Morphological Variation of the Five Vole Species of the Genus Microtus

Acta Zoologica (Stockholm) 90: 254–264 (July 2009) doi: 10.1111/j.1463-6395.2008.00348.x

MorphologicalBlackwell Publishing Ltd variation of the ﬁve vole species of the genus Microtus (Mammalia, Rodentia, Arvicolinae) occurring in Greece Stella E. Fraguedakis-Tsolis, Basil P. Chondropoulos, Costas V. Stamatopoulos and Sinos Giokas

Abstract Section of Animal Biology, Department of Fraguedakis-Tsolis, S.E., Chondropoulos, B.P., Stamatopoulos, C.V. and Biology, University of Patras, GR-26500 Giokas, S. 2009. Morphological variation of the five vole species of the genus Patras, Greece Microtus (Mammalia, Rodentia, Arvicolinae) occurring in Greece. — Acta Zoologica (Stockholm) 90: 254–264 Keywords: Greece, Microtus, morphometrics, Morphometric data for the five vole species of the genus Microtus living in Rodentia, Terricola Greece are old, sparse, poor and insufficiently analysed. This work aims to give the first comprehensive morphometric analysis of body and skull inter- Accepted for publication: and intraspecific variation for M. (M.) guentheri, M. (M.) rossiaemeridionalis, 13 June 2008 M. (Terricola) subterraneus, M. (T.) felteni and M. (T.) thomasi, applying multivariate statistics to 28 linear morphometric variables. It was based on ample material (202 adult individuals) using samples from localities that adequately cover the entire distributional range of each species in Greece. The five species and the two subgenera (Microtus and Terricola) were morphometrically clearly distinguished and discriminating variables were revealed. However, morphometrics did not provide robust criteria to infer phylogenetic relations among species. Furthermore, three species, M. (M.) guentheri, M. (M.) rossiaemeridionalis and M. (T.) thomasi, exhibited considerable intraspecific size or shape variation, which was mostly random and not associated with geographical proximity. Comparisons with data in the literature, mainly concerning populations of these species from adjacent areas, indicate that the Greek M. (M.) guentheri, M. (M.) rossiaemeridionalis and M. (T.) thomasi tend to be smaller than their conspecifics, while M. (T.) subterraneus and M. (T.) felteni are about equal in size to their Balkan relatives. Sinos Giokas, Section of Animal Biology, Department of Biology, University of Patras, GR-26500 Patras, Greece. E-mail: [email protected]

makes it ideal for evolutionary studies examining inter/ Introduction intraspecific geographical variability, adaptive convergence Microtus Schrank 1798 is one of the most speciose, differentiated and divergence, and identification of sibling species. and widespread mammalian genera (Mitchell-Jones et al. According to recent knowledge (Mitchell-Jones et al. 1999; Musser and Carleton 2005). However, the phylogenetic 1999), five vole species of the genus Microtus live in Greece relationships within Microtus and its closest relatives, based belonging to the European subgenera Microtus and Terricola initially on dental and skull morphology and karyotypes, are (although there are objections to the status of Terricola – uncertain and some difficulties remain both in delimiting Kryßtufek et al. 1996); these are Microtus (Microtus) guentheri species and in defining subgenera even with the use of mole- (Danford & Alston 1880), M. (M.) rossiaemeridionalis Ognev cular approaches (Jaarola et al. 2004). Yet, it is noteworthy 1924, M. (Terricola) subterraneus (de Sélys-Longchamps 1836), that the genus Microtus displays a number of features that M. (T.) felteni Malec & Storch 1963 and M. (T.) thomasi

Acta Zoologica (Stockholm) 90: 254–264 (July 2009) Fraguedakis-Tsolis et al. • Morphometrics of the Greek Microtus voles

previous morphometric attempts did not partition efficiently size from shape components. The present work aims to give the first comprehensive morphometric analysis of body and skull variation for these five vole species in Greece. This analysis, using multivariate statistics, is based on ample material consisting of samples from localities that adequately cover the entire distributional range of each species in Greece. An effort was also made to compare our morphometric data with those available in the literature. This approach allows us not only to investigate the morphometric and geographical variation of these Microtus species in Greece, and to examine if such morphometrics are sufficient in providing a reliable identity for each species or population, but also to identify possible morphological size and shape adaptations, useful in understanding the diversifica- tion processes of these small mammals.

