Geographic phenetic variation of two eastern-Mediterranean non-commensal mouse species, Mus macedonicus and M. cypriacus (Rodentia: Muridae) based on traditional and geometric approaches to morphometrics

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Zoologischer Anzeiger 247 (2008) 67–80 www.elsevier.de/jcz

Geographic phenetic variation of two eastern-Mediterranean non-commensal mouse species, Mus macedonicus and M. cypriacus (Rodentia: Muridae) based on traditional and geometric approaches to morphometrics Milosˇ Machola´na,b,, Ondrˇ ej Mikulaa, Vladimı´ r Vohralı´ kc a

Laboratory of Mammalian Evolutionary Genetics, Institute of Animal Physiology and Genetics, AS CR, 602 00 Brno, Czech Republic b Institute of Botany and Zoology, Masaryk University, Brno, Czech Republic c Department of Zoology, Faculty of Sciences, Charles University, 128 44 Prague 2, Czech Republic Received 19 March 2007; received in revised form 4 July 2007; accepted 4 July 2007 Corresponding editor: D.G. Homberger

Abstract We tested the hypothesis that skull shape within the genus Mus may vary with geographic location by assessing the extent and spatial distribution of phenotypic skull variation within and among two wild mouse species, M. macedonicus and M. cypriacus, using traditional and geometric morphometrics including a rather novel application of sliding semilandmarks. Shape was shown to be significantly correlated both with longitude and latitude in M. macedonicus, yet the correlation between morphometric and geographic distances was not significant, and morphometric differences between Asian and European populations were not higher than those within the particular continents. The phylogenetic signal was found to be stronger in dental characters than in cranial ones, however, overall concordance between the pattern of morphometric variation and the presumed history of M. macedonicus was rather weak. In both species, the dorsal and ventral sides of the skull were shown to covary in many aspects though there were also some differences between them, making the functional interpretation of these differences difficult. Discrimination between M. cypriacus and M. macedonicus as well as discrimination between two M. macedonicus subspecies was highly reliable using both traditional and geometric morphometric tools to analyze skull measurements. r 2007 Elsevier GmbH. All rights reserved. Keywords: Mus macedonicus; Mus cypriacus; Phenotypic variation; Multivariate analysis; Geometric morphometrics; Thin-plate spline

1. Introduction In the last decade, data have accumulated broadening our knowledge of the systematics and phylogenetic Corresponding author. Laboratory of Mammalian Evolutionary Genetics, Institute of Animal Physiology and Genetics, AS CR, Veverˇ ı´ 97, 602 00 Brno, Czech Republic. Fax: +420 532290138. E-mail address: [email protected] (M. Machola´n).

0044-5231/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.jcz.2007.07.003

relationships in mice of the genus Mus (Prager et al. 1998; Boursot et al. 1993; Lundrigan et al. 2002; Chevret et al. 2005). New findings have also appeared regarding the group of west-Palearctic non-commensal mouse species. This group, called ‘aboriginal’ by Sage (1981), was shown to consist of three species: The western Mus spretus Lataste, 1883, from southern France, Spain, and north-western Africa (Machola´n 1999a); and the two eastern species M. spicilegus Pete´nyi, 1882, from the

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Panonian plains and lowlands of eastern Europe (Machola´n 1999b) and M. macedonicus Petrov and Ruzˇic´, 1983, from the Balkan Peninsula and the Middle East (Machola´n 1999c). Regardless of whether these species form a monophyletic group or not (cf. e.g., She et al. 1990; Sage et al. 1993; Prager et al. 1996), they all have been considered genetically rather uniform in contrast to the commensal house mouse Mus musculus (Bonhomme et al. 1984; Prager et al. 1996). However, in the late 1990s, a morphologically distinct population of M. spicilegus was reported from the vicinity of the town of Ulcinj, Montenegro, and described as a new subspecies, Mus spicilegus adriaticus (Krys˘ tufek and Machola´n 1998). At the same time, a close morphometric similarity of populations from Albania (Lake Shkode¨r, Tirane¨) and western Greece (Vlaherna) to the M. s. adriaticus was reported by Machola´n and Vohralı´ k (1997). More recently, two deep mitochondrial DNA (mtDNA) lineages were revealed in M. macedonicus, one from Israel and the other comprising populations from the rest of the species range (Orth et al. 2002). This result, together with the differentiation of nuclear genes (though slightly lower than for mtDNA), led Orth et al. (2002) to describe the Israeli clade as a separate subspecies, M. macedonicus spretoides. Finally, a new mouse species, formerly referred to as M. macedonicus, was discovered on the Mediterranean island of Cyprus (Cucchi et al. 2002). The new taxon, called Mus cypriacus (the Cyprus

mouse), was shown to be genetically close to M. macedonicus, but clearly distinct from it and almost equidistant to M. spicilegus (Bonhomme et al. 2004; Cucchi et al. 2006). In this paper, we present results of traditional and geometric morphometric analyses aimed at an assessment of the extent and spatial distribution of the phenotypic variation within and among two wild mouse species, M. macedonicus and M. cypriacus. More specifically, we have focused on the following questions: Is the morphometric variation in M. macedonicus and M. cypriacus random or is it correlated with the physical distances between the populations? Are there any geographic or other correlates (e.g., altitude, geographic location) of size and shape variation? Is there any apparent difference between European and Asian populations? What are the morphometric differences between the taxa?

2. Material and methods 2.1. Mouse specimens Specimens of M. macedonicus were collected from 37 sites scattered across the majority of the species range (Fig. 1 and Table 1). The samples were pooled according to their geographic proximity into 10 groups, hereafter referred to as ‘populations’: M. m. macedonicus – (1)

Fig. 1. Location of the sampling sites in the Balkan Peninsula and the Middle East. Geographically close localities were pooled into 11 groups or populations: M. m. macedonicus: BLG ¼ Bulgaria; EPI ¼ Epirus (Greece); MAC ¼ Macedonia (Greece); THR ¼ Thrace (Greece); SMT ¼ Samothraki (Greece); CAN ¼ central Anatolia (Turkey); CIL ¼ Cilicia (Turkey); GIA ¼ Georgia; SYR ¼ Syria; M. m. spretoides: ISR ¼ Israel; M. cypriacus: Cyprus.

