Stuart, B.L. and Fritz, U. 2008. Historical DNA from Museum Type

Home , Hans Fruhstorfer, Molecular phylogenetics

Biological Journal of the Linnean Society, 2008, 94, 131–141. With 1 ﬁgure

Historical DNA from museum type specimens clariﬁes diversity of Asian leaf turtles (Cyclemys)

BRYAN L. STUART1* and UWE FRITZ2

1The Field Museum, Department of Zoology, Division of Amphibians & Reptiles, 1400 S. Lake Shore Drive, Chicago, IL 60605-2496, USA 2Museum of Zoology, Natural History State Collections Dresden, A. B. Meyer Building, Königsbrücker Landstr. 159, D-01109 Dresden, Germany

Received 22 February 2007; accepted for publication 9 July 2007

Species boundaries in Asian leaf turtles of the genus Cyclemys are difficult to define on the basis of morphology, primarily because many populations exhibit considerable ontogenetic variation in shell and head coloration. Two recent molecular phylogenetic hypotheses of Cyclemys species relationships, based largely on market and pet-trade samples of uncertain provenance, were highly incongruent. We used historical DNA methods to sequence fragments of the mitochondrial cytochrome b gene from eight type specimens of Cyclemys (including one collected by Alfred Russel Wallace), and phylogenetically placed these type sequences into the context of published cytochrome b variation. Our phylogenetic hypothesis supports the recognition of four named species (Cyclemys atripons, Cyclemys dentata, Cyclemys oldhamii, and C. pulchristriata), as well as a fifth species of unknown geographical provenance obtained from the Hong Kong pet trade. The type sequences show that previous molecular phylogenetic studies were hampered by misidentifications, supporting the notion that Cyclemys of unknown provenance are not reliably identified to species solely on the basis of morphology. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 94, 131–141.

ADDITIONAL KEYWORDS: Geoemydidae – historical DNA – museum specimens – species delimitation.

INTRODUCTION into valuable sources of genetic material for systematic studies on rare and extinct populations and species Phylogenetic analyses of DNA sequence data are now (e.g. Roy et al., 1994; Sorenson et al., 1999; Austin & regularly incorporated into taxonomic studies, and are Arnold, 2001; Payne & Sorenson, 2002; Austin, Arnold especially useful in cases when species boundaries and & Jones, 2004; Kearney & Stuart, 2004; Parham et al., evolutionary relationships are obscured by a high 2004; Wisely, Maldonado, & Fleischer, 2004; Fuchs, degree of morphological homoplasy. The most accurate Fjeldså & Pasquet, 2005; Asher & Hofreiter, 2006; means of matching DNA sequences to Linnaean names Fleischer et al., 2006; Kohlsdorf & Wagner, 2006). entail obtaining DNA sequence data from the type Relatively few systematic studies have attempted to specimens on which the original descriptions of species obtain DNA from old type specimens (e.g. Krings et al., were based (Tautz et al., 2003; Austin & Melville, 1997; Amato et al., 1999; Hughey, Silva & Hommer- 2006). However, most existing type specimens were sand, 2001; Austin, Arnold & Bour, 2002; Austin, collected prior to the molecular revolution in system- Arnold & Bour, 2003; Hassanin & Ropiquet, 2004; atic biology, and so lack associated tissue samples that Johnson, Watson & Mindell, 2005; Stuart, Inger & were specially preserved for genetic analysis. Methods Voris, 2006a). to retrieve DNA from degraded samples remain chal- Asian leaf turtles of the genus Cyclemys provide an lenging (Handt et al., 1994; Wayne, Leonard & Cooper, excellent example of a taxonomic group in which 1999; Cooper & Poinar, 2000; Hofreiter et al., 2001), species boundaries and their evolutionary relation- but have transformed traditional museum specimens ships are obscured by a high degree of morphological homoplasy. Species of Cyclemys are primarily diag- *Corresponding author. E-mail: [email protected] nosed by shell and head coloration, but many