Fig. 1—Map showing the distributional borders of the five Materials and Methods Microtus species in Greece...... M. (M.) guentheri, ------M. (M.) rossiaemeridionalis, __ __M. (Terricola) subterraneus, Sampling and variables ___M. (T.) felteni and ___.. M. (T.) thomasi. A total of 202 adult individuals of the five Microtus species studied were collected from 40 localities, in an effort to adequately cover their variation. Because of the small sample (Barrett-Hamilton 1903). The geographical distribution size of M. (M.) rossiaemeridionalis and M. (T.) thomasi of these species in mainland Greece shows considerable collected from some of these localities, we pooled the material differences in the extent of their distribution and sympatry coming from the same physiographic/geographical unit to (Fig. 1). Specifically, M. (T.) thomasi is the most common form larger groups for the purposes of the statistical analysis. and widespread species, living from Western Makedonia in Information about the location (geographical and regional the north to the southernmost part of Peloponnisos; origin) and the size of each sample are presented in Table 1. M. (M.) guentheri occurs in most parts of the northern The following 28 linear morphometric variables (four and eastern Greek mainland except Peloponnisos; M. (M.) external and 24 cranial) were analysed: head and body length rossiaemeridionalis is distributed in the northern parts of the (HB), tail length (TL), ear length (EL), hind foot length country (Ipeiros, Makedonia, Thraki); the remaining two (HFL), condylobasal length (CBL), basal length (BL), species, M. (T.) subterraneus and M. (T.) felteni, have a condyloincisive (condylobasilar) length (CIL), occipitonasal restricted range confined to the mountainous north-central length (ONL), upper diastema length (UDL), incisive continental part of Greece (Ondrias 1966; Niethammer foramen length (IFL), nasal length (NL), upper molar row 1982a,b,c,d; Petrov and RuΩic 1982; Vohralík and Sofianidou length (at the alveoli level) (UMR), upper molar row length 1987, 1992; Sofianidou and Vohralík 1991; Mitchell-Jones (at the crowns level) (UMRc), tympanic bulla length (TBL), et al. 1999). The only Greek islands inhabited by voles are interorbital width (interorbital constriction) (IOW), occipital Lesvos, where M. (M.) guentheri is known to occur (Stam- width (OW), palatal length (PL), zygomatic width (ZW), atopoulos and Ondrias 1995; Mitchell-Jones et al. 1999), brain-case height (from the tympanic bulla) (BCHb), brain-case and Evvoia, where M. (T.) thomasi is distributed (Niethammer height (from the foramen of the tympanic bulla) (BCHf), 1982d; Mitchell-Jones et al. 1999). It is noteworthy that the brain-case width (BCW), rostral height (at the alveolus of the southernmost distributional borders of these five Microtus M1 (ROH1), rostral height (at the middle of the upper species are found in Greece. Furthermore, M. (T.) felteni molars) (ROH), mastoid width (MW), mandibular length and M. (T.) thomasi are endemic to the southern Balkans (ML), lower molar row length (at the alveoli level) (LMR), (Niethammer 1982c,d; Mitchell-Jones et al. 1999). lower molar row (at the molar crowns) (LMRc) and articular The morphometric data for these vole species in Greece height (AH). The above variables are commonly used in are rather old, sparse, limited and insufficiently analysed morphometric analyses of voles and are illustrated in (Ondrias 1965, 1966; Spitz 1978; Niethammer and Krapp Niethammer and Krapp (1978) and Ventura and Gosálbez 1982; Brunet-Lecomte and Nadachowski 1994; Tsekoura (1989). The four external variables were measured with a et al. 2002), mainly because of the small numbers of specimens ruler to the nearest 0.5 mm while the remaining variables examined and variables measured, and the few populations were measured using a digital vernier calliper (Mitutoyo Ltd, sampled within each species range. Furthermore, these UK) to the nearest 0.01 mm. All measurements for our

Morphometrics of the Greek Microtus voles • Fraguedakis-Tsolis et al. Acta Zoologica (Stockholm) 90: 254–264 (July 2009)

Table 1 Sampling localities and the respective number of individuals examined for each Microtus species

Species Sampling localities and number of individuals (in parentheses)

M. (M.) guentheri Paranesti (Drama Pref.), Eastern Makedonia (4); Amyntaio (Florina Pref.), Western Makedonia (7); Sykourio (Larisa Pref.), Thessaly (8); Gravia (Fokida Pref.), Sterea Ellada (15). M. (M.) rossiaemeridionalis Protokklisi (Evros Pref.) and Kremasti (Xanthi Pref.), Thrace (8); Lagkadikia (Thessaloniki Pref.) and Olynthos (Chalkidiki Pref.), Central Makedonia (6); Sevastiana (Pella Pref.) and Kopanos (Imathia Pref.), Western Makedonia (27); Arnissa (Pella Pref.) and Kato Vermio (Imathia Pref.), Western Makedonia (8); Pisoderi (Florina Pref.), North-western Makedonia (3). M. (T.) subterraneus Lailias, Vrontous Mts., Eastern Makedonia (6); Vorras Mt, Western Makedonia (18); Vermio Mt, Western Makedonia (10). M. (T.) felteni Pisoderi (Florina Pref.), Western Makedonia (7). M. (T.) thomasi Edessa (Pella Pref.), Agios Georgios (Imathia Pref.) and Skra (Kilkis Pref.), Central-Western Makedonia (14); Vradeto, Baldouma and Dodoni (Ioannina Pref.), and Gorgomylos (Preveza Pref.), Central Ipeiros (13); Kalodiki (Preveza Pref.) and Kalamas river estuaries (Thesprotia Pref.), Western Ipeiros (4); Elati and Pertouli (Trikala Pref.), and Neraida (Karditsa Pref.), Western Thessalia (8); Stanos, Prodromos, Astakos, Agrinio and Paravola (Aitoloakarnania Pref.), Western Sterea Ellada (10); Karpenisi (Evrytania Pref.), Teichio (Fokida Pref.) and Pavliani (Fthiotida Pref.), Central Sterea Ellada (13); Panachaiko Mt and Chelmos Mt (Achaia Pref.), Northern Peloponnisos (6); Taygetos Mt (Lakonia Pref.), Southern Peloponnisos (7).

