However, it is worth noting that the sample of R. ferrumequinum analyzed herein is restricted geographically, and consequently patterns of longitudinal and latitudinal size variation were difficult to detect. In some bat species, e.g. Pipistrellus pipistrellus (Stebbings, 1973), Eptesicus fuscus (Burnett, 1983), Myotis daubentonii (Bogdanowicz, 1990), Cynopteryx sphinx (Storz et al., 2001) and Rhinolophus mehelyi (Dietz et al., 2006) larger individuals were found in the cooler areas. Such a pattern of morphological variation could be related to the influence of climatic factors on bat growth and development, according to predictions of Bergmann’s rule. On the other side, Kryˇstufek (1993) found that specimens of R. ferrumequinum from the warmer areas were the largest. Our results are concordant with his findings, cranial size was increasing with higher maximal temperature of the warmest month. Other authors gave possible explanations of such trend. Temperatures in April and May determine pregnancy duration, birth timing and postnatal development of greater horseshoe bats (Ransome, 1973). Therefore, specimens from cooler areas (northern parts and mountains) are born later and have a shorter time to finish postnatal development and enter hibernation compared to those from warmer areas. Moreover, juveniles must deposit fat reserves before winter (Kunz et al., 1998) and prey availability depends on climatic factors, i.e. lower temperatures and strong precipitation decrease insect densities (Taylor, 1963). Thus, bats living in such environments are more likely to be smaller (Dietz et al., 2007). Additionally, Burnett (1983) found that precipitation had a greater influence on wing and skull size of E. fuscus than did the temperature, while Marchán-Rivadeneira et al. (2012) reported that skull size in A. lituratus increased as the amount of precipitation during the driest season increased. On the other hand, we found that four climatic factors related to temperature have statistically significant effects on the skull size variation. Therefore, temperature had a greater influence on R. ferrumequinum skull size than did the precipitation. Although multivariate analysis of variance disclosed statistically significant cranial shape variation in greater horseshoe bats from the analyzed geographic regions, subsequent multivariate analyses revealed only a tendency for geographic region separation. In addition, we found no statistically significant cranial shape differences between bats from the Internal and External Dinarides. Compared to specimens from the Carpatho–Balkanides, bats from the other two regions have crania wider in the temporal and lengthened in the facial region, while the basicranial region is smaller. Although similar, these shape differences are more pronounced between bats from the External Dinarides and the Carpatho–Balkanides. Analysis of size variation showed no significant differences between the sexes, while multivariate analysis disclosed statistically significant sexual shape dimorphism. However, we found absence of sexual shape dimorphism within each geographic region. Our results for sexual size dimorphism are in agreement with those reported by DeBlase (1980) and De Paz (1995a). DeBlase (1980) failed to find statistically significant differences between the sexes for nine cranial characters. De Paz (1995a) documented a significant difference between males and females of the greater horseshoe bat for only one (ramus mandibulae) out of 48 morphological traits. On the other hand, examination of linear wing measurements showed that females of R. ferrumequinum were significantly larger than males in all five characters (Dietz et al., 2006).