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Table 1. List of sampling sites, sample sizes (numbers refer to specimens analyzed with traditional morphometric methods), and geographic location and approximate altitude of each site Mus macedonicus macedonicus 1. Bulgaria: Krumovo (N ¼ 7; 421160 N; 261240 E; 150 m a.s.l.); Banya (N ¼ 5; 421460 N; 271490 E; 70 m); 2. Epirus: Konitsa (N ¼ 2; 401030 N; 201460 E; 480 m); Perama (N ¼ 3; 391420 N; 201510 E; 480 m); 3. Macedonia: Gephyra (N ¼ 16; 401440 N; 221400 E; 20 m); Lagadas (N ¼ 3; 401440 N; 231050 E; 100 m); Lagadikia (N ¼ 2; 401390 N; 231130 E; 100 m); Mandres (N ¼ 1; 401520 N; 221550 E; 90 m); Metalliko (N ¼ 3; 411030 N; 221500 E; 180 m); Rapsomaniki (N ¼ 2; 401340 N; 221220 E; 20 m); Serres (N ¼ 1; 411060 N; 231330 E; 150 m); Strymonikon (N ¼ 3; 411030 N; 231210 E; 20 m); Thessaloniki (N ¼ 2; 401350 N; 221580 E; 10 m); 4. Thrace: Komara (N ¼ 11; 411360 N; 261140 E; 60 m); Ladi (N ¼ 6; 411280 N; 261160 E; 50 m); Mikro Derio (N ¼ 7; 411200 N; 261070 E; 170 m); Monastirakio (N ¼ 8; 401510 N; 261060 E; 30 m); Porto Lagos (N ¼ 6; 411010 N; 251080 E; 0 m); Rizia (N ¼ 6; 411370 N; 261250 E; 50 m); 5. Samothraki, Greece: Kamariotissa (N ¼ 17; 401270 N; 251310 E; 0 m); Makrilies (N ¼ 2; 401270 N; 251310 E; 50 m); 6. Central Anatolia: Bardakci (N ¼ 12; 391070 N; 281310 E; 600 m); Karabulut (N ¼ 1; 381280 N; 311290 E; 1000 m); Suludere (N ¼ 10; 371390 N; 301100 E; 950 m); 7. Cilicia: Adana (N ¼ 10; 371000 N; 351190 E; 150 m); Tarsus (N ¼ 8; 361550 N; 341540 E; 150 m); 8. Georgia: Alazani (N ¼ 8; 411370 N; 451580 E; 290 m); Chardakhi (N ¼ 4; 411520 N; 441350 E; 320 m); Krtzanissi (N ¼ 2; 411360 N; 441570 E; 250 m); Lake Lissi (N ¼ 3; 411440 N; 441470 E; 250 m); 9. Syria: Qattinah (N ¼ 34; 341400 N; 361370 E; 550 m); M. m. spretoides 10. Israel: Anavim (N ¼ 4; 311490 N; 351080 E; 320 m); Dalyhiat ah Carmel (N ¼ 1; 321440 N; 351020 E; 300 m); Haifa (N ¼ 1; 321490 N; 351000 E; 50 m); Mt. Carmel (N ¼ 11; 321440 N; 351030 E; 330 m); Ortal (N ¼ 1; 331050 N; 351450 E; 380 m); Poriyya (N ¼ 1; 321420 N; 351370 E; 150 m); M. cypriacus 11. Cyprus: Neo Chorio (N ¼ 2; 351010 N; 321200 E; 300 m); Apsiou (N ¼ 4; 341480 N; 331010 E; 400 m); Paramytha (N ¼ 5; 341460 N; 331000 E; 300 m); Kornos (N ¼ 1; 341560 N; 331240 E; 305 m); St. Hilarion (N ¼ 1; 351190 N; 331170 E; 570 m); Zeytinlik, Girne (N ¼ 2; 351190 N; 331180 E; 160 m).

Bulgaria (BLG; N [traditional morphometrics/dorsal landmarks/ventral landmarks] ¼ 12/11/11); (2) Epirus, Greece (EPI; N ¼ 5/4/4); (3) Macedonia, Greece (MAC; N ¼ 33/33/33); (4) Thrace, Greece (THR; N ¼ 44/35/ 33); (5) Samothraki, Greece (SMT; N ¼ 19/19/19); (6) Central Anatolia, Turkey (CAN; N ¼ 23/19/19); (7) Cilicia, southern Turkey (CIL; N ¼ 18/11/10); (8) Georgia (GIA; N ¼ 17/18/18); (9) Syria (SYR; N ¼ 34/ 32/32); M. m. spretoides; (10) Israel (ISR; N ¼ 19/19/ 19). M. cypriacus was sampled at six sites located throughout Cyprus, and the samples were pooled into a single population (Fig. 1): and (11) Cyprus (CYP; 15/15/

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15). The whole data set, thus, consisted of 239, 216 and 213 specimens, respectively. Mus cypriacus is characterized by large overall size, elongated ears, and large and protruding eyes, which makes it markedly different from M. macedonicus. The dorsal side is darker and more brownish than that of the latter species, with a slight reddish tinge (Fig. 2A and C). The belly of the Cyprus mouse adults is whitish and clearly demarcated contrary to the rather grayish belly of M. macedonicus (Fig. 2B and D). Voucher specimens were deposited in the collections of: The Department of Zoology, Charles University, Prague, Czech Republic (Nos. BB3526–27, 3532–33, 3538, 3543, 3940; BG1859, 1934–35, 1937, 1943–44, 1950, 1952, 1972, 1977, 1980, 1988, 2013, 2068–69, 2097, 2113, 2138, 2140, 2151, 2331–34, 2337, 2406–07, 2534, 2963–64, 2966–67, 2970, 3004, 3030, 3036–37, 3062–63, 3083, 3091–92, 3108–09, 3401, 3406–07, 3410–12, 3415–16, 3419, 3422–23, 3427, 3429–31, 3434–3435, 3437–38, 4074–75, 4077, 4079, 4085, 4094–95, 4098, 4100, 4103–05, 4107, 4115, 4123, 4128, 4139, 4141, 4143–46, 4148–61, 4168–69, 4173–74, 4184, 4187–88, 4194, 4202–03, 4206, 4210; TU536, 544–46; TU73, 106, 123, 134–35, 140, 152, 156–58; MISC151–153, 155, 158, 160, 163, 167, 169–71, 173–74, 178, 184, 186, 188–90, 192, 196, 198, 200–03, 205, 207–209, 211, 213, 219, 226; CYP5–7, 9–11, 20–24, 35, 37–39); the Slovenian Museum of Natural History, Ljubljana, Slovenia (Nos. T167, 169, 180–84, 190–91); the Institut des Sciences de l’Evolution de Montpellier, Universite´ Montpellier II, France (Nos. 1, 6–8, 19–21, 24, 382–84, 386–91, 393–94, 6157–6160, 6162, 10471, 10473, 10475, 10478–80, 10486, 10520–22, 10522, 10525, 10528–30, 10539–40); and the Smithsonian Institution, National Museum of Natural History, Washington, DC, USA (Nos. USNM327690–97).