samples were taken by the same researcher. To reduce errors of sexual dimorphism and geographical location for each because of cranial asymmetries, measurements on bilaterally species. For that reason sampling localities from the same symmetric structures were taken separately for each of them broader geographical area were pooled and eight geographical and their mean value was used. Descriptive statistics (mean, areas were used, i.e. Central-Western Makedonia, Central range, standard deviation) of these 28 variables for each Ipeiros, Western Ipeiros, Western Thessalia, Western Sterea Microtus species studied are presented in Appendix S1. Ellada, Central Sterea Ellada, Northern Peloponnisos and Southern Peloponnisos (see also Table 1). To reveal possible geographical patterns, the morphometric squared Maha- Analyses lanobis distances derived from CVA were used for a cluster To study the size-free shape differences between species (i.e. analysis (unweighted pair group method with arithmetic to remove the effect of a within-population size factor from mean; upgma) of the pooled populations of each species. The the between-population group morphometric comparison), goodness of fit of the cluster analysis (upgma) to the data was the morphometric data were analysed applying the Burnaby’s examined by an analysis of cophenetic values (Rohlf and size correction method (Burnaby Principal Component Sokal 1981). Finally, a Mantel-test was used to detect possible Analysis BPCA; Burnaby 1966; Rohlf and Bookstein 1987) correlation between the morphometric (Mahalanobis) and using the program burnaby pca (available from N. MacLeod, geographical distances (between broader distributional Natural History Museum, London http://www.nhm.ac.uk/ geographical areas) for each species. hosted sites/palaeonet/ftp/ftp.html). In the BPCA approach, The BPCA and CVA were also performed to compare the size and shape components are separated and a multivariate morphometric data of our study with those of the same species analysis of shape is accomplished, eliminating the contribution (except M. (M.) guentheri – see below) given in Niethammer of size to the second and following principal components (1982a,b,c,d) and Petrov and RuΩic (1982). These were the (shape components), so restricting the size component to only bibliographic data available in raw form. Only 13 common PC1 (PC, principal component). This procedure is considered characters were found between these literature sources and the most effective traditional morphometric method for our study, and so these were used for this comparison (HB, isolating shape from size variation (Bookstein et al. 1985; TL, HFL, CBL, UDL, IFL, NL, UMR, TBL, OW, ZW, ML Rohlf and Bookstein 1987). Furthermore, variability among and IOW). The M. (M.) guentheri data in Niethammer taxa and groups was tested using Canonical Variates Analysis (1982a) were not used because of the insufficient number of (CVA) on the adjusted data set. Morphological relationships examined characters held in common with our characters between taxa were also evaluated by Mahalanobis distances and the small number of specimens for which raw data are between means (Dillon and Goldstein 1984). given. In the latter analyses we used 279 specimens (202 of Multivariate analysis of variance (manova) and analysis of our survey plus 77 from the literature). For the statistical variance (anova) tests, using the resultant species-specific methods used in this study see Reyment et al. (1984), Morri- size and shape PCs, were used to identify possible differences son (1990), and Quinn and Keough (2002). Before the between species and between the two subgenera of Microtus. analyses, the data were examined for departures from statistical The manova tests, using also the resultant species-specific assumptions, and were adjusted when necessary. All analyses size and shape PCs, were further used to examine the effect were performed with SPSS for Windows v.13.

© 2008 The Authors 256 Journal compilation © 2008 The Royal Swedish Academy of Sciences Acta Zoologica (Stockholm) 90: 254–264 (July 2009) Fraguedakis-Tsolis et al. • Morphometrics of the Greek Microtus voles

Table 2 Standardized canonical variates analysis coefﬁcients for the 28 variables examined, using our samples of the ﬁve Microtus species

Variable CVI CV2

TL –0.636 0.096 ROH 0.512 –0.165 OW 0.451 0.322 ML 0.372 0.149 BCHB 0.352 –0.260 BCHF 0.244 –0.162 MW 0.237 –0.054 UDL 0.217 0.164 LMR 0.182 0.101 IFL 0.130 0.047 ZW 0.433 0.495 HFL –0.028 –0.413 TBL –0.054 –0.259 BCW 0.231 0.214 ONL 0.026 –0.303 ROH1 0.280 –0.198 CIL 0.159 –0.191 IOW 0.130 0.072 LMRC 0.203 0.141 PL 0.137 0.131 EL –0.221 –0.184 UMRC 0.097 0.154 UMR 0.037 0.091 NL –0.054 –0.055 CBL 0.147 –0.032 BL 0.112 –0.052 Fig. 2—A. BPCA plots, —B. size-free CVA plots for the ﬁve HB –0.088 0.084 Microtus species studied, based on our material only. AH 0.042 0.047

