Phylogenetic hypotheses for the turtle family Geoemydidae☆
Introduction
Currently, the turtle family Geoemydidae is composed of 23 genera and approximately 73 species. It is the largest turtle family in the world, accounting for about 25% of the total species-level diversity of turtles (Iverson, 1992). Geoemydids are predominantly freshwater aquatic and semi-aquatic turtles, and are widely distributed from Europe and North Africa, to India and southern Russia, to Indonesia, and the Philippines. Although geoemydids are often referred to as “Old World pond turtles,” one genus, Rhinoclemmys, is found in the New World from Mexico south to Ecuador, Venezuela, and Brazil (Ernst and Barbour, 1989; Iverson, 1992).
The Geoemydidae (the names Batagurinae/Bataguridae are junior synonyms of Geoemydidae (Bour and Dubois, 1986; McCord et al., 2000) ) has been the subject of several recent morphological and molecular phylogenetic studies, and its taxonomy is in flux. Based on seven morphological characters, McDowell (1964) subdivided what was then the Emydidae into two subfamilies, Emydinae and Batagurinae, and further subdivided the Batagurinae into four implicitly monophyletic generic complexes: Batagur, Geoemyda, Hardella, and Orlitia (Table 1a). Based on a similar mechanism for closing the anterior part of the shell, Bramble (1974) hypothesized that Cuora, Cyclemys, and Pyxidea form a closely related phyletic assemblage (his Cyclemys group). Bramble further postulated that the Cyclemys group was probably derived from a Heosemys-like ancestor and therefore all four genera could be united into a Heosemys complex (Table 1b). Using chromosomal data, Carr and Bickham (1986) concluded that the genus Malayemys was distinct enough to warrant elevating it to its own generic-level complex along with the other five complexes (Table 1c). The combined complexes of McDowell (1964) and Bramble (1974) are further supported by chromosomal data (Bickham, 1975), while weak support for Carr and Bickham’s (1986) Malayemys complex comes from allozyme data (Sites et al., 1984).
Hirayama (1984) produced the first fully resolved generic level phylogenetic hypothesis for the Geoemydidae (Fig. 1) based on 82 morphological and four chromosomal characters. Hirayama proposed a novel phylogenetic hypothesis for the group that recognized a basal, sister-group relationship between two previously unrecognized clades. One, equivalent to the Batagur, Hardella, and Orlitia complexes, was highly aquatic, including herbivorous turtles with an extensive secondary palate (his broad-jawed group). The other, equivalent to McDowell’s Geoemyda complex plus the tortoises, were relatively terrestrial turtles with a less extensive secondary palate (the narrow-jawed group) (Hirayama, 1984). Gaffney and Meylan (1988) elevated the Batagurinae and Emydinae to family status (Bataguridae and Emydidae, respectively), and recognized Hirayama’s broad-jawed and narrow-jawed clades at the subfamilial level (Geoemydinae and Batagurinae, respectively).
Recently, both morphological and molecular analyses have been conducted on various subsets of the Geoemydidae. Yasukawa et al. (2001) completed a morphological (35 characters) phylogenetic analysis of 28 species of the subfamily Geoemydinae, and their results were largely in agreement with those of Hirayama (1984). Like Hirayama (1984), Yasukawa et al. (2001) found that Rhinoclemmys was not monophyletic and therefore partitioned it into two genera: Rhinoclemmys, which included R. areolata, R. diademata, R. funerea, R. melanosterna, R. nasuta, R. pulcherrima, and R. punctularia, and Chelopus, which was resurrected for annulata and rubida. Hirayama (1984) and Yasukawa et al. (2001) also recognized the division of Cuora into Cuora (containing C. amboinensis, C. aurocapitata, C. mccordi, C. pani, C. trifasciata, C. yunnanensis, and C. zhoui) and Cistoclemmys (containing flavomarginata and galbinifrons; reviewed in Ernst and Barbour, 1989).
Honda et al. (2002a) analyzed phylogenetic relationships among 17 geoemydine (sensu Hirayama, 1984; including all genera except Melanochelys and Rhinoclemmys) and four batagurine genera (four species) based on 882 base pairs (bp) of combined 12S and 16S ribosomal mitochondrial DNA (mtDNA). The primary goal of their study was to reconstruct phylogenetic relationships within the genus Cuora (the Asian box turtles). Based on their discovery that the monotypic Pyxidea was nested within Cuora, Honda et al. (2002a) recommended synonymizing Cistoclemmys and Pyxidea with Cuora, and this recommendation has been followed by some recent authors (Stuart and Parham, in press). Honda et al., 2002a, Honda et al., 2002b further noted that Mauremys appeared to be paraphyletic with respect to Chinemys and Ocadia, but made no taxonomic recommendations.
Additional intrageneric phylogenetic analyses have been completed for four geoemydid genera. Sites et al. (1981) produced a phylogeny for a subset of Rhinoclemmys (five out of eight species) based on isozyme data. In their results, R. pulcherrima is the sister taxon to the group (R. rubida (R. punctularia (R. funerea, R. areolata))). Iverson et al. (1989) (using morphometric data) and Barth et al. (2003) (using 871 bp of cytochrome b [cytb] mtDNA sequence data), produced phylogenies for the three species of Chinemys (C. megalocephala, C. nigricans [=kwangtungensis], and C. reevesii). Both analyses found C. reevesii paraphyletic with respect to C. megalocephala. Guicking et al. (2002) produced a phylogeny for all five species of Cyclemys based on 982 bp cytb and anonymous nuclear (inter simple sequence repeats [ISSR]) DNA data. They found strong support for the non-monophyly of three species including C. pulchristriata, C. atripons, and C. oldhamii. They also identified two genetically distinct lineages within Cyclemys that may represent undescribed species. Finally, Stuart and Parham (in press) analyzed phylogenetic relationships within Cuora and found support for elevating all three subspecies of C. galbinifrons (C. g. boureti, C. g. galbinifrons, and C. g. picturata) to full species status. They also found paraphyly of Cuora with respect to Pyxidea, and followed Honda et al. (2002a) in subsuming Pyxidea within Cuora. Here, we follow the taxonomic revisions proposed by Honda et al. (2002a) and Stuart and Parham (in press) in considering mouhotii a species of Cuora.