2.2. Morphometric methods Skull morphology was quantified with traditional and geometric morphometric methods. The traditional morphometrics were represented by a set of 16 cranial and 14 dental measurements. The variables were the same as those described in Krysˇ tufek and Machola´n (1998) except for the distance between the medial margins of the Foramina lacerta rostralia (referred to as Fissurae petrotympanicae, or LPT, in Krysˇ tufek and Machola´n 1998), which was omitted in this study, because too many specimens had the relevant part of the skull damaged (see Krysˇ tufek and Machola´n 1998 for details on the measurements). In the broadest sense, geometric morphometrics is a class of multivariate methods developed for capturing the geometry of a structure and is usually based on an analysis of the Cartesian coordinates of selected points

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Fig. 2. Skins of M. cypriacus (A, C) from Paramytha, Cyprus (CY-19, female, leg. M. Machola´n) and M. m. macedonicus (B, D) from Lagadikia, Greece (BG-2135, female, leg. V. Vohralı´ k) from dorsal (A, B) and ventro-lateral (C, D) view. Note the large body and ears, reddish tinge of the fur on the back, and the clearly demarcated whitish belly of the Cyprus mouse (photo: M. Sˇandera).

called landmarks (Rohlf and Marcus 1993). We used 14 landmarks digitized on the dorsal side and 22 landmarks on the ventral side of the skull as shown in Fig. 3. The points correspond to the landmarks of types 1 (discrete juxtaposition of three structures) and 2 (maxima of curvature or other morphogenetic processes) according to Bookstein (1991). The images of the skulls were taken using a digital camera and then landmarks were digitized only on the left side to minimize potential influence of asymmetry. To digitize the ventral side, the skulls were attached to a mat with a piece of Plasticine, and its horizontal orientation was then checked visually. In addition, 20 ‘sliding semilandmarks’ (Bookstein 1997; Rohlf 2005a) were digitized in more or less regular intervals along the outline of the skull in the dorsal projection: Five sliding semilandmarks between the landmarks 10 and 11, and 15 sliding semilandmarks between the landmarks 8 and 14 as indicated in Fig. 3. Unlike true landmarks, sliding semilandmarks are allowed to move along a curve so as to minimize the amount of shape change between each of the specimens analyzed and the average of all specimens. For this purpose, the curves were approximated by the direction of a chord drawn between the adjacent points, and the points were positioned iteratively, so as to minimize the bending energy (see below). Both the landmarks and

semilandmarks were digitized with the TpsDig2 program (Rohlf 2005b). All configurations of landmarks and semilandmarks were superimposed using the generalized least-squares procedure (generalized Procrustes analysis (GPA); Rohlf and Slice 1990) implemented in the tpsRelw program (Rohlf 2005a).

2.3. Data analyses All cranial and dental linear measurements were logtransformed before processing them in order to decrease differences in variances between variables. Variation in size and shape, together with morphometric and phylogenetic relationships among populations, were then assessed using standard morphometric tools. As skull size is inherently a multivariate character, principal component analysis was used to detect a size vector. In order to avoid potential bias caused by confounding variation within and between different groups due to pooling character relationships irrespective of group, multiple-group principal components analysis (MGPCA) was used (Thorpe 1983). Canonical variates analysis (CVA) was carried out to reveal morphometric relationships among the populations studied while both ordinary and stepwise discriminant function analysis (DFA) was

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used to assess discrimination between species and/or subspecies. The whole set of 30 cranial and dental measurements was used (the variables were not logtransformed in this case). Relationships among the populations were further summarized through clustering methods: Unweighted pair-group method using arithmetic averages (UPGMA) and neighbor-joining (NJ). The Epirus (EPI) population was excluded from both UPGMA and NJ analyses due to its small sample size. The analyses were performed with Statistica (StatSoft, Inc. 2004; DFA), NTSYS-pc (Rohlf 1997; CVA) and PHYLIP (Felsenstein 2004; UPGMA, NJ). In order to assess the variation among the landmark configurations, thin-plate spline relative warp analysis (TPSRW; Rohlf 1993) was carried out using tpsRelw(Rohlf 2005d). This method is equivalent to PCA with variation weighted by so-called ‘bending energy’. The procedures involved in the analysis start by creating a bending energy matrix, which is a function of the amount of transformation in shape and the degree of localization of this transformation (i.e., the closer two points between which the shape change takes place, the higher the bending energy). Subsequently, partial warps are created by projecting each specimen onto eigenvectors, called principal warps, extracted from the bending energy matrix. The zeroth partial warp corresponds to an affine (uniform) shape component whereas all other partial warps correspond to non-affine (non-uniform) shape changes. The matrices of partial warp scores (including the affine component) for the dorsal and ventral sides, respectively, were used for the standard multivariate analyses listed above.