Most signiﬁcant weights in bold. Abbreviations deﬁned in Materials and Methods section. Results

Size and shape discrimination among species and subgenera important characters responsible for this discrimination are Both BPCA and size-free CVA discriminated the five TL, ROH, OW, ZW, ML and BCHb. The CV2, accounting Microtus species well (Figs 2 and 3) and resulted in similar for 29.9% of the within-species variation, discriminates the groupings, albeit more evident in CVA. In BPCA the first two subgenera of Microtus, that is M. (T.) thomasi, M. (T.) three axes (size PC1, and the size-free PC2 and PC3) subterraneus and M. (T.) felteni from M. (M.) rossiaemeridiona- accounted for 42.26%, 11.82% and 9.39% of the variance, lis and M. (M.) guentheri. The most important characters for respectively. The first two variates of CVA on the adjusted this separation were ZW, HFL, OW, ONL and BCHb data described an acceptable representation of population (Table 2, column CV2). From the between-species squared variation because of the high cumulative explained variance Mahalanobis distances presented in Table 3, we can see that (88.9%). Standardized canonical coefficients of the first two M. (T.) subterraneus and M. (T.) felteni were the most similar canonical variates (pooled within class) are given in Table 2. (d = 15.54). This was because of their size (and not shape) Figure 2(B) gives the plot of the first size-free canonical similarity (anova on size and shape PCs, Bonferroni post hoc variate (CV1) of each individual considered versus the test, P = 0.894) (Fig. 4a). second size-free one (CV2). It shows a marked morphological The a posteriori cross-validation classification matrix obtained, divergence between species that are well separated along the using linear discriminant functions in CVA, showed a correct first and the second discriminant axes. Most of the species classification in the expected species, ranging from 85.7% differentiation was accounted for by the CV1 (59.0%), which to 100% (mean 97.5%). An examination of each species separates M. (T.) thomasi and M. (M.) guentheri from M. (M.) revealed a correct specimens classification (100%) for M. (M.) rossiaemeridionalis, M. (T.) subterraneus and M. (T.) felteni. rossiaemeridionalis and M. (M.) guentheri. The absolute values of the standardized canonical coefficients anova tests, using the resultant species-specific size and shown in column CV1 (Table 2) indicate that the most shape PCs, revealed significant (P < 0.001) size and shape

© 2008 The Authors Journal compilation © 2008 The Royal Swedish Academy of Sciences 257 Morphometrics of the Greek Microtus voles • Fraguedakis-Tsolis et al. Acta Zoologica (Stockholm) 90: 254–264 (July 2009)

Table 3 Squared Mahalanobis distances among the centroids of the ﬁve Microtus species studied, based on our material only

M. (T.) thomasi M. (T.) felteni M. (T.) subterranaeus M. (M.) rossiaemeridionalis

M. (T.) felteni 44.85 M. (T.) subterranaeus 62.25 15.54 M. (M.) rossiaemeridionalis 75.83 54.65 40.19 M. (M.) guentheri 37.77 72.41 90.43 62.97

differences between the two subgenera (Microtus and Terricola) of Microtus. For all species there was no effect of sex on size and shape PCs (manova, Wilk’s Lambda: P > 0.05).

Intraspecific geographical variation In three species, M. (T.) thomasi, M. (M.) rossiaemeridionalis and M. (M.) guentheri, significant intraspecific differences (size or shape) were found, among broader distributional areas (Wilk’s Lambda: P < 0.05 in manova and anova tests). In particular, for M. (T.) thomasi we found significant size differences (F7,67 = 3.656, P = 0.002) between the population from Southern Peloponnisos and the samples from Central- Western Makedonia and Central Ipeiros (Sidak post hoc test), and significant shape differences (F7,67 = 5.284, P < 0.001) between the Central-Western Makedonia populations and samples from Western Thessalia, Northern Peloponnisos and Southern Peloponnisos. For M. (M.) rossiaemeridionalis significant size differences (F3,48 = 3.277, P = 0.029) between the Central-Western Makedonia and Western Ipeiros populations, and significant shape differences (F3,48 = 4.389, P = 0.008) between the Central- Western Makedonia populations and samples from Central Ipeiros and Western Thessalia were found. Finally, for

