Herpetologica,52(1), 1996, 89-97 ? 1996 by The Herpetologists' League, Inc. MOLECULAR SYSTEMATICSOF THE GENUS CLEMMYS AND THE INTERGENERIC RELATIONSHIPSOF EMYDID TURTLES JOHN W. BICKHAM12, TRIP LAMB3,PATRICK MINX4, AND JOHN C. PATTON2 'Department of Wildlife and Fisheries Sciences, Texas A&M University, College Station, TX 77843, USA 2LGLEcological Genetics, Inc., 1410 Cavitt St., Bryan, TX 77801, USA 3Departmentof Biology, East Carolina University, Greenville, NC 27858, USA 4GenomeSequencing Center, Washington University School of Medicine, 4444 Forest Park Avenue, St. Louis, MO 63108, USA ABSTRACT: Nucleotide sequence data from a 556 bp segment of the mitochondrial16S ribosomal RNA gene supportthe partitionof emydid turtlesinto two distinctsubfamilies. Phylogenetic analysis identifies two major clades and corroboratesprevious morphologicalassignments of genera to the subfamilies Deirochelyinae and Emydinae. Within the subfamily Deirochelyinae, Deirochelys ap- pears to be the sister taxon to all other genera in the subfamily, and support is found for a clade that includes Trachemys, Graptemys, and Malaclemys. However, the other generic relationships within this subfamily are not well resolved. Within the Emydinae, the genus Terrapene is mono- phyletic. Conversely,the genus Clemmys is paraphyletic,necessitating a new generic arrangement of the species now considered to be in Clemmys, Emydoidea, and Emys. Key words: Systematics;Ribosomal DNA; Turtles;Emydidae; Nucleotide sequences THE status of the genus Clemmys and ican distribution include Emys, found in its proposed relationships to other genera Europe, North Africa, and parts of the within the family Emydidae have had a Middle East, and Trachemys, the range of varied taxonomic history. Prior to 1964, which encompassesportions of both North Clemmys included four North American and South America (Iverson, 1992). The and four Eurasian species. McDowell family clearly is monophyleticbut has been (1964) recognized the polyphyletic nature divided into two subfamilies by Gaffney of this group and partitioned the genus and Meylan (1988). The Deirochelyinae such that New World forms were retained includes the predominantly aquatic in Clemmys and the Old World forms were Chrysemys, Deirochelys, Graptemys, Ma- relegated to Mauremys and Sacalia. At the laclemys, Pseudemys, and Trachemys, time, emydid turtles were considered a whereas the Emydinae includes both subfamily (Emydinae) of the Testudini- aquatic and terrestrialspecies in the gen- dae. McDowell (1964) subdivided this sub- era Emys, Emydoidea, Clemmys, and family by reassigning the predominantly Terrapene. This arrangementhas received Old World emydine turtles (including support from Seidel and Adkins (1989) Mauremys and Sacalia) to a new subfam- based on isoelectric focusing. ily, the Batagurinae. The three testudinid Relationshipsamong the four genera of subfamilies recognized by McDowell emydine turtles have been the subject of (1964) now represent the three testudinoid recurring debate. Clemmys (McDowell, families Testudinidae, Emydidae, and Ba- 1964), Emys (Milstead, 1969), and Emy- taguridae (Gaffney and Meylan, 1988). doidea (Bramble, 1974; Gaffney and Mey- As currently recognized, the family lan, 1988) all have been hypothesized to Emydidae comprises 10 genera and 37 be the sister taxon to Terrapene. The po- species (Gaffney and Meylan, 1988). It is sition of Emydoidea is particularly con- thought to have originated in North Amer- troversial, as several investigatorshave in- ica where nine of the 10 genera currently ferred a close relationship to Deirochelys occur. Two exceptions to this North Amer- (Loveridge and Williams, 1957; McDow- 89 This content downloaded on Tue, 19 Feb 2013 13:06:40 PM All use subject to JSTOR Terms and Conditions 90 HERPETOLOGICA [Vol. 52, No. 1 ell, 1964; Tinkle, 1962; Zug, 1966) whereas and Emydoidea blandingii. Representa- others have shown a close relationship be- tives of the Deirochelyinae included Dei- tween Emydoidea and the other emydine rochelys reticularia, Trachemys scripta, genera (Bramble, 1974; Frair, 1982; Gaff- Graptemys geographica, Malaclemys ter- ney and Meylan, 1988; Seidel and Adkins, rapin, Pseudemys concinna, and Chrys- 1989). emys picta. Orlitia borneensis and Ma- Within the Emydinae, only Clemmys layemys subtrijuga were chosen as out- and Terrapene contain more than one spe- groups because they (along with Sieben- cies. Milstead (1969) subdivided Terra- rockiella crassicollis) are the only pene into two species groups (i.e., T. car- batagurids to have a karyotype (2N = 50) olina and T. coahuila; T. nelsoni and T. like that of emydids (Bickham, 1975; Bick- ornata), an interpretationthat generally is ham and Baker, 1976; Bickham and Carr, well accepted. The relationshipsof the four 1983; Carr and Bickham, 1981) and be- species of Clemmys are not as well estab- cause the Bataguridae is considered the lished. Some studies have suggested that sister taxon of the Emydidae (Gaffney and C. insculpta is basal (the sister taxon) to Meylan, 1988). the other three species (Merkle, 1975;) and Genomic DNA