TheBlackwell Publishing Ltd. freshwater turtle genus Mauremys (Testudines, Geoemydidae) — a textbook example of an east–west disjunction or a taxonomic misconcept? DANA BARTH, DETLEF BERNHARD, GUIDO FRITZSCH & UWE FRITZ Accepted: 9 July 2003 Barth, D. Bernhard, D. Fritzsch, G. & Fritz, U. (2004): The freshwater turtle genus Mauremys — a textbook example of an east–west disjunction or a taxonomic misconcept? — Zoologica Scripta, 33, 213–221. We compare 1036 bp of the mitochondrial cytochrome b gene (cyt b) from all six Mauremys species with 16 other taxa, representing both currently recognized subfamilies of the Geo- emydidae (Geoemydinae and Batagurinae) to contribute a comprehensive dataset towards resolving the conflicting Mauremys taxonomy and phylogeography. Mauremys, a representa- tive of the Geoemydinae, is thought to be an example of a taxon with an east–west disjunction due to Pleistocene glacial extinction, with species occurring in the western Palearctic and spe- cies in the eastern Palearctic and Oriental regions. Our results contradict this traditional zoo- geographical scheme and the current taxonomy of the Geoemydidae. Mauremys is paraphyletic with respect to two East Asian genera belonging to the Batagurinae: Chinemys and Ocadia. Therefore, Mauremys, as currently understood, clearly represents a taxonomic misconcept. Mauremys + Chinemys + Ocadia contains four well supported clades, two of which — M. japonica + Chinemys + Ocadia and M. annamensis + M. mutica — are confined to eastern Asia. The other two — M. caspica + M. rivulata and M. leprosa — occur in the western Palearctic. Mauremys lep- rosa may represent an ancient lineage which differentiated before the split between the other western and eastern species occurred. The patchy distribution of the four clades is likely the result of several ancient radiations rather than of a Pleistocene extinction. The sister-group of Mauremys + Chinemys + Ocadia is Cuora, a morphologically highly specialized genus with a complicated shell hinging mechanism. Dana Barth & Detlef Bernhard, University of Leipzig, Institute of Zoology, Molecular Evolution & Animal Systematics, Talstr. 33, 04103 Leipzig, Germany Guido Fritzsch, University of Leipzig, Interdisciplinary Centre for Bioinformatics (IZBI), Kreuzstr. 7b, 04103 Leipzig, Germany Uwe Fritz, Zoological Museum, Natural History State Collections Dresden, A. B. Meyer Building, Königsbrücker Landstr. 159, 01109 Dresden, Germany. E-mail: [email protected] Introduction (Fig. 1). It is currently thought to consist of six species, The freshwater turtle genus Mauremys belongs to the Geo- three in the west and three in the east (Fritz 2001). The emydidae, a family consisting of approximately 60 species in three western Palearctic species are M. leprosa, M. rivulata and 25 genera. Geoemydid turtles are mainly distributed in east- M. caspica. M. leprosa inhabits western North Africa (Morocco ern Asia. Only three Mauremys species occur in the western to western Libya) and the Iberian Peninsula (Keller & Busack Palearctic, and one genus (Rhinoclemmys) is distributed within 2001). M. rivulata is distributed, in Europe, along the Adriatic central and northern South America (Ernst et al. 2000; Fritz coast southwards from central Dalmatia over Albania and 2001). Until recently, the family Geoemydidae Theobald, Greece to Bulgaria; it is also found in many islands in the 1868 was better known under its junior synonym Bataguridae Ionic and Aegean Seas and in Crete and Cyprus. In Asia Minor, Gray, 1869. However, Bour & Dubois (1986) demonstrated it is confined to the coastal regions of western and southern that Geoemydidae is the nomenclaturally valid name. Turkey and stretches southwards along the Levantine coast Mauremys has a patchy distribution, including parts of the to Israel (Fritz & Wischuf 1997; Wischuf & Busack 2001). western Palearctic region and, separated by a huge disjunc- M. caspica occurs in central Anatolia, the eastern Caucasus tion, parts of the Oriental and eastern Palearctic regions and Transcaucasus, Syria, Iraq, Iran, and western Turkmenistan. © The Norwegian Academy of Science and Letters • Zoologica Scripta, 33, 3, May 2004, pp213–221 213 Taxonomy of Mauremys • D. Barth et al. Fig. 1 Distribution of all currently accepted Mauremys species based on Iverson (1992), Fritz & Wischuf (1997), Keller & Busack (2001) and Wischuf & Busack (2001). Its southernmost outposts are in the southern Persian Gulf, Here we compare 1036 bp of the mitochondrial cytochrome and include Bahrein and Saudi Arabia (Fritz & Wischuf 1997; b gene (cyt b) from all six Mauremys species with 16 other taxa, Wischuf & Fritz 2001). The eastern species are M. mutica, representing both currently recognized subfamilies of the M. japonica