M. (M.) guentheri we found significant shape differences (F3, 30 = 12.518, P < 0.001) between the populations from Western Fig. 3—A. BPCA plots, —B. size-free CVA plots for the five Thessalia and samples from Central Ipeiros and Western Microtus species studied, based on both literature (white symbols) upgma Ipeiros. The dendrograms (not shown), based on the and our data (black symbols). resulting Mahalanobis distances after CVA on the adjusted data sets for the above mentioned three species, exhibited a good fit with the data (e.g. for M. (T.) thomasi: r = 0.665, P = 0.0002), and did not reveal any reasonable geographical of species and greater similarities between the conspecific pattern, i.e. clustering of pooled populations (of the same samples (Fig. 3). That discrimination is more evident in CVA broader distributional area). Furthermore, the resulting because in that analysis differences between groups rather Mahalanobis distances among the pooled populations (of the than individuals are maximized, as in the case with PCA same broader distributional area) of each species were not (Quinn and Keough 2002). In that BPCA the first three axes correlated with their linear geographical distances (e.g. for (size PC1, and the size-free PC2 and PC3) account for M. (T.) thomasi: Mantel test, r = 0.225, t = 0.914, P = 0.210). 42.42%, 38.99% and 5.66% of the variance, respectively. Moreover, in CVA, the CV1 accounts for 62.2% of the variation, and the CV2, accounting for 19.1% of the within- Discrimination using additional literature data samples variation, discriminates the two subgenera of Microtus. BPCA and especially size-free CVA performed on the nine The most important characters for the latter separation of species samples, i.e. the five samples of the present study and the nine samples were HFL, TL, CBL, ZW and NL (Table 4, the four samples of the same species (except M. (M.) guentheri) column CV2). The a posteriori cross-validation classification taken from bibliographic sources, revealed a clear distinction matrix obtained, using linear discriminant functions in CVA,

© 2008 The Authors 258 Journal compilation © 2008 The Royal Swedish Academy of Sciences Acta Zoologica (Stockholm) 90: 254–264 (July 2009) Fraguedakis-Tsolis et al. • Morphometrics of the Greek Microtus voles

Table 4 Standardized canonical variates analysis coefficients for the coefficients shown in Table 2, we can suggest that a meaningful 13 variables, common in our study and in the analysed literature data discrimination of the two subgenera is possible if we take into of the five Microtus species account HFL (of the external body variables) and ZW, OW, ONL and BCHb (of the cranial variables). Variable CV1 CV2 The absence of any sex effect on shape (and size) differ-

TL –0.662 0.366 ences indicates a weak role for sexual selected morphological OW 0.653 0.206 adaptations and variation. Moreover, it gives more support ML 0.644 –0.210 to the interspecific or intergeneric discrimination suggested ZW 0.619 0.268 by BPCA and CVA. UDL 0.401 –0.001 Our results (see Figs 2 and 3 and Tables 3 and 5) do not HFL –0.012 –0.679 agree with the established (Jaarola et al. 2004) phylogenetic CBL 0.341 –0.342 relations between the Microtus species. In their comprehen- TBL –0.148 –0.213 IFL 0.178 –0.129 sive work Jaarola et al. (2004), presented a robust molecular NL 0.166 –0.233 phylogeny of the genus Microtus that clearly discriminated HB 0.034 –0.125 the two subgenera, and showed that within the subgenus UMR 0.250 –0.066 Terricola, M. (T.) felteni and M. (T.) thomasi are sister species. IOW 0.137 0.095 This discrepancy is not unexpected because morphological variation is more affected by the processes of adaptive Most significant weights in bold. Abbreviations defined in Materials and Methods section. convergence and divergence (Scotland et al. 2003; Wiens et al. 2003; Wiens 2004). Yet, our morphometric analysis revealed a considerable inter- and intraspecific variation subject to showed a 92.8% correct classification in the expected future research on its adaptive significance. samples, ranging from 70% (Niethammer’s sample of M. (T.) felteni ) to 100% (samples of M. (M.) guentheri and Niet- Intra- and interspecific geographical variation and convergence hammer’s M. (T.) subterraneus). From the between-samples Mahalanobis distances, The statistically significant intraspecific size and shape presented in Table 5, we can observe that the Greek samples variation that was found for M. (T.) guentheri, M. (M.) M. (T.) subterraneus and M. (T.) felteni were the most similar rossiaemeridionalis and M. (T.) thomasi did not show a clear (d = 7.02). That affinity resulted from size (and not from geographical pattern. Especially for M. (T.) thomasi, these shape) similarities between these two species (anova on size results are corroborated by other morphometric, allozyme and shape PCs, Bonferroni post hoc test, P > 0.3) (Fig. 4B). and mitochondrial DNA data (Tsekoura et al. 2002; Kornilios et al. 2005; Thanou et al. 2005; Tryfonopoulos et al. in press), all of which indicate that the affinities among the Discussion studied Greek populations are independent of their linear geographical distances. This may indicate that morphological Size and shape discrimintation between Microtus species variation and differentiation of this species in Greece is rather and subgenera stochastic, or at least is not correlated with obvious factors, Both BPCA and CVA showed that the five studied species such as geographical isolation. are morphometrically clearly distinguished (Fig. 2). It is well Comparing our raw data with those reported by Niet- established (James and McCulloch 1990; Jolliffe 2002) that hammer (1982a,b,c,d) and Petrov and RuΩic (1982) (see in the latter analysis, especially after removing the effect of a Table 6), we can see that in the case of M. (T.) thomasi there within-population size factor from the between-population is an extensive overlap of the ranges of the variable values and group morphometric comparison (Rohlf and Bookstein that the morphometric profile of the Niethammer’s sample 1987), the first CV indicates differences mainly attributed to falls under the marked overall variation found for this species the ‘size’ while the subsequent CVs are more indicative for in the present study, probably because the Niethammer ‘shape’ differences of the compared taxa. Considering this (1982d) material (20 specimens) was also collected from rule and the widely accepted subgeneric status of Microtus Greece. On the contrary, in the cases of M. (M.) rossiaeme- and Terricola (Chaline et al. 1988; Brunet-Lecomte 1990; ridionalis and M. (T.) subterraneus this overlapping is much Brunet-Lecomte and Chaline 1992; Jaarola et al. 2004), our smaller, as those samples (21 and 24 specimens, respectively) results showed that for the Greek voles this subgeneric used for comparison with ours, were collected from the division is well verified by shape rather than size difference, Former Yugoslavian Republic of Macedonia (FYROM) since the species belonging to the subgenus Microtus (M. thomasi, (Petrov and RuΩic 1982) and Germany (Niethammer 1982b), M. guentheri and M. rossiaemeridionalis) are grouped together respectively. and are clearly separated from the Terricola species along the A result worth mentioning is the morphometric affinity size-free CV2 axis. Therefore, according to the standardized between the Greek samples of M. (T.) subterraneus and

