Org Divers Evol (2014) 14:279–293 DOI 10.1007/s13127-014-0169-3 ORIGINAL ARTICLE A phylogeny of softshell turtles (Testudines: Trionychidae) with reference to the taxonomic status of the critically endangered, giant softshell turtle, Rafetus swinhoei Minh Le & Ha T. Duong & Long D. Dinh & Truong Q. Nguyen & Peter C. H. Pritchard & Timothy McCormack Received: 18 July 2013 /Accepted: 12 February 2014 /Published online: 6 March 2014 # Gesellschaft für Biologische Systematik 2014 Abstract Several important aspects of the evolution of a well resolved and strongly supported phylogeny. The the softshell turtle (family Trionychidae) have not been results of our time-calibration and biogeographic opti- addressed thoroughly in previous studies, including the mization analyses show that trionychid dispersals out of pattern and timing of diversification of major clades and Asia took place between 45 and 49 million years ago in species boundaries of the critically endangered Shanghai the Eocene. Interestingly, the accelerated rates of diver- Softshell Turtle, Rafetus swinhoei. To address these sification and dispersal within the family correspond issues, we analyzed data from two mitochondrial loci surprisingly well to global warming periods between (cytochrome b and ND4) and one nuclear intron (R35) the mid Paleocene and the early Oligocene and from for all species of trionychid turtles, except Pelochelys the end of the Oligocene to the mid Miocene. Our study signifera, and for all known populations of Rafetus also indicates that there is no significant genetic diver- swinhoei in Vietnam and one from China. Phylogenetic gence among monophyletic populations of Rafetus analyses using three methods (maximum parsimony, swinhoei, and that previous taxonomic revision of this maximum likelihood, and Bayesian inference) produce species is unwarranted. Electronic supplementary material The online version of this article (doi:10.1007/s13127-014-0169-3) contains supplementary material, which is available to authorized users. M. Le (*) T. Q. Nguyen Department of Environmental Ecology, Faculty of Environmental Department of Terrestrial Ecology, Cologne Biocenter, University of Science, Hanoi University of Science, VNU, 334 Nguyen Trai Cologne, Zülpicher Strasse 47b, 50674 Cologne, Germany RoadThanh Xuan District Hanoi, Vietnam e-mail: [email protected] M. Le Centre for Natural Resources and Environmental Studies, VNU, 19 P. C. H. Pritchard Le Thanh Tong Street, Hanoi, Vietnam Chelonian Research Institute, 402 South Central Avenue, Oviedo, FL 32765, USA M. Le Department of Herpetology, Division of Vertebrate Zoology, American Museum of Natural History, New York, NY 10024, USA H. T. Duong : L. D. Dinh T. McCormack Department of Genetics, Faculty of Biology, Hanoi University of Asian Turtle Program, Cleveland Metroparks Zoo, No. 1302 Thanh Science, VNU, 334 Nguyen Trai RoadThanh Xuan District Hanoi, Cong Building, 57 Lang Ha Street, Hanoi, Vietnam Vietnam Present Address: T. Q. Nguyen L. D. Dinh Institute of Ecology and Biological Resources, Vietnam Academy of Department of Fundamental Sciences, VNU-School of Medicine and Science and Technology, 18 Hoang Quoc Viet, Hanoi, Vietnam Pharmacy, 144 Xuan Thuy RoadCau Giay District Hanoi, Vietnam 280 M. Le et al. Keywords Trionychidae . Rafetus swinhoei . Systematics . period of overexploitation, most populations in China and in Evolution . Africa . Asia . Europe . North America . ND4 . Vietnam appear to be extinct (Pritchard 2001;Leand cytb . R35 Pritchard 2009; Wang and Shi 2011). Taxonomically, al- though previous molecular and morphological comparisons show that this is a single species (Le and Pritchard 2009; Introduction Farkas et al. 2011), Le et al. (2010) produced radically differ- ent results, and described populations in Vietnam as a new Softshell turtles of the family Trionychidae are characterized by species, R. vietnamensis.Farkasetal.(2011)sheddoubton highly derived morphological characters, which have evolved theanalysesofLeetal.(2010) by highlighting sources of to adapt to an almost entirely aquatic environment. These potential errors. Despite this, it is likely that populations from special features include smooth leathery skin covering reduced Vietnam and China constitute independent evolutionary line- bony shell, flattened body shape, and webbed toes (Meylan ages given the distance and river systems separating them 1987; Ernst and Barbour 1989). Trionychid turtles, consisting (Fig. 1). To test this hypothesis, we employed a phylogenetic of 31 species and 13 genera (Van Dijk et al. 2012), are distrib- approach, and used the phylogenetic species concept as an uted widely, occurring in Africa, Asia (including New Guinea), operational definition. the Mediterranean, and North America (Iverson 1992). Fossil Moreover, the diversification pattern of this interesting records documented in Australia, Europe, and South America group has not been addressed adequately in previous studies. (Wood and Patterson 1973; Gaffney and Bartholomai 1979; Because the most primitive fossils have been found in Asia, Danilov 2005;Headetal.2006; Scheyer et al. 2012)indicate the continent has been widely regarded as the ancestral area of that, historically, the group was even more widespread. the group (Hirayama et al. 2000; Joyce and Lyson 2010; Since the first computer-aided analysis of trionychid phylo- Scheyer et al. 2012). However, the timing and pattern of genetic relationships using morphological data (Meylan 1987), dispersal events out of Asia to other continents, including many subsequent works have selected molecular data, both the Americas, Europe, and Africa, have not been investigated mitochondrial and nuclear markers, as a means to address comprehensively. In particular, a time-calibrated phylogeny in phylogenetic relationships among different species of the fam- combination with explicit biogeographic optimizations, which ily (Weisrock and Janzen 2000;Engstrometal.2002, 2004; can be used to test different diversification scenarios of the Praschag et al. 2007; McGaugh et al. 2008).Asaresult,afairly family, was lacking in earlier efforts. well resolved and robust molecular phylogeny of trionychids To resolve these issues, we reconstructed a phylogeny for has been established, e.g., Engstrom et al. (2004). In addition, all softshell turtle species, except Pelochelys signifera,using species boundaries within a number of widely distributed spe- two mitochondrial genes (cytochrome b and NADH dehydro- cies or species complexes have been clarified (Weisrock and genase subunit 4 - ND4) and a nuclear intron, G protein- Janzen 2000; Engstrom et al. 2002; Praschag et al. 2007; coupled receptor R35 (R35), and multiple outgroups, Caretta McGaugh et al. 2008; Fritz et al. 2010;Praschagetal.2011; caretta, Carettochelys insculpta, and Pelomedusa subrufa. Stuckas and Fritz 2011;Yangetal.2011). Samples from all known populations of Rafetus swinhoei in However, to date the taxonomic status of the critically Vietnam, and from living individuals in China were also endangered Shanghai Softshell Turtle, Rafetus swinhoei,is included in the analyses. We calibrated time divergence of still a matter of debate (Le and Pritchard 2009; Le et al. the phylogeny using the Bayesian relaxed clock method, and 2010;Farkasetal.2011). Ranked as one of 100 most endan- optimized biogeographic patterns using the statistical gered species in the world, only four live individuals of this dispersal-vicariance (S-DIVA) and Bayesian Binary MCMC species are recognized globally: two in Vietnam and two in (BBM) methods to infer the historical diversification of this China (Baillie and Butcher 2012). A captive breeding pro- turtle group. gram has been launched in the Suzhou Zoo, China, for the two individuals residing in China. Nonetheless, these efforts have been unsuccessful in producing offspring, apparently due to Materials and methods the age of the male (Kuchling 2012). To improve the proba- bility of success, the captive breeding program needs to in- Taxonomic sampling clude additional individuals of this species from other popu- lations. It is therefore critical to assess the taxonomic status of For Rafetus swinhoei, we sequenced four new samples, in- populations within its range. cluding fresh tissue from the individual in Hoan Kiem Lake Historically, this species had a large distribution range, located in downtown Hanoi and three bone samples from Ba including the Yellow River, Yangtze River, and their tribu- Vi Town near Hanoi and from Yen Bai and Phu Tho Prov- taries in China and the Red River system, as well as Ma River inces, northern Vietnam. These three bone samples are rela- and associated wetlands in Vietnam (Fig. 1). After a long tively young, ranging from 12 to just over 20 years old. We A phylogeny of softshell turtles (Testudines: Trionychidae) 281 Fig. 1 River systems where Rafetus swinhoei has been recorded. Locations of the type specimen and Vietnam’s samples used in this study are shown in yellow and red,respectively also added published data from the individual inhabiting Dong Bone samples were first cleaned with 10 % chlorox and then Mo Lake in the suburb of Hanoi (Le and Pritchard 2009), from placed on a clean surface to dry in order to eliminate the risk of samples collected in Ba Vi Town, Hoan Kiem Lake, and contamination on the surface of the samples. Bone or tissue Thanh Hoa Province (Le et al. 2010), and from Chinese samples were then extracted following protocols specified in samples (Table 1). Since the sequences