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When extracting relative warps from partial warps, scaling with a ¼ 0 was used, giving the same weight to all partial warps, as there was no a priori reason to give greater weight to either large-scale variation (i.e., variation among specimens in the relative positions of widely separated landmarks; a40) or small-scale variation (i.e., in the positions of landmarks that are close to each other; ao0). This approach seems appropriate for most systematic studies (Rohlf and Corti 2000), including the present one. Fig. 3. Positions of the landmarks (dots) on the dorsal (A) and ventral (B) side of the mouse skull with approximate positions of 20 semilandmarks (open circles). (A) Dorsal side: LM1 ¼ rostralmost point of the nasal bone (rhinion); LM2 ¼ intersection of the naso-frontal suture in the midline (nasion); LM3 ¼ intersection of the coronal and sagittal sutures (bregma); LM4 ¼ intersection of the sagittal and parietal-interparietal sutures (lambda); LM5 ¼ caudal end of the curvature of the occipital (opistocranion); LM6 ¼ intersection of the rostral curvature of the nasal process of the incisive bone (Processus nasalis ossis incisivi) and the nasal bone in the dorsal projection; LM7 ¼ point of maximum curvature of the rostro-lateral part of the maxilla; LM8 ¼ rostral end of the zygomatic plate; LM9 ¼ caudal end of the intersection of the zygomatic process of the maxilla and the upper limb of this process; LM10 ¼ lateral end of the naso-frontal suture in the dorsal projection; LM11 ¼ rostralmost point of the parietal bone; LM12 ¼ rostral end of the zygomatic process of the temporal bone (Processus zygomaticus partis squamosae ossis temporalis); LM13 ¼ intersection of the parietal-interparietal and interparietal-occipital sutures; LM14 ¼ caudolateral end of the occipital bone in the dorsal projection. (B) Ventral side: LM1 ¼ rostralmost point of the upper incisor in the midline; LM2 ¼ lateral end of the upper incisor in the dorsal projection; LM3 ¼ rostral end of the rostral palatine fissure (Fissura palatina); LM4 ¼ caudal end of the rostral palatine fissure; LM5 ¼ intersection of the maxillo-palatine suture in the midline; LM6 ¼ center of the Foramen palatinum majus; LM7 ¼ rostral end of the curvature of the palatine bone (Lamina horizontalis ossis palatini); LM8 ¼ lateral end of the basisphenoid-basioccipital suture in the dorsal projection; LM9 ¼ rostral end of the occipital foramen in the midline (basion); LM10 ¼ caudal end of the occipital foramen in the midline (opisthion); LM11 ¼ caudal end of the medial edge of the occipital condyle; LM12 ¼ rostral end of the zygomatic plate; LM13 ¼ medial end of the zygomatic plate in the ventral projection; LM14 ¼ rostral end of the first upper molar in the dorsal projection; LM15 ¼ lingualmost point of contact of the first and second upper molars in the ventral projection; LM16 ¼ lingualmost point of contact of the second and third upper molars in the ventral projection; LM17 ¼ caudal end of the third upper molar in the ventral projection; LM18 ¼ rostral end of the Foramen lacertum rostrale; LM19 ¼ caudal internal maximum curvature of the zygomatic process of the temporal bone (Processus zygomaticus partis squamosae ossis temporalis); LM20 ¼ point of juxtaposition of the tympanic bulla and the muscular process (Processus muscularis); LM21 ¼ caudal end of the external opening of the auditory channel; and LM22 ¼ styloglossal process of the tympanic bulla.

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The null hypothesis that the shape of the skull changes isometrically with an increase of size was tested by regressing each partial warp on size using the TpsRegr program (Rohlf 2005c). If the test were significant, we would conclude that the skull growth is allometric, i.e., the shape of the skull changes during growth. As a measure of the size of an object (i.e., a landmark configuration), the centroid size was used, defined as the square root of the sum of squared distances of all landmarks to their centroid (Slice et al. 1996). Subsequently, the homogeneity of regression slopes among the three groups, i.e., M. m. macedonicus, M. m. spretoides, and M. cypriacus, was tested as described in Rohlf (2005c). In M. macedonicus, isolation by distance was assessed as the correlation between geographic and morphometric distance matrices by the Mantel test (Mantel 1967) with 5000 permutations using the NTSYS-pc program. Mahalanobis and Procrustes distances were used as measures of morphometric divergence in the traditional and geometric approaches, respectively. Minimum geographic distances between population centroids were measured using the Microsoft Encarta World Atlas (1998), avoiding large bodies of water as described in Machola´n et al. (2001). Population centroids were estimated as weighted means of longitudes and latitudes of individual sampling sites within each group. Finally, a two-block partial least-squares (2B-PLS) analysis was carried out to explore patterns of shape covariation between the dorsal and ventral sides as described by Rohlf and Corti (2000), using the tpsPLS program (Rohlf 2005d). That is, we tried to assess what covariation there was between the relative positions of landmarks on the two projections of the skull. The 2BPLS method constructs pairs of variables that are linear combinations of the original variables within each of the two sets of variables (i.e., for the dorsal and ventral side, respectively). The combinations are constructed so that the new variables account for as much as possible of the covariation between the two original sets of variables. Landmark coordinates of Procrustes-aligned specimens were used as shape variables for M. macedonicus and M. cypriacus separately. An advantage of this approach is that the Procrustes shape variables are already standardized for a subsequent 2B-PLS analysis. Semilandmarks were not used for these analyses.

3. Results 3.1. Measurements The first multiple-group principal component (PC1), based on 16 cranial measurements, accounted for 43.3%

of the total morphometric variation and seemed to express the overall size of the skull, with the populations from Bulgaria, Syria, Cilicia, and Israel being the smallest and the population from Samothraki the largest. However, PC1 coefficients differed both in sign and magnitude, so that this eigenvector could not be regarded as a size component, and the usual technique of size correction by excluding PC1 would have resulted in loss of part of the shape information. Subsequent analyses were, thus, based on all principal components (‘size-in’ analysis). ‘Size-in’ CVA revealed a largely random pattern. The second canonical axis (CV2; 25.1% of amonggroup variation) was probably at least partly related to size as it contrasted the small skulls of M. m. spretoides from Israel against the large skulls of M. m. macedonicus from Samothraki and M. cypriacus from Cyprus. CV1 (31.2% of among-group variation), however, is more difficult to interpret. Finally, CV3 (15.0%) contrasted the two island populations, namely the one from Samothraki and the one from Cyprus (M. cypriacus). Importantly, although Mahalanobis distances between M. cypriacus and all populations of M. macedonicus were significant, CVA did not reveal any substantial morphometric difference between the two species in the size and shape of the skull. In contrast to the cranial variables, the first multiplegroup principal component extracted from 14 dental measurements (46.8% of the total morphometric variation) can be considered a size vector owing to the same sign and very similar magnitude of PC1 coefficients. According to this component, mouse specimens from Syria, Israel, and Epirus possess by far the smallest molars, whereas the largest molars are characteristic of populations from Thrace, Cyprus, Samothraki, and Macedonia. This result is corroborated by the length of the upper and lower tooth rows, which is highest in M. cypriacus, as well as by the length of M1 (largest in Samothraki and Cyprus) and M1 (largest in Thrace and Samothraki). Results of both ‘size-in’ and ‘size-out’ CVA tended to group geographically close M. macedonicus populations together and showed a rather distinct position for M. cypriacus as displayed in the NJ tree based on ‘size-out’ Mahalanobis distances between population centroids (Fig. 4A). However, the branching pattern differed between the ‘size-in’ and ‘size-out’ analyses, and the position of the Israeli subspecies did not correspond to its distinct taxonomic and phylogenetic status. Within M. macedonicus, the Mantel test revealed a significant correlation between geographic and morphometric distances based on the cranial variables (R ¼ 0.464, p[rand zXobs z] ¼ 0.0128) as well as the dental ones (R ¼ 0.464, p[rand zXobs z] ¼ 0.0116). However, both the cranial and dental morphometric distances between the European and Asian M. m.