© 2008 The Authors Journal compilation © 2008 The Royal Swedish Academy of Sciences 259 Morphometrics of the Greek Microtus voles • Fraguedakis-Tsolis et al. Acta Zoologica (Stockholm) 90: 254–264 (July 2009)

Fig. 4—Means and 95% conﬁdence intervals for the size and shape principal components (PC) scores of the ﬁve (A, B) and the nine (C, D) Microtus samples.

M. (T.) felteni, the latter represented by both Niethammer’s the Mahalanobis distance between our M. (T.) felteni sample and our material. The Mahalanobis distances between these and its conspecifics studied by Niethammer (1982c) is two species are 7.02 and 12.91, respectively (Table 5), rather high (9.79), although the Niethammer’s material (10 and these distance values are remarkably lower than those specimens) was also collected from Greece. That indicates calculated when the German M. (T.) subterraneus material considerable intraspecific morphological variation in M. (T.) (Niethammer 1982b) is used for the same comparisons felteni as well, despite its much smaller range compared with (30.62 and 20.07, respectively). This is obviously a result of that of M. (T.) subterraneus. That variation possibly can be the relatively high distance value (18.65) between the Greek attributed to the discontinuous distribution of this vole on and German M. (T.) subterraneus samples, and is possibly high mountainous Greek areas. This morphometric affinity justified by the known geographical variation of this vole over between M. (T.) felteni and M. (T.) subterraneus may reflect its broad Eurasian range (Niethammer 1974, 1982b; Corbet convergent size-related morphological adaptations of these 1978; Petrov 1992; Kryßtufek and Vohralík 2005). Likewise, two species to similar mountainous conditions in the Greek

© 2008 The Authors 260 Journal compilation © 2008 The Royal Swedish Academy of Sciences Acta Zoologica (Stockholm) 90: 254–264 (July 2009) Fraguedakis-Tsolis et al. • Morphometrics of the Greek Microtus voles

Table 5 Squared Mahalanobis distances among the centroids of the ﬁve Microtus species studied, based on material from the literature and the current study

M. (T.) M. (T.) M. (T.) M. (M.) M. (M.) M. (T.) M. (M.) M. (T.) thomasi felteni subterraneus rossiameridionalis guentheri thomasi (Nd) rossiameridionalis (P&R) subterraneus (Nb)

M. (T.) felteni 15.76 M. (T.) subterraneus 34.44 7.02 M. (M.) rossiaemeridionalis 65.01 29.27 21.28 M. (M.) guentheri 23.04 29.78 52.54 51.91 M. (T.) thomasi (Nd) 10.55 20.18 34.86 67.88 34.99 M. (M.) rossiameridionalis (P&R) 109.56 64.81 50.73 25.91 83.25 102.57 M. (T.) subterraneus (Nb) 61.59 30.62 18.65 40.99 73.71 64.18 44.64 M. (T.) felteni (Nc) 26.95 9.79 12.91 39.29 35.52 18.74 56.99 20.07

No literature data were used for M. (M.) guentheri – see in the text. P&R: Petrov and RuΩic (1982); Nb: Niethammer (1982b); Nc: Niethammer (1982c); Nd: Niethammer (1982d).