In spite of these analyses, phylogenetic relationships and the taxonomy derived from those relationships within the Geoemydidae remain uncertain. The widespread confusion regarding the phylogenetic content and relationships of the Geoemydidae stems from at least three issues. First, no analyses have included a broad enough sampling of geoemydid turtles and appropriate outgroups to draw firm conclusions on intrafamilial relationships. Second, there is a lack of even the most rudimentary knowledge of the natural history, distribution and ecology of most species in the wild (Ernst and Barbour, 1989; Lau et al., 2000; Lau and Shi, 2000; Thirakhupt and van Dijk, 1994). Third, a number of studies have identified potential widespread hybridization among species and genera, which has greatly confounded recent efforts to clarify species boundaries and taxonomic status of several taxa (Parham et al., 2001; Stuart and Parham, in press; Wink et al., 2001). In part, all of these stem from the same potential cause—many key species of Asian turtles have been commercially over-exploited in the food and medicine trade during the last several decades (Engstrom et al., 2002; Stuart and Parham, in press; van Dijk et al., 2000), forcing systematists to rely on specimens derived solely from market vendors as a source of material. Recent economic change in China has led to a staggering increase in the numbers of turtles imported for food and traditional Chinese medicine (TCM) (Gibbons et al., 2000; IUCN Asian Turtle Workshop, 2001; van Dijk et al., 2000), and wild populations of many geoemydid species have been over-harvested to the point where they are commercially extinct. The extremely high demand and value of turtles and turtle products for food and TCM has led to a large and growing turtle-farming industry in China and southeast Asia. Turtle farmers typically keep turtles of many species in multi-species ponds (Shi and Parham, 2001), and Parham et al. (2001) asserted that these conditions produced hybrids that went to markets and were purchased and described as new species.
To work toward a stronger resolution of the phylogeny of the diverse, poorly known, and frequently endangered geoemydid turtles, we present a comprehensive molecular phylogeny for almost the entire family (and appropriate outgroups) based on cytb and 12S ribosomal mtDNA as well as nuclear DNA sequence data from a novel intron (Fujita et al., in press). Using the resultant phylogenetic trees, we address three key issues for geoemydid turtles. First, we derive a new phylogeny for almost the entire group (59 of 73 species and all 23 genera), and use rigorous statistical tests to compare our tree with those proposed by previous, primarily morphological analyses. Second, we briefly address the origin and validity of several potentially hybrid species. We note that clearly-identified hybrid taxa do provide important insights into the hybridization potential between long-recognized species and genera, but they should not be considered valid species. Finally, we propose several taxonomic revisions within this diverse group of turtles to reflect the emerging consensus on their phylogenetic relationships.
Section snippets
Choice of taxa and genes
Due to the rarity of many geoemydid turtles in the wild, much of the material currently used in phylogenetic studies (including ours) comes from turtles collected from food markets in Asia and from the pet trade (Guicking et al., 2002; Hirayama, 1984; Honda et al., 2002a, Honda et al., 2002b; Parham et al., 2001; Stuart and Parham, in press; Yasukawa et al., 2001, and see below). Our tissue samples were obtained from live animals (66 geoemydids and four tortoises) from the private collection of
80-taxon phylogenetic results
Given our goal of producing a comprehensive tree for all geoemydid taxa, we first asked whether there was any significant conflict within our mtDNA or between our mtDNA and nDNA data partitions. Currently, the ILD test is often used to assess data combinability, although recent work indicates that this test has limited ability to detect incongruence (Darlu and Lecointre, 2002; Dolphin et al., 2000; Dowton and Austin, 2002), and at least some authors (Yoder et al., 2001) go so far as to suggest
Discussion
As in other studies, our cytb sequence data are more variable than the 12S or nuclear intron data (Engstrom et al., unpublished; Giannasi et al., 2001; Palkovacs et al., 2002; Prychitko and Moore, 2000; Shaffer et al., 1997). For the 41 geoemydid taxa with complete cytb and 12S data, cytb has a mean uncorrected pairwise sequence divergence of 13.7% while the corresponding 12S data are 8.3% divergent Background data text, Background data text. For the 24 geoemydid taxa with complete
Conclusions
With the large number of molecular data sets and analyses now available, researchers can tailor genes to particular phylogenetic questions. In our analyses, cytb as well as 12S and R35 sequence data analyzed separately and in combination provided relatively clear resolution for the intrafamilial phylogenetics of the Geoemydidae. Both MP and ML analyses recovered similar topologies with many well-supported clades, although resolution for some of the deeper nodes in our analyses remain elusive or
Acknowledgements
For supplying tissue samples, we thank S. Chancellor (Cuora picturata), I. Das (Indotestudo travancorica), J. Parham (Cuora trifasciata), P. Vander Schouw (Mauremys rivulata), and R. Wicker (Rhinoclemmys rubida). T. Engstrom, T. Near, J. Parham, and D. Starkey provided helpful comments on the manuscript, and the Shaffer lab group provided useful discussion of many of the issues in this paper. This material is based upon work supported by the NSF under grants 9727161/0213153, the UC Davis
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Supplementary data associated with this article can be found, in the online version, at doi: 10.1016/j.ympev.2003.12.015.