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Fig. 4. Neighbor-joining trees based on Mahalanobis distances between population centroids from the canonical variates analysis of 14 dental measurements with Burnaby size adjustment (A) and 22 ventral landmarks and 20 sliding semilandmarks (B). Population abbreviations: BLG ¼ Bulgaria; EPI ¼ Epirus (Greece); MAC ¼ Macedonia (Greece); THR ¼ Thrace (Greece); SMT ¼ Samothraki (Greece); CAN ¼ central Anatolia (Turkey); CIL ¼ Cilicia (Turkey); GIA ¼ Georgia; SYR ¼ Syria; M. m. spretoides: ISR ¼ Israel; M. cypriacus: Cyprus. Epirus was excluded from the analyses due to small sample size.

macedonicus populations were not significantly higher than those between populations within the continents. DFA revealed all three taxa to be highly significantly differentiated, and the classification was correct in all but one case – the only exception being a single M. m. spretoides specimen incorrectly classified as M. m. macedonicus. When only the M. m. macedonicus sample from Syria, i.e., the closest population to the Israeli M. m. spretoides, was considered, the classification was 100% correct in all cases. The forward stepwise DFA reduced the number of variables used for discrimination to 17, whereas the backward analysis used four measurements only. Since both procedures yielded 100% correct classification and both shared the same four variables, we can use the reduced set of four measurements for discriminating between the taxa according to the following classification functions: 49.35*LAR+53.31*LAZ+102.41*HCB+371.84*LM13I1180.01 for macedonicus, 67.70*LAR+ 64.34*LAZ+85.10*HCB+383.29*LM13I1140.54 for spretoides, and 79.04*LAR+70.74*LAZ+97.20*HCB+ 413.04*LM13I1361.14 for M. cypriacus (abbreviations: LAR ¼ rostral width, LAZ ¼ zygomatic width, HCB ¼ height of the braincase, LM13I ¼ length of the lower toothrow).

3.2. Landmarks 3.2.1. Size The centroid size was computed both for the dorsal (CSD) and ventral (CSV) sides. In agreement with the results of traditional morphometrics, the largest CS by far was found in M. cypriacus (Fig. 5A). In M.

macedonicus, the results confirmed the small size of the Israeli M. m. spretoides; however, the average size of this subspecies was not significantly different from that of the M. m. macedonicus populations nor was it the lowest value within the species (Fig. 5B). Since CS did not discriminate between M. m. macedonicus and M. m. spretoides (and, hence, the size differences could not be attributed to phylogeny), potential correlates of the skull size were searched for. We tested the significance of the regression of CSD and CSV of individual mouse specimens on the altitude, longitude, and latitude of particular collecting sites. It was found that size decreased significantly with altitude (CSD: R ¼ 0.200, p ¼ 0.0046; CSV: R ¼ 0.144, p ¼ 0.0442), whereas it increased with latitude, i.e., from the south to the north (CSD: R ¼ 0.269, p ¼ 0.0001; CSV: R ¼ 0.253, p ¼ 0.0004). Conversely, the regression with longitude was not significant. 3.2.2. Dorsal shape The pattern of morphological variation of the dorsal side of the skull described by the first two relative warps is shown in Fig. 5a. The shape differences explained by the RW1 axis are located predominantly in the orbital area. As we move from negative to positive RW1 values, the zygomatic arch becomes more convex and protrudes rostrally, whereas the rostral extremity of the squamosal process (landmark 12) moves towards the median axis. Thus, the dorsal projection of the orbit has a rather rounded shape. Moreover, the rostral extremity of the fronto-parietal suture (landmark 11) shifts caudally with increasing RW1 scores. While the second relative warp is also partly related to a broadening of the zygomatic arches, the main shape

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Fig. 5. (A) Relative warp 1 scores plotted against the centroid size for the ventral side of the skull; black dots ¼ M. m. macedonicus; open circles ¼ M. m. spretoides; gray triangles ¼ M. cypriacus. (B) Box plot showing differences in the centroid size for the dorsal side of the skull among the M. macedonicus populations. Population abbreviations: BLG ¼ Bulgaria; EPI ¼ Epirus (Greece); MAC ¼ Macedonia (Greece); THR ¼ Thrace (Greece); SMT ¼ Samothraki (Greece); CAN ¼ central Anatolia (Turkey); CIL ¼ Cilicia (Turkey); GIA ¼ Georgia; SYR ¼ Syria; M. m. spretoides: ISR ¼ Israel.

change described by the RW2 axis is the relative size of the braincase (landmarks 13 and 14). Hence, individuals with high RW2 scores (e.g., mice from Georgia [GIA] in Fig. 6A) are characterized by relatively broad zygomatic arches and a compressed occipital part of the neurocranium. The skull of M. cypriacus (CYP) looks quite robust, with broad zygomatic arches and braincase (Fig. 5A, bottom left). The first relative warp was significantly correlated with the geographic position of individual collecting sites in M. macedonicus, both with longitude (R ¼ 0.229, p ¼ 0.0035) and latitude (R ¼ 0.475,

p ¼ 0.0000). Moreover, RW1 was significantly negatively correlated with altitude (R ¼ 0.382, p ¼ 0.0000). This means that from the south-east to the north-west and towards lower altitudes, the skulls of M. macedonicus acquire broad and convex zygomatic arches with the rostral part of the squamosal process shifted medially. On the other hand, the correlation between geographic and morphometric (Procrustes) distances was not significant (Mantel test: R ¼ 0.161, p[rand zXobs z] ¼ 0.2148). In addition, morphometric distances between the European and Asian populations were not significantly higher than those within the continents. MANOVA revealed highly significant differences between populations (Wilks’ Lambda: 1.5424  104, p ¼ 1.8440  1074). The classification matrix is shown in Table 2 (left columns). The rate of correct classification into predicted groups ranged from 84% in M. m. spretoides to 100% in M. cypriacus (average: 98.15%). When only M. m. spretoides from Israel and M. m. macedonicus from Syria (the nearest population studied) were compared, the discrimination was 100% correct, hence individuals from these two Near Eastern populations can be correctly classified using the 14 dorsal landmarks and 20 sliding semilandmarks. The first canonical variate appeared to separate M. cypriacus from the M. macedonicus populations. The ordination showed a rather central position for the Thrace population within M. macedonicus, as this was connected with all M. macedonicus populations except that from Cilicia with the minimum-length spanning tree (MST). Similarly, M. cypriacus was rather distinct in the UPGMA and NJ trees, yet the branching pattern within M. macedonicus appeared rather random (except for a sister relationship between the populations from Thrace and Macedonia).

