Mammalia 2015; 79(3): 293–303 Boris Kryštufek, Toni Koren, Simon Engelberger, Győző F. Horváth, Jenő J. Purger, Atilla Arslan, Gabriel Chişamera* and Dumitru Murariu Fossorial morphotype does not make a species in water voles Abstract: Phenetic and ecological plasticity in Arvicola has caused a long-standing dispute over the number of species Introduction within the genus, which is currently thought to consist of Water voles (genus Arvicola Lacépède, 1799) are extraor- two aquatic (sapidus, amphibius) and one fossorial species dinary among the Palaearctic rodents for their outstand- (scherman). We used mitochondrial cytochrome b (cytb) ing phenetic and ecological plasticity. Two morphological gene sequences to reconstruct phylogenetic relationships types can be distinguished, the aquatic and the fossorial, between the fossorial and the aquatic water voles from each with different living habits (Meylan 1977). This varia- the various regions of their European and Asiatic range. bility has caused a long-standing dispute over the number These two types differed morphologically and exhibited of species within the genus. Whereas Ellerman and Mor- allopatric ranges. Our study provided 50 new haplotypes, rison-Scott (1951) clumped a wide array of morphological generating a total dataset of 70 different water vole cytb forms under one nominal species, A. terrestris (Linnaeus, haplotypes. Phylogenetic reconstructions retrieved two 1758), Hinton (1926) recognized four species and Miller major lineages that were in a sister position to A. sapidus: (1912) listed seven species for Western Europe only. Since a fossorial Swiss lineage and a widespread cluster, which 1970, the majority of authors have accepted a division into contained aquatic and fossorial water voles from Europe two species: A. sapidus Miller, 1908 with a small range in and western Siberia. The phylogeographic architecture in Western Europe, and a widespread and polytypic A. ter- water voles is explained by Quaternary climatic dynamics. restris (Corbet 1978, Niethammer and Krapp 1982 Gromov Our results show that A. scherman in its present scope is and Polyakov 1992, Shenbrot and Krasnov 2005). Within not a monophyletic taxon. the latter, Ognev (1964) distinguished two species, the fos- sorial A. scherman (Shaw, 1801) and the aquatic A. terrestris. Keywords: Arvicola amphibius; Arvicola scherman; cyto- Ognev’s taxonomic arrangement was largely ignored, but chrome b; molecular phylogeny; Quaternary refugia. gained credit after being adopted in the influential compi- lation by Musser and Carleton (2005). The genus Arvicola DOI 10.1515/mammalia-2014-0059 is currently regarded as consisting of two aquatic (sapidus, Received April 15, 2014; accepted July 3, 2014; previously published amphibius) and one fossorial species (scherman). Musser online August 5, 2014 and Carleton (2005) even anticipated “that future revision- ary research will […] converge towards Miller’s (1912) rec- *Corresponding author: Gabriel Chişamera, Department of ognition of biodiversity”, i.e., in an increase in the species Terrestrial Fauna, “Grigore Antipa” National Museum of Natural History, Sos. Kiseleff no. 1, Bucharest 1, RO-011341, Romania, of water voles. Evidently, our understanding of species e-mail: [email protected] limits in the genus Arvicola is still far from final. Boris Kryštufek: Slovenian Museum of Natural History, Prešernova Arvicola sapidus, which is endemic to the Pyrenean 20, SI-1000 Ljubljana, Slovenia Peninsula and France (Shenbrot and Krasnov 2005), pos- Toni Koren: Science and Research Centre, University of Primorska, sesses a unique set of chromosomes (Matthey 1956) and Garibaldijeva 1, SI-6000 Koper, Slovenia is therefore well defined taxonomically. We shall subse- Simon Engelberger: Museum of Natural History Vienna, Burgring 7, 1010 Wien, Austria quently focus on two species that result from splitting A. Győző F. Horváth and Jenő J. Purger: Institute of Biology, Faculty of terrestris (sensu Corbet 1978) into A. amphibius (Linnaeus, Sciences, Department of Ecology, University of Pécs, H-7624 Pécs, 1758) and A. scherman (Musser and Carleton 2005). The Hungary names amphibius and terrestris are synonymous and the Atilla Arslan: Faculty of Science, Department of Biology, Selçuk former has priority based on the principle of first reviser University, 42031 Konya, Turkey Dumitru Murariu: Department of Terrestrial Fauna, “Grigore Antipa” (Corbet 1978). Both names were used interchangeably in National Museum of Natural History, Sos. Kiseleff no. 1, Bucharest 1, the past. To avoid confusion, we shall subsequently use RO-011341, Romania amphibius sensu stricto (s.s.) in the sense of Musser and 294 B. Kryštufek et al.: Fossorial morphotype does not make a species in water