and M. annamensis. M. mutica occurs in Vietnam, Geoemydidae (Geoemydinae and Batagurinae; Gaffney & southern China and the Japanese Ryukyu Islands. M. japonica Meylan 1988) with the aim of providing a comprehensive is confined to the main islands of Japan, while M. annamensis, dataset which may help resolve the conflicts between the which was transferred from the monotypic genus Annamemys taxonomy and phylogeography of Mauremys. to Mauremys by Iverson & McCord (1994), is restricted to Vietnam (Iverson & McCord 1994; McCord 1997). Two further Materials and methods taxa described as new Mauremys species in the 1990s were Sampling demonstrated to be of hybrid origin. M. iversoni Pritchard Tissue or blood samples were obtained from 25 specimens & McCord, 1991 originated from hybridization of M. mutica belonging to 22 species (Table 1). These samples represent with Cuora trifasciata (Parham et al. 2001; Wink et al. 2001), all currently recognized species of the genera Mauremys, M. pritchardi McCord, 1997 from hybridization of M. mutica Chinemys and Sacalia, and representative species of other with Chinemys reevesii (Wink et al. 2001). major groups of the Geoemydidae. Tissue samples from Other taxa which share similarly disjunct distributions are thigh muscles were obtained by dissecting freshly killed Cyanopica cyanus (Aves; Sedlag 1995; Fok et al. 2002), Misgur- animals. All specimens were identified by two specialists. nus fossilis (Osteichthyes; Lattin 1967; Sedlag 1995), and Complete alcohol-preserved specimens are deposited in the among the class Amphibia the Rana ‘esculenta’−nigromaculata herpetological collection of the Zoological Museum Dresden complex, the genus Bombina and the Old World representa- (= Museum für Tierkunde Dresden, MTD) under the catalogue tives of Hyla (Borkin 1984, 1986; Zug 1993). The phenome- numbers listed in Table 1. Blood samples were acquired by non is generally thought to be the result of glacial extinctions coccygeal vein puncture as described in Haskell & Pokras during the Pleistocene (Lattin 1967; Sedlag 1995; Lapparent (1994). Blood and tissue samples were stored at −70 °C in de Broin 2001). ethanol or EDTA buffer (Arctander 1988). Virtually all previous studies dealing with the systematics of Mauremys have been based on osteology and general mor- DNA Extraction, PCR amplification and sequencing phology (McDowell 1964; Busack & Ernst 1980; Hirayama Total genomic DNA was extracted from thigh muscle tissues et al. 1985; Pritchard & McCord 1991; Iverson & McCord following the protocol of Gustincich et al. (1991), while DNA 1994; Yasukawa et al. 1996, 2001; Fritz & Wischuf 1997; from blood samples was isolated using the QIAamp Blood McCord 1997). The monophyly of the genus was, until Mini Kit (Qiagen). recently, never questioned and the East Asiatic genus Sacalia Slightly modified versions of the primers mt-A (Lenk & was generally accepted as its sister-group (McDowell 1964; Wink 1997) and H15909 (Lenk et al. 1999) were used to Hirayama 1985; Yasukawa et al. 2001). The first molecular amplify a fragment of approximately 1080 bp containing studies, although based on only a limited dataset, could not 1036 bp of cyt b and part of the tRNA threonine (Table 2). confirm a close relationship between Mauremys and Sacalia PCR conditions were as follows: 5 min at 95 °C, then 40 (Wu et al. 1999; McCord et al. 2000; Honda et al. 2002a), cycles of 1 min at 95 °C, 1 min at 50 °C, 2 min at 72 °C, and while a close relationship between M. japonica and the East a single extension step of 10 min at 72 °C. Asiatic genus Chinemys was demonstrated, suggesting a Sequencing reactions were performed with the 7-deaza- paraphyletic Mauremys (Honda et al. 2002a). dGTP sequencing kit (Amersham Pharmacia) and separated 214 Zoologica Scripta, 33, 3, May 2004, pp213–221 • © The Norwegian Academy of Science and Letters D. Barth et al. • Taxonomy of Mauremys Table 1 Specimens examined in this study. (MTD, Museum für Tierkunde Dresden). Taxon Locality Voucher number/Origin EMBL acc. no. SUBFAMILY GEOEMYDINAE Mauremys annamensis (Siebenrock, 1903) Vietnam MTD live collection AJ564456 M. caspica caspica (Gmelin, 1774) Turkey: Birecik MTD 42628 AJ564453 M. caspica siebenrocki Wischuf & Fritz, 1997 Bahrain live collection, Breeding Centre for AJ564454 Endangered Arabian Wildlife, Sharja, UAE M. japonica (Temminck & Schlegel, 1835) Unknown MTD 42498 AJ564458 M. leprosa (Schweigger, 1812) Spain: Doñana wild specimen AJ564457 Biological Reserve M. mutica mutica (Cantor, 1842) Unknown MTD 41973 AJ564459 M. mutica cf. kami Yasukawa, Ota & Iverson, 1996 Unknown MTD live collection AJ564460 M. rivulata (Valenciennes, 1833) Turkey: Izmir MTD 41974 AJ564455 Sacalia bealei (Gray, 1831) Unknown MTD 41583 AJ519501 S. quadriocellata