Table 6 Combined range values for morphometric variables, which are common in other studies and ours (see text in Discussion) concerning the Greek voles

M. (M.) guentheri M. (M.) rossiaemeridionalis M. (T.) subterraneus M. (T.) felteni M. (T.) thomasi

HB 83–127 76–125 – 87–98 71–111 TL 16–36 30–50 – 21–28.5 17–30 EL 10–16 9–16 – 7.0–9.0 7–11 HFL 17–22 16–21 – 14.5–16.0 14–18 CBL 26.7–29.61 21.1–28.2 – 21.6–23.55 20.7–27.29 BL 22.34–27.9 21.1–25.92 ––– UDL 6.0–9.8 6.0–8.2 5.6–7.53 6.4–7.18 6.0–9.05 IFL – 3.52–5.02 – 3.43–4.2 3.6–5.22 NL – 6.03–8.72 – 6.0–7.2 6.0–8.5 UMR 5.2–7.2 5.4–6.82 – 5.44–6.2 5.4–6.99 TBL – 5.86–8.2 – 5.52–7.0 5.34–6.87 IOW 3.0–4.0 3.26–3.92 – 3.5–4.0 3.52–4.50 OW – 9.87–12.3 – 10.71–11.9 10.7–13.79 ZW 13.8–17.8 11.5–16.2 – 12.90–14.8 13.2–17.67 BCHb – – – – 8.17–10.61 BCW – 8.7–11.18 ––– ML – 13.36–16.72 – 13.3–15.6 16.14–18.27 LMR 5.4–7.33 5.32–6.89 – 5.38–6.37 5.4–7.11

Limits derived from the present study are in bold. Abbreviations deﬁned in Materials and Methods section.

mainland, where the southernmost margins of both species For M. (M.) guentheri, Niethammer (1982a) gives very few distribution areas exist. and incomplete raw data, making any multivariate statistical Similarly, in the case of M. (M.) rossiaemeridionalis the analysis and comparisons unreliable. Mahalanobis distance between the specimen series of Petrov and RuΩic (1982) and ours (25.91), is higher than the dis- Comparisons with Balkan Microtus representatives using tances between our series of this species and the series of our non-comprehensive literature data M. (T.) subterraneus (21.28). This affinity is the result of shape similarities (anova on PC2, Bonferroni post hoc test, Apart from the publications mentioned above, all the P = 0.998). This may imply high intraspecific variation in morphometric data available from other literature sources M. (M.) rossiaemeridionalis in the southernmost part of its for the Greek Microtus species always concern fewer variables overall range that results in the morphometric affinity of the and are given in forms (i.e. means, range) not suitable for above two species in Greece. comprehensive comparative statistical treatment. Therefore,