I. Budinski et al. / Zoologischer Anzeiger 254 (2015) 8–14

Analyses of wing area and wing loading measurements in European horseshoe bats (Dietz, 2007) and S. lilium (De Camargo and de Oliveira, 2012) unveiled that females have a larger handwing area than males. However, a study of 50 dental and cranial measurements in R. euryale (Popov and Ivanova, 2002) revealed the occurrence of slight but significant sexual dimorphism, all characters being larger in males. Biometric investigation of M. myotis and M. blythii showed that the cranial measurements were, on average, larger in males than in females, whereas forearm length was greater in females (Benda, 1994). Longer forearms in females were also noted for Myotis lucifugus and Myotis volans (Saunders and Barclay, 1992). Qumsiyeh (1985) and Romero (1990) found that bat females from the family Rhinolophidae tended to be larger than males. The same pattern of sexual dimorphism has been described for vespertilionid bats, where females had a larger body size or larger wings in species in which significant differences between the sexes had been found (De Camargo and de Oliveira, 2012; Findley and Traut, 1970; Myers, 1978; Solick and Barclay, 2006; Williams and Findley, 1979). According to the “Big mother” hypothesis, bat females need to fly with and nourish large fetuses and sometimes newborn bats (Myers, 1978; Ralls, 1976). This could be the most common selective pressure favoring larger size in females compared to males. In spite of data on sexual size dimorphism of body and wing measurements in R. ferrumequinum, we did not obtain size differences between males and females for cranium. Thus, we assume that in this species, developmentally and functionally different morphological structures, such as skulls and wings, are under different sex specific selective pressures. Likewise, after analyzing size and shape dimorphism in body regions (e.g. cranium and pelvis) with different sex specific functions (e.g. display in mating, locomotion and reproduction) of two fox species within the genus Urocyon, Schutz et al. (2009) showed that sexual dimorphism patterns were not uniform throughout an organism. Allometry could contribute significantly to overall shape variation of morphological structures. Thus, one of our goals was to estimate whether allometric growth has an influence on variation of cranial shape in R. ferrumequinum. We found that allometry accounted for a small, but significant percentage of the total cranial shape variation. Investigating geographic variation and sexual dimorphism in M. redmani, Mancina and Balseiro (2010) detected no significant influence of size on mandibular shape. De Camargo and de Oliveira (2012) analyzed sexual dimorphism in S. lilium and they also found no significant allometric effect on wing length, shape and measurements of wing area. In addition, we showed that bats from distinct geographic regions follow the same allometric scaling pattern, i.e. they have uniform regression slopes. The main cranial shape features associated with allometry (larger specimens are characterized by crania with a reduced basicranial region, an elongated facial and a wider temporal region) are similar to cranial shape differences observed among bats from different geographic regions. These allometric shape changes also correspond to the common shape changes during the postnatal skull development in tetrapods (Emerson and Bramble, 1993). We showed that size varies with temperature, allometry contributes to overall shape variation and there is a covariation between cranial shape and climate variables. However, after removing the effect of allometry, shape does not covary with climatic factors. Therefore, it seems that climate does not have size-independent effect on cranial shape variation, i.e. allometry somehow contributes to covariation between cranial shape and environmental variables. Geographic variation and sexual dimorphism in R. ferrumequinum have been studied previously, but, as far as we know, the allometric effect in this species was tested for the first time herein. In the light of the presented results and related literature, as well as