3.2.3. Ventral shape The first relative warp extracted from positions of landmarks on the ventral side of the skull explains differences in the position of the zygomatic process of the temporal bone and tympanic bulla together with the size of the occipital foramen (Fig. 6B). Specimens with higher RW1 scores have relatively broad skulls with landmarks 19 and 22 shifted laterally; the basion (landmark 9) is shifted rostrally while the opisthion (landmark 10) moves caudally so that the occipital foramen becomes large. In addition, the size of the molars increases with increasing RW1 scores. The second relative warp reflects shape differences similar to those of RW1, but the changes are more complex: The skulls characterized by higher scores have a relatively long rostral part (including a longer rostral palatine fissure), and the tympanic bulla has shifted in a rostro-medial direction (most apparent in landmark 20).

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Fig. 6. Projection of the first two relative warps based on the dorsal (A) and ventral (B) landmark configurations of the skull: (black dots) M. m. macedonicus; gray dots ¼ M. m. spretoides; and open square ¼ M. cypriacus. Population abbreviations: BLG ¼ Bulgaria; EPI ¼ Epirus (Greece); MAC ¼ Macedonia (Greece); THR ¼ Thrace (Greece); SMT ¼ Samothraki (Greece); CAN ¼ central Anatolia (Turkey); CIL ¼ Cilicia (Turkey); GIA ¼ Georgia; SYR ¼ Syria; M. m. spretoides: ISR ¼ Israel; M. cypriacus: Cyprus. In the right column, thin-plate spline deformation grids correspond to positive and negative displacements along the first and second relative warp axes, respectively (deformations arbitrarily magnified). Underneath each plot, deformations corresponding to the position of M. cypriacus in the morphospace are shown.

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Table 2. Classification table derived from the discriminant function analysis based on the partial warp scores for dorsal and ventral landmark configurations of the skull Dorsal side of skull Ventral side of skull M. m. macedonicus 99.45% 181 1 0 99.44% 177 0 1 M. m. spretoides 84.21% 3 16 0 100.00% 0 15 0 M. cypriacus 100.00% 0 0 15 94.74% 1 0 18 Total

98.15% 184 17 15 99.06%

178 15 19

Rows are observed groups and columns are predicted groups.

M. cypriacus has a rather average shape of the ventral side of the skull (Fig. 6B, bottom left). Within M. macedonicus, the scores of RW1 based on ventral landmarks were significantly negatively correlated with longitude (R ¼ 0.157, p ¼ 0.0276), but not with latitude (R ¼ 0.074, p ¼ 0.3041) or altitude (R ¼ 0.028, p ¼ 0.6996). In other words, towards the west, the skulls tend to have a broader braincase and a larger occipital foramen. This result seems to contradict the plot in Fig. 5B, where such a geographic gradient along the RW1 axis is hardly detectable. However, it should be noted that the regression between RW1 and longitude is rather weak and is based on individual scores rather than on the population centroids shown in Fig. 5. As in the case of the dorsal side of the skull, the correlation between geographic and Procrustes distances was not significant (Mantel test: R ¼ 0.233, p[rand zXobs z] ¼ 0.1576), and the European and Asian populations were not more differentiated than those within the continents. Differences between populations were shown to be highly significant in MANOVA (Wilks’ Lambda: 1.5107  103, P ¼ 3.724  1081). Also, the accuracy of the classification was better than that based on the dorsal landmarks (average: 99.06%). Interestingly, only a single individual of M. cypriacus was misclassified within the group of M. m. macedonicus populations (Table 2, right columns). Again, when only M. m. spretoides from Israel and M. m. macedonicus from Syria were compared, the discrimination based on 22 ventral landmarks was 100% correct. Similar to the dorsal ordination, CVA based on ventral shape variables showed the Thracian population to be connected with most of the M. macedonicus populations (Bulgaria, Samothraki, central Anatolia, Georgia, Syria). The unrooted NJ tree is shown in Fig. 4B. In contrast to the measurements for the shape of the dorsal side of the skull, M. cypriacus does not appear to be morphologically divergent from M. macedonicus in the shape of the ventral side (cf. also Fig. 6B). Again, the branching pattern is rather random even though the M. m. spretoides population (ISR) is correctly placed as the sister taxon to all the M. m. macedonicus populations and some geographically close populations tend to be grouped

together (e.g., the populations from Macedonia, Thrace, and Bulgaria; Fig. 4B). 3.2.4. Allometry and shape covariation For both the dorsal and ventral sides, the regression of partial warps on the centroid size was highly significant (dorsal side: Wilks’ Lambda ¼ 0.0199, p ¼ 1.256  1057; ventral side: Wilks’ Lambda ¼ 0.4701; p ¼ 1.686  1013), suggesting that both the dorsal and ventral sides grow allometrically (i.e., there is a change in shape during growth). The test of homogeneity of regression slopes among the three groups (M. m. macedonicus, M. m. spretoides, and M. cypriacus) showed that there were no significant differences in allometries on the ventral side (Wilks’ Lambda ¼ 0.6424, p ¼ 0.297). However, the test was significant for the dorsal side (Wilks’ Lambda ¼ 0.4053, p ¼ 0.032), suggesting heterogeneity of regression slopes. Subsequent tests revealed that the result was caused by the different allometries between M. macedonicus and M. cypriacus (p ¼ 0.011) rather than between the two M. macedonicus subspecies (p ¼ 0.406). Table 3 shows the results of the 2B-PLS analysis using 999 permutations (only the first 10 dimensions are shown). In M. macedonicus, the first pair of latent variables (squared singular values) accounts for 62% of the squared covariance (no other permutation resulted in a higher value for the first dimension), whereas all other singular values were much smaller and not significant. The correlation for the first pair of variables was 0.751 (again, with no other permutation resulting in a higher value). The other correlations were lower, even though most of them were significant. In M. cypriacus, the covariance between the dorsal and ventral sets of landmarks revealed a pattern similar to Table 3. analysis