voles Carleton (2005), and amphibius sensu lato (s.l.) to contain Musser and Carleton’s amphibius s.s. and scherman. The range of Arvicola amphibius s.s. stretches from Western Europe to the River Lena in eastern Siberia (Bat- saikhan et al. 2008). The geographic scope of A. scherman is more restricted, but it is also poorly resolved. Hinton (1926) believed that its range extends over “Central Europe from the Baltic southwards to the Pyrenees”, also encompassing the Alps. Panteleyev (2001) and Amori et al. (2008a) mapped A. scherman for the mountainous regions in northern Spain and the Pyrenees, the Alps, the mountains of Central Europe, and the Carpathians. The two species are presumably allopatric in the south, but show overlapping ranges in the north (e.g., Meylan 1977). Arvicola voles, which typically possess rootless molars, presumably evolved from the extinct rhizodont Mimomys Forsyth Major, 1902, and have been known in the Figure 1 Distribution of water vole samples sequenced for cyto- chrome b gene. Approximate ranges of Arvicola amphibius s.s. fossil record since the beginning of the Early Pleistocene and A. scherman follow Batsaikhan et al. (2008) and Amori et al. (Gromov and Polyakov 1992). Therefore, the taxonomic (2008a), respectively. Symbols for morphotypes (Δ – fossorial; □ richness in Arvicola results from a relatively recent radia- – aquatic) correspond to those in Figure 4 and Table 1. Samples of tion and the species may still be in an ongoing speciation unknown morphotype are denoted by circles. process. In such cases, which are common in arvicolines (e.g., Jaarola et al. 2004), molecular data provide vital insight into past cladogenetic events and current species of Biology, Selçuk University (Konya, Turkey). Tissue limits. In the present study, we used mitochondrial cyto- samples are deposited in the PMS and NMW. chrome b (cytb) gene sequences to reconstruct phyloge- netic relationships between the fossorial and the aquatic water voles from various regions of their European and Morphotypes Asiatic range. Our aim was to test the taxonomic arrange- ment of A. amphibius s.l. Our null hypothesis predicted We examined museum specimens of water voles for their the reciprocal monophyly of the aquatic and the fossorial morphology. Included in the study were individuals morphotypes. If our hypothesis would not be rejected, sequenced for cytb but also museum vouchers of unknown the results will support the current tripartite taxonomic genetic makeup from the same site as the genotyped mate- arrangement of the genus Arvicola. rial. Swiss and German sequences were downloaded from GenBank and their morphotype was not reported (Pfunder et al. 2004, Schlegel et al. 2012b). Swiss samples were from the range of a subspecies scherman (Meylan and Saucy Materials and methods 1995) and were assigned on this ground to the fossorial morphotype. German water voles were not classified into Samples the morphotype. Identification was based on character states and five We studied the mitochondrial genetic makeup of water linear measurements (three external and two cranial) that voles from Switzerland, Austria, Hungary, Slovenia, are used in determination keys (see references in the next Bosnia and Herzegovina, Serbia, Romania, Turkey, and paragraph). Measurements were scored only in full grown Russia (Figure 1, Table 1). Voucher specimens (skins and voles (age groups V and VI of Hinton 1926) with complete skulls, or wet specimens) were preserved for the majority skulls: H&B – length of head and body; TL – length of of sequenced individuals. They are deposited in the Slo- tail; HF – length of hind foot (without claws); condyloba- venian Museum of Natural History (PMS; Ljubljana, Slo- sal length of skull (CbL); and length of molar tooth-row venia), Naturhistorisches Museum Wien (NMW; Vienna, (MxT). External measurements were obtained from speci- Austria), “Grigore Antipa” National Museum of Natural men tags and skull dimensions were measured using a History (Bucharest, Romania), and the Department dial calliper to the nearest 0.1 mm. B. Kryštufek et al.: Fossorial morphotype does not make a species in water voles 295 Table 1 Water voles included in the phylogenetic analysis, arranged according to morphotypes (Δ – fossorial; □ – aquatic). Names of coun- tries are abbreviated as follows: A – Austria; BH – Bosnia and Herzegovina; CH – Switzerland; D – Germany; H – Hungary; RO – Romania; RU – Russia; SL – Slovenia; SR – Serbia; TR – Turkey. Location Sample locality Longitude/latitude N Morphotype Haplotype GenBank access. no. 1 Hagen, D 51°11′/07°58′ D1 JF318969a 2 Eckhardtshausen, D 50°52′/10°16′ D2 HQ728473b D3 HQ728474b 3 Dürrweitzschen, D 51°10′/12°43′ D4 HQ728469b D5 HQ728470b 4 Scharfenberg, D 51°09′/13°29′ D6