was extracted from 30- that C. muhlenbergii and C. guttata are 50,ul of blood by incubation in 450 ,1 of closest relatives (Parsons,1962; Zug, 1966). STE buffer (5.0 M NaCl, 2.0 M Tris-HCI, Merkle (1975) hypothesized that C. gut- 0.5 M EDTA, pH 7.5), 25 ,ul of 20% SDS, tata and C. marmorata were the most and 25 Al of proteinase-Kat 10 ,ug/ml for closely related species, with C. muhlen- 1 h. We then added 5M NaCl (150 ,u) and bergii being their sister taxon and C. in- placed the solution on ice for 1 h, centri- sculpta being the most distantly related fuged it at 9000 RPM for 15 min on a taxon. However, Lovich et al. (1991) con- Savant HSC 10K Microcentrifuge, and ex- cluded that C. muhlenbergii shares fewer tracted the supernatant with a phenol- similarities with the other three species. methylene chloride-isoamyl alcohol mix In this study, we examine phylogenetic (25:24:1). DNA was precipitated with an relationships among emydid turtles using equal volume of 100% isopropyl alcohol, nucleotide sequence from the mitochon- centrifuged at 9000 RPM for 1 min, and drial 16S ribosomal gene. We employ a washed in 70% isopropyl alcohol. Precip- phylogenetic analysis to address three ma- itated DNA was resuspended in 200 ,Alof jor issues. (1) We test Gaffney and Mey- TE (1 mM Tris, 100liM EDTA, pH 7.5). lan's (1988) hypothesis regarding the status We amplified a 598 bp portion of the 16S and generic composition of the subfamilies gene of the mitochondrial DNA by PCR Deirochelyinae and Emydinae. (2) We with the primers LGL 381 (5'-ACC CCG concentrate specifically on relationships CCT GTT TAC CAA AAA CAT-3') and within the genus Clemmys, and (3) in so LGL 286 (5'-AGA TAG AAA CCG ACC doing, we examine the phylogenetic status TGG AT-3'). These primers correspond to of Clemmys relative to the other emydine positions 2487-2510 and 3104-3085 of the genera. human mitochondrial genome map, re- spectively. These primers are "universal" and were constructed based on similarities MATERIALS AND METHODS between published mtDNA sequences of We obtained blood samples from a sin- diverse vertebrate taxa. Samples were am- gle individual for each of 15 emydid spe- plified by 50 ,l reactions which consisted cies and two batagurid species. Represen- of the following: 0.1-0.5 ,ug genomic DNA; tatives of the Emydinae included all four 5,Il lOX buffer (0.1 M Tris-HCI pH 8.5, currently recognized species of Clemmys 0.025 M MgCl2, 0.5 M KCI), 5 jil dNTP (C. marmorata, C. muhlenbergii, C. gut- mix (2 mM dATP, dTTP, dCTP, dGTP, tata, and C. insculpta), three (of the four) in 0.1 M Tris-HCl, pH 7.9), 5 jil of a 10 species of Terrapene (T. coahuila, T. or- jiM solution of each primer, 0.025-0.5 jil nata, and T. carolina), Emys orbicularis, Taq DNA polymerase, and brought up to This content downloaded on Tue, 19 Feb 2013 13:06:40 PM All use subject to JSTOR Terms and Conditions March 1996] HERPETOLOGICA 91 volume with deionized water. Amplifica- nucleotides are present (N = 105), the tran- tions were done by 32 cycles of 95 C for sition : transversionratio is 4.25:1. 45 s of denaturing, 50 C for 30 s of an- Phylogenetic analysisof all species using nealing, 70 C for 2.5 min of extension, and a heuristic search (Swofford, 1990) iden- 4 s of auto-extension. The PCR products tified four most-parsimonioustrees of 323 were used to sequence a total of 566 bp steps. In each of these trees, the two sub- using an Applied Biosystems 373A Auto- families of emydid turtles were readily dis- mated DNA sequencer employing dye la- tinguished (Fig. 2). In the bootstrap anal- belled terminators (Ferl et al., 1991). Se- ysis, the emydine clade occurred in 94% quence alignments were made using the of the trees and the deirochelyine clade computer software package SeqEd from occurred in 99%of the trees (Fig. 3). Thus, Applied Biosystems Inc., Foster City, Cal- monophyly for these two taxa, as suggested ifornia. by Gaffney and Meylan (1988), is strongly Phylogenetic relationships were recon- supported in this study and is consistent structed using Paup 3.1 (Swofford, 1990). with another molecular data set obtained Transversionswere weighted 3:1 relative by isoelectric focusing (Seidel and Adkins, to transitions and gaps were treated as 1989). Synapomorphies that define the missing data. A heuristic analysis using 100 Deirochelyinae were observed at positions searches with the random addition of taxa 78, 79, 166, 245, 263, 336, 377, 443, and was used to search for trees of the shortest 500. Synapomorphies that defined the length. We estimated the reliability of the Emydinae were found at positions212,231, branches of the shortest trees by using a 253, 256, 260, 362, 385, and 435. These bootstrapanalysis with 250 replicates. Sub- were all transitions. sequently, an exhaustive search was per- In order to explore more fully the re- formed separatelyon justthe emydine taxa, lationships of the emydine genera, we using Deirochelys as the outgroup, and on reanalyzed the data for the nine emydine justthe deirochelyine taxa, using Clemmys species using only Deirochelys as an out- guttata and Terrapene coahuila as the group.