Results of the two-block partial least-squares

Dim. M. macedonicus P 2 ri li P 1 2 3 4 5 6 7 8 9 10

0.623 0.119 0.059 0.042 0.039 0.030 0.025 0.016 0.010 0.009

0.001 0.989 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000

0.751 0.531 0.536 0.518 0.542 0.450 0.457 0.360 0.438 0.374

P

M. cypriacus P 2 li P ri

P

0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.098 0.002 0.011

0.511 0.167 0.115 0.062 0.044 0.036 0.023 0.018 0.009 0.005

0.237 0.375 0.256 0.289 0.040 0.784 0.167 0.503 0.456 0.444

0.035 0.826 0.762 0.984 0.97 0.873 0.919 0.763 0.941 0.989

0.857 0.857 0.880 0.873 0.916 0.789 0.878 0.797 0.811 0.826

P Only the first 10 dimensions are shown; li2 is the sum of squared singular values, ri is the correlation for the ith pair of latent variables. Probabilities P are based on 999 random permutations (plus one observed value) of the association between the dorsal and ventral sets of landmarks. See text for details.

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that in M. macedonicus. The first pair of latent variables accounted for about 51% of the squared covariance (with 34 random permutations exceeding the observed value), whereas contributions of the other dimensions were negligible. None of the correlations were significant. Fig. 7 shows the shape changes corresponding to the positive ends of the ordination along the axis of the first pair of dorsal and ventral latent variables, respectively (the ordination scatterplot is not shown). More specifically, the figure shows what aspects of the shape of the two sides of the mouse skull covary. Obviously, the shape changes of the dorsal and ventral landmark configurations are very similar: The most apparent aspect is the broader braincase and zygomatic arches and narrower rostral part, as shown by the lateral shift of the dorsal landmarks 12–14 and the ventral landmark 19, and the medial shift of the dorsal landmarks 6–10 and ventral landmarks 12–13. Another feature common to both sides of the skull is the relative position of the dorsal landmarks 7 and 8 and ventral landmarks 12 and 13, revealing differences in the shape of the rostral margin of the zygomatic plate (i.e., convex in the positive direction vs. straight in the negative direction along the first 2B-PLS dimension). However, there is probably more involved than these features, since the position of the ventral landmark 13 (the medial extremity of the zygomatic plate in the ventral projection, see Fig. 3) would be expected to be placed more caudally in the rounded zygomatic plates. Moreover, there is also an apparent contrasting effect between the dorsal and ventral sides in the interorbital part of the skull, as the frontal bone (dorsal landmarks 2 and 3) is slightly elongated relative to the shortened palatal part (ventral landmarks 4–8), suggesting different evolutionary histories in the shape of these two components.

4. Discussion 4.1. Interspecific variation Recent discoveries in the western-Palearctic group of non-commensal (‘aboriginal’) mouse species seem to be

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inconsistent with the common view of these species as genetically rather homogeneous, especially when compared with M. domesticus (Prager et al. 1996, 1998; Gu¨ndu¨z et al. 2000). At the same time, these findings have highlighted the need for thorough analyses of the

Fig. 7. Thin-spline visualization of dorsal (A) and ventral (B) shapes of the skull of M. macedonicus as a deformation of the average landmark configuration. The shapes correspond to the positive ends of the ordination along the first 2B-PLS dimension. Note that the ventral side is flipped horizontally relative to Fig. 2. The numbers denote individual landmarks: (A) Dorsal side: LM1 ¼ rostralmost point of the nasal bone (rhinion); LM2 ¼ intersection of the naso-frontal suture in the midline (nasion); LM3 ¼ intersection of the coronal and sagittal sutures (bregma); LM4 ¼ intersection of the sagittal and parietal-interparietal sutures (lambda); LM5 ¼ caudal end of the curvature of the occipital (opistocranion); LM6 ¼ intersection of the rostral curvature of the nasal process of the incisive bone (Processus nasalis ossis incisivi) and the nasal bone in the dorsal projection; LM7 ¼ point of maximum curvature of the rostro-lateral part of the maxilla; LM8 ¼ rostral end of the zygomatic plate; LM9 ¼ caudal end of the intersection of the zygomatic process of the maxilla and the upper limb of this process; LM10 ¼ lateral end of the nasofrontal suture in the dorsal projection; LM11 ¼ rostralmost point of the parietal bone; LM12 ¼ rostral end of the zygomatic process of the temporal bone (Processus zygomaticus partis squamosae ossis temporalis); LM13 ¼ intersection of the parietal-interparietal and interparietal-occipital sutures; LM14 ¼ caudo-lateral end of the occipital bone in the dorsal projection. Ventral side: LM1 ¼ rostralmost point of the upper incisor in the midline; LM2 ¼ lateral end of the upper incisor in the dorsal projection; LM3 ¼ rostral end of the rostral palatine fissure (Fissura palatina); LM4 ¼ caudal end of the rostral palatine fissure; LM5 ¼ intersection of the maxillopalatine suture in the midline; LM6 ¼ center of the Foramen palatinum majus; LM7 ¼ rostral end of the curvature of the palatine bone (Lamina horizontalis ossis palatini); LM8 ¼ lateral end of the basisphenoid-basioccipital suture in the dorsal projection; LM9 ¼ rostral end of the occipital foramen in the midline (basion); LM10 ¼ caudal end of the occipital foramen in the midline (opisthion); LM11 ¼ caudal end of the medial edge of the occipital condyle; LM12 ¼ rostral end of the zygomatic plate; LM13 ¼ medial end of the zygomatic plate in the ventral projection; LM14 ¼ rostral end of the first upper molar in the dorsal projection; LM15 ¼ lingualmost point of contact of the first and second upper molars in the ventral projection; LM16 ¼ lingualmost point of contact of the second and third upper molars in the ventral projection; LM17 ¼ caudal end of the third upper molar in the ventral projection; LM18 ¼ rostral end of the Foramen lacertum rostrale; LM19 ¼ caudal internal maximum curvature of the zygomatic process of the temporal bone (Processus zygomaticus partis squamosae ossis temporalis); LM20 ¼ point of juxtaposition of the tympanic bulla and the muscular process (Processus muscularis); LM21 ¼ caudal end of the external opening of the auditory channel; and LM22 ¼ styloglossal process of the tympanic bulla.