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from the perspective of future studies, we would like to emphasize the following. First, morphological differences among bats from the analyzed geographic regions could indicate variation at the molecular level and warrant further conservation and management of this strictly protected bat species. Second, sexual dimorphism patterns throughout different body regions (e.g. skull and wing) of the greater horseshoe bat could be examined per se. Finally, we suggest that the effect of allometry should be taken into consideration in any future morphological study in this species. Acknowledgements This study was supported by the Ministry of Education, Science and Technological Development of Serbia, Grant No. 173003. The authors are grateful to Tanja Vukov for preparation of Fig. 1 and ˇ Aleksa Cepi c´ for provided climatic data of the analyzed geographic regions. Appendix. Specimens analyzed in this study. The following information is presented for each specimen: geographic region, locality, geographic coordinates, sex (m: male; f: female). Carpatho–Balkanides: Golubac 44◦ 39 N, 21◦ 40 E (11f); Dankina ´ ´ pecina 44◦ 32 N, 22◦ 14 E (4f); Gradaˇsniˇcka pecina 44◦ 27 N, 22◦ 13 ◦  ◦  ˇ ´ E (3 m); Pecina kod Strbaˇcke sˇ kole 44 33 N, 22 15 E (2f); Duboˇcka ´ pecina 44◦ 4 N, 21◦ 16 E (3f); Ceremoˇsnja 44◦ 22 N, 21◦ 38 E (3m,1f); ´ Peˇstera Mare 44◦ 16 N, 21◦ 59 E (6m, 1f); Piˇstolj pecina 44◦ 22 N, ´ 21◦ 54 E (1f); Rajkova pecina 44◦ 26 N, 21◦ 57 E (1f); Jabukovac ´ 44◦ 5 N 21◦ 37 E (4m, 2f); 44◦ 20 N, 22◦ 22 E (2f); Radoˇseva pecina ◦  ◦  ´ ´ Suvajska pecina 44 5 N, 21 38 E (1f); Vlaˇska pecina 44◦ 4 N, 21◦ 42 E (1m, 1f); Vernjikica 44◦ 1 N, 21◦ 58 E (7m, 1f); Markova reka 44◦ 5 N, 22◦ 1 E (1f); Ravna pec´ 43◦ 23 N, 22◦ 20 E (1m, 1f); Prekonoˇska ´ pecina 43◦ 23 N, 22◦ 6 E (1f); Kalna 43◦ 24 N, 22◦ 25 E (1f). ˇ Internal Dinarides: Topˇcider 44◦ 47 N, 20◦ 26 E (3m, 19f); Suplja ˇ c´ u Crvenom bregu 44◦ 34 N, stena 44◦ 41 N, 20◦ 31 E (1m, 11f); Stoli 20◦ 37 E (1f); Ripanj 44◦ 38 N, 20◦ 32 E (2m); Ljuta Strana 44◦ 37 N, 20◦ 28 E (2m, 2f); Mali sˇ tol kod rudnika Kosmaj 44◦ 31 N, 20◦ 32 E (3m, 2f); Guberevac 44◦ 32 N, 20◦ 28 E (1m, 4f); Risovaˇca 44◦ 18 N, 20◦ 34 E (2f); Rudarski potkop Jezero 44◦ 8 N, 20◦ 31 E (5m, ´ 2f); Rudnik olova i cinka 44◦ 8 N, 20◦ 33 E (1f); Valjevska pecina ´ ´ 44◦ 16 N, 19◦ 53 E (3m, 1f), Tmuˇsa 44◦ 9 N, 19◦ 52 E (6m, 3f); Cebi ca ´ ´ ´ pecina 44◦ 15 N, 19◦ 49 E (3m, 1f); Bacina pecina 44◦ 13 N, 19◦ 51 E ´ ´ 44◦ 14 N, 19◦ 55 E (2m, 1f); Visoka pecina (1m, 1f); Petniˇcka pecina ´ 44◦ 13 N, 19◦ 51 E (2m); Krevetara pecina 44◦ 13 N, 19◦ 51 E (1m, ´ ´ 44◦ 14 1f); Varoˇs pecina 44◦ 14 N, 19◦ 52 E (1f); Tunel kod Degurica ◦  ◦  ◦ N, 19 53 E (1m); Tunel u kanjonu Suˇsice 44 9 N, 19 47 E (1f); ˇ ckovcu 44◦ 8 N, 19◦ 56 E (1f); Prozorˇcara pecina ´ 44◦ 9 Tunel u Ciˇ ◦  ◦  ◦  ´ N, 19 53 E (1m); Ploˇcara pecina 44 8 N, 19 56 E (1m); Ribniˇcka ´ ´ 44◦ 12 N, 20◦ 5 E (3m, 1f); Megara pecina 43◦ 50 N, 19◦ 45 E pecina (1m, 1f). ´ 41◦ 54 N, 19◦ 13 (2m, 6f); External Dinarides: Sumporna pecina ´ ´ Obodska pecina 42◦ 21 N, 19◦ 1 E (1m, 1f); Pecina na ulazu u Rugov´ na izvoru Belog Drima sku klisuru 42◦ 39 N, 20◦ 15 E (1m); Pecina 42◦ 41 N, 20◦ 16 E (8m, 3f). References Benda, P., 1994. Biometrics of Myotis myotis and Myotis blythii: age variation and sexual dimorphism. Folia Zool. 43, 297–306. Benda, P., Horaˇcek, I., 1995. Geographic variation in three species of Myotis (Mammalia, Chiroptera) in South of the Western Palearctic. Acta Soc. Zool. Bohem. 59, 17–39. Birch, J.M., 1995. Comparing wing shape of bats: the merits of principal components analysis and relative warp analysis. J. Mammal. 78, 1187–1198. Bogdanowicz, W., 1990. Geographic variation and taxonomy of Daubenton’s bat, Myotis daubentoni, in Europe. J. Mammal. 71, 205–218.

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