© 2008 The Authors Journal compilation © 2008 The Royal Swedish Academy of Sciences 261 Morphometrics of the Greek Microtus voles • Fraguedakis-Tsolis et al. Acta Zoologica (Stockholm) 90: 254–264 (July 2009) only simple comparisons of means and ranges can be variables measured in a total of 60 Turkish individuals give attempted. In this context, combining the available information means that are mostly higher than ours but with narrower based on the Greek material for the five species studied, we ranges. Consequently, all the above data for M. (T.) subter- give the ranges for the common examined variables, between raneus allow us to say that this taxon is morphometrically other studies and ours, in Table 6. In our data, values extend variable, having smaller individuals in Germany and larger in the lower range limit for 10 variables in M. (M.) guentheri, central Europe and Turkey while Balkan specimens (Greek for 11 in M. (M.) rossiaemeridionalis, for 11 in M. (T.) felteni ones included) have an intermediate size. and for seven in M. (T.) thomasi, as well as the upper range Finally, regarding M. (T.) thomasi, there are data for 14 limit for 11, 10, 6 and 15 variables, respectively. For M. (T.) variables, yet measured in only few specimens from Mon- subterraneus the ranges of all variables have been derived only tenegro, Yugoslavia (Petrov and ¸ivkovic 1972). For 10 of from the present study because the morphometrics of only these characters (HB, HFL, CBL, ZW, OW, NL, UDL, ML, one Greek specimen of this taxon is available in the literature UMR and LMR) the mean values are higher than ours. The (Ondrias 1966). tendency of individuals of M. (T.) thomasi from the former Furthermore, we compared our results to those concerning Yugoslavia to be larger than the Greek voles, inferred by the samples of the same species coming from non-Greek comparison of the mean values, is also mentioned by parts of their ranges. For M. (M.) guentheri, this comparison Niethammer (1974, 1982d) and Petrov (1992). The ranges to material from the former Yugoslavia and Bulgaria, of the same 14 variables measured in our material are all mentioned by Niethammer (1974, 1982a), and from Turkey wider, but this is reasonable because of the large difference (Kryßtufek and Vohralík 2005) indicates that the Greek in the sample size of the two series studied (5 vs. 75 individuals, Günther’s voles inhabiting the western margin of this species respectively). range are the smallest. This is mainly evidenced by the means and/or ranges of HB, TL, CBL, ZW, UMR and TBL. Conclusions Our specimens of M. (M.) rossiaemeridionalis tend to be smaller than those from Bulgaria and Romania (15 specimens) In conclusion, our results showed that the Microtus species studied by Král et al. 1981), as inferred by the lower means that occur in Greece are characterized by considerable of HB, TL, EL, CBL, ZW, UDL, UMR and LMR (only intraspecific variation. Consequently, there was some overlap HFL and IOW have means higher in our material). However, between inter- and intraspecific morphometric variation. ranges of the values are broader in our material for all of these Comparisons with literature data, mainly concerning popu- variables, except for CBL and ZW, for which they are shifted lations of these species from adjacent areas, indicate that the to lower values. Data given by Kryßtufek and Vohralík (2005) Greek M. (M.) guentheri, M. (M.) rossiaemeridionalis and for 17 specimens of the same species from Turkey, indicate M. (T.) thomasi tend to be smaller than their conspecifics, higher means than ours in all seven variables presented in while M. (T.) subterraneus and M. (T.) felteni are about that work (HB, TL, HFL, EL, CBL, ZW and UMR), equal-sized with their Balkan relatives. whereas ranges either fall into our ranges or have shifted to Even though morphometric analysis did not provide higher values. Turkish specimens also tend to be larger than robust criteria to infer phylogenetic relations, it was proved Bulgarian and Romanian ones. Therefore, as in M. (M.) adequate for delimiting these closely related species and guentheri, the Greek individuals of M. (M.) rossiaemeridionalis subgenera. Moreover, that variation between and within seem to be the smallest in the southwestern margin of this currently recognized species, shows that speciation and species range. differentiation is not fixed in Microtus and that there is a A sample of 13 individuals of M. (T.) felteni from Serbia promising potential for research on the adaptive significance and FYROM (Petrov and ¸ivkovic 1979) shows ranges of this morphological variation. either shifted to higher values (for HB, TL and CBL) or to lower values (for EL). Acknowledgements Krapp and Winking (1976) studied 60 specimens of M. (T.) subterraneus from areas of central Europe (Switzerland, We thank an anonymous reviewer for comments and sugges- Austria and northernmost Italy) and only seven specimens tions for improving the manuscript, and Panayotis Kornilios from the Balkans (Kroatia and FYROM). The comparison of for linguistic and grammatical suggestions. our material with the former of these two specimen series, on the basis of seven variables, showed that Greek specimens have similar or slightly lower means and broader ranges for References most of the variables examined. On the other hand, the Bookstein, F., Chernoff, B., Elder, R., Humphries, J., Smith, G. and comparison to the Balkan series of Krapp and Winking Strauss, R. 1985. Morphometrics in evolutionary biology: the (1976) reported higher mean values for UDL, IFL, ZW and geometry of size and shape change, with examples from fishes. – BCHb but their ranges are in all cases broader in our Academy of Natural Sciences of Philadelphia Special Publication 15: material. Kryßtufek and Vohralík (2005) data for seven 1–277.

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speciation in Microtus (T.) thomasi (Arvicolidae, Rodentia) in Supplementary material Greece. – Biological Journal of the Linnean Society. Tsekoura, N., Fraguedakis-Tsolis, S., Chondropoulos, B. and The following supplementary material is available for this Markakis, G. 2002. Morphometric and allozyme variation in article: central and southern Greek populations of Microtus (Terricola) thomasi. – Acta Theriologica 47: 137–149. Appendix S1 Sample size (N), minimum, maximum, Ventura, J. and Gosálbez, J. 1989. Taxonomic review of Arvicola mean values and their standard deviation of the variables terrestris (Linnaeus, 1758) (Rodentia, Arvicolidae) in the Iberian examined for each Microtus species. Peninsula. – Bonner Zoologische Beiträge 40: 227–242. Vohralík, V. and Soﬁanidou, T. 1987. Small mammals (Insectivora, This material is available as part of the online article from: Rodentia) of Macedonia, Greece. – Acta Universitatia Carolinae http://www.blackwell-synergy.com/doi/abs/10.1111/ Biologica 11: 319–354. j.1463-6395.2008.00348.x Vohralík, V. and Soﬁanidou, T. 1992. Small mammals (Insectivora, (This link will take you to the article abstract). Rodentia) of Thrace, Greece. – Acta Universitatia Carolinae Biologica 36: 341–369. Please note: Blackwell Publishing are not responsible for the Wiens, J. J. 2004. The role of morphological data in phylogeny content or functionality of any supplementary materials reconstruction. – Systematic Biology 53: 653–661. Wiens, J. J., Chippindale, P. T. and Hillis, D. M. 2003. When are supplied by the authors. Any queries (other than missing phylogenetic analyses misled by convergence? A case study in material) should be directed to the corresponding author for Texas cave salamanders. – Systematic Biology 52: 501–514. the article.