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genetic and morphological variation within and among aboriginal mouse species. Although some morphometric analyses were presented in connection with the description of M. macedonicus spretoides (Orth et al. 2002) and M. cypriacus (Cucchi et al. 2006), we have extended these studies by analyzing populations sampled from the major part of the species range (M. macedonicus) and from the entire Island of Cyprus including the northern Turkish areas (M. cypriacus). We used both traditional and geometric morphometric tools including a rather novel application of sliding semilandmarks. One of the tasks of morphometric studies is to search for possible phylogenetic, geographical and ecological correlates of phenotypic variation within species. In the

context of the present study, we asked whether the pattern of morphometric variation among populations of the Balkan short-tailed mouse, M. macedonicus, reflected the presumed phylogenetic history of the species or, rather, geological events, such as the separation of Europe and Asia. An alternative hypothesis might by a clinal character of variation, following, for example, Bergman’s rule. The latter was suggested by Orth et al. (2002), who found the southern subspecies M. m. spretoides to be smaller than the northern populations of M. m. macedonicus from Bulgaria and Georgia, and concluded this to be in agreement with Bergman’s rule. Even though we showed a significant positive correlation between centroid size (CS) and latitude, as predicted by Bergman’s rule, CS of the Israeli M. m. spretoides population was not the smallest among the samples analyzed in this study. Moreover, the correlation between CS and altitude was found to be negative, indicating that the mice were smaller at higher altitudes. One possible explanation of this phenomenon may be better nutritional conditions in lowlands relative to hilly areas. The clinal character of morphometric variation in M. macedonicus is not limited to size as was shown above (see Results). Conversely, significant correlations between geographic and morphometric distances were found only in traditional measurements, both cranial and dental, but not in dorsal and ventral landmark configurations of the skull. In addition, morphometric differences between Asian and European M. macedonicus populations were not significantly higher than those within the continents. The weak concordance between the pattern of morphometric variation and the pattern expected from the presumed history of M. macedonicus (Krysˇ tufek and Machola´n 1998; Machola´n et al. 2007) seems to corroborate the results of molecular studies suggesting weak phylogeographic structuring in this species Fig. 8. Skulls of M. cypriacus (A–C) from Paramytha (CY-20, male, leg. M. Machola´n) and M. m. macedonicus (D–F) from Gephyra, Greece (BG-1943, male, leg. V. Vohralı´ k) in dorsal (A, D), ventral (B, E) and lateral (C, F) views. In comparison with M. macedonicus, the M. cypriacus skull is, besides its bigger size, characterized by a robust appearance with broad and angular zygomatic arches that provide large ocular orbits, especially in the squamosal part. The external occipital crest is noticeably pronounced. In the lateral view, the ventral wing of the parietal is usually wedge-shaped, more or less regular, not tortuous, but rarely smooth. The rostral edge of the zygomatic plate is straight, similar to that of M. macedonicus (note that since the M. macedonicus skull is very pale it seems to be slightly out of focus). The skulls of M. m. macedonicus and M. m. spretoides (not shown) are very similar, without any apparent qualitative differences, and can be distinguished only through multivariate morphometric methods (photo: M. Machola´n).

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(Machola´n et al. 2007). In contrast, the phylogenetic signal (‘phylogenetic inertia’) was found to be stronger in dental characters than in cranial ones in agreement with the results of Machola´n (2006), who found a significant correlation between phylogenetic and morphometric distances indicating that phylogenetic history contributes about 80% to the shape differences in the first upper molar within the genus Mus. Both in M. macedonicus and M. cypriacus, the pattern of morphometric covariance between the dorsal and ventral side of the skull was largely one-dimensional, i.e., the first dimension of the 2B-PLS analysis described the major part of this pattern. A similar result was reported in house mice by Rohlf and Corti (2000), who revealed a contrasting effect in the rostral and palatal parts of the skull and suggested a differing evolution in shape in these components, resulting in different functionality. In the present study, both sides of the skull were shown to covary in many aspects (i.e., the width of the braincase, zygomatic arches and rostral part of the skull, as well as the shape of the zygomatic plate). In contrast, there were also some differences between the two sides, for instance, in the zygomatic plate or interorbital part of the skull, suggesting different rates or directions in shape change in these components. However, a functional interpretation of these differences is not easy.

4.2. Differences between taxa The observed close morphometric similarity among populations of M. macedonicus agrees with Orth et al. (2002), who found a great similarity in shape between M. m. spretoides and M. m. macedonicus and concluded that, while the former subspecies could be distinguished from the latter mainly according to its smaller size, reliable discrimination of the two taxa was possible only through the multivariate morphometric approach described by Gerasimov et al. (1990). However, we have shown in this study that reliable discrimination of M. m. spretoides and M. m. macedonicus is possible with as few as four measurements describing the general shape of the skull (i.e., rostral width, zygomatic width, height of the braincase, and length of the lower tooth row). Likewise, the percentage of correct classification based on dorsal and ventral landmark configurations of the skull was very high, reaching 100% when only Near East populations were used as representatives of the two subspecies (i.e., M. m. macedonicus from Syria and M. m. spretoides from Israel). As expected, morphometric differentiation of M. cypriacus from M. macedonicus is much easier than between the two subspecies of M. macedonicus. The most diagnostic trait of M. cypriacus is the great size of its skull. This phenomenon can be ascribed to island gigantism, as

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acknowledged also by Cucchi et al. (2006). Besides its bigger size, the skull is characterized by a general robustness with broad and angular zygomatic arches, this trait being more pronounced in the caudal, squamosal part. The zygomatic plate is wide and rostrally displaced (Fig. 8). In contrast, the shape of the ventral side of the skull appears rather average among the samples analyzed in this study. Thus, the original description by Cucchi et al. (2006) was largely confirmed by our analysis of materials sampled from six sites covering not only the Troodos Mountains in central Cyprus and adjacent areas as in Cucchi et al. (2002), but also from the north-western and northern Turkish parts of the island, as well as by using two sets of semilandmarks describing parts of the skull that do not have reliable landmarks.

Acknowledgements We are grateful to J.-C. Auffray (Universite´ Montpellier II), B. Krysˇ tufek (Slovenian Museum of Natural History, Ljubljana), and the National Museum of Natural History (Smithsonian Institution, Washington, DC) for kindly providing skeletal materials; to D. Hardekopf (University of California at San Diego) for language corrections; and to D.G. Homberger and an anonymous reviewer for useful suggestions. This study was supported partly by Grant no. A6045307 of the Grant Agency of the Academy of Sciences of the Czech Republic and partly by the Czech Science Foundation Grant nos. 206/06/0707 and 206/05/2334.

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