© 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 94, 131–141 131 132 B. L. STUART and U. FRITZ populations exhibit considerable ontogenetic varia- et al., 2002). Within the dark-plastron group, only tion in these characters (Fritz, Gaulke & Lehr, 1997; adults of C. oldhamii and C. s. shanensis are reliably Fritz & Ziegler, 1999). Furthermore, some species distinguished from C. s. tcheponensis,asC. oldhamii of Cyclemys were originally described from juvenile and C. s. shanensis lose head and neck stripes during specimens, and others were described from adult ontogeny, but C. s. tcheponensis does not (Fritz et al., specimens (Fritz et al., 1997; Iverson & McCord, 1997; Guicking et al., 2002). No morphological char- 1997), making comparisons difficult. As a result, acters have been reported that can reliably distin- species boundaries in Cyclemys are contentious and guish C. oldhamii from C. s. shanensis. Within the notoriously difficult to define, and the genus has a yellow plastron group, C. atripons and C. pulchris- complicated taxonomic history. triata have immaculate throats, but C. dentata has a The genus Cyclemys is widely distributed from striped throat (Fritz et al., 1997; Iverson & McCord, north-eastern India to the Philippines (Fritz et al., 1997). No morphological characters have been 1997; Iverson & McCord, 1997; Fritz & Ziegler, 1999). reported that can reliably distinguish C. atripons Historically, only one species, Cyclemys dentata from C. pulchristriata. Fritz et al. (2001) proposed (Gray, 1831), or sometimes two species, C. dentata that the number of ventral neck stripes differed and Cyclemys tcheponensis (Bourret, 1939) were between C. atripons and C. pulchristriata, but the thought to encompass this vast range. In 1997, a range of neck stripes reported by Stuart & Platt taxonomic revision (Fritz et al., 1997) and a new (2004) in just two turtles collected together in south- species description (Iverson & McCord, 1997) brought western Cambodia (near to the type locality of C. atri- the number of recognized species to five (Artner, 1998; pons) exceeded the range reported for both species by Fritz & Ziegler, 1999): Cyclemys atripons Iverson & Fritz et al. (2001). Because of their morphological McCord, 1997, with a type locality of Krat, south- similarity, some authors have suggested that C. atri- eastern Thailand; Cyclemys dentata (Gray, 1831), pons and C. pulchristriata may be conspecific (Fritz & with a type locality of Java, Indonesia; Cyclemys Obst, 1999; Iverson in Guicking et al., 2002). oldhamii Gray, 1863, with a type locality of Mergui, Defining species boundaries in Cyclemys is more Myanmar; Cyclemys pulchristriata Fritz, Gaulke & than academic. Asian turtles are overexploited for Lehr, 1997, with a type locality of Phuc-Son in central food, for traditional medicine, and for pets (van Dijk, Vietnam; and Cyclemys tcheponensis (Bourret, 1939), Stuart & Rhodin, 2000), and now more than half of with a type locality of Haute Sé-Bang-Hien on the the Asian species are listed as endangered or criti- border of southern Laos and central Vietnam. In the cally endangered (IUCN, 2004). The conservation most recent taxonomic treatment, Guicking et al. status of Cyclemys has been evaluated as a single, (2002) resurrected Cyclemys dhor shanensis Annan- widespread taxon, C. dentata, owing to the uncer- dale, 1918 out of synonymy with C. oldhamii, tainty in the limits and geographical distributions of elevated shanensis to full species status, referred the other named species (IUCN/SSC Tortoise and populations from Myanmar and Thailand to the sub- Freshwater Turtle Specialist Group and Asian Turtle species Cyclemys shanensis shanensis Annandale, Trade Working Group, 2000). However, the genus 1918, and referred C. tcheponensis to the subspecies Cyclemys contains multiple, highly divergent mito- Cyclemys shanensis tcheponensis (Bourret, 1939). chondrial DNA lineages (Guicking et al., 2002; Four additional described species of Cyclemys are Spinks et al., 2004), each of which implicitly have currently considered to be junior synonyms of C. den- smaller geographical ranges that are more vulnerable tata or C. s. tcheponensis (reviewed in Fritz et al., to extinction than a single evolutionary lineage of 1997). Specifically, Cyclemys orbiculata Bell, 1834 and Cyclemys. A more realistic assessment of species Cyclemys bellii Gray, 1863, with uncertain type locali- boundaries in Cyclemys will allow for a more accu- ties, and Cyclemys ovata Gray, 1863, with a type rate, and probably direr, assessment of the conserva- locality of Sarawak are treated as junior synonyms of tion status of these turtles. C. dentata,andCyclemys tiannanensis Kou, 1989, An independent molecular dataset is highly desir- with a type locality of Yunnan, China, is treated as a able given the uncertainties in defining species of junior synonym of C. s. tcheponensis. Cyclemys solely on the basis of morphology. Two Despite the uncertainties in the number of taxa previous hypotheses of phylogenetic relationships of (species and subspecies), Cyclemys are readily classi- Cyclemys species have been proposed, both based on fied into two distinct morphological groups on the the mitochondrial cytochrome b (cyt b) gene. Guicking basis of plastron (ventral part of the shell) coloration: et al. (2002) found samples of C. oldhamii, C. atri- C. oldhamii, C. s. shanensis, and C. s. tcheponensis pons, and C. pulchristriata to be paraphyletic, and have mostly dark brown or black plastra, whereas interpreted a lineage morphologically resembling C. atripons, C. dentata, and C. pulchristriata have C. oldhamii, and a lineage morphologically resem- mostly yellow plastra (Fritz et al., 1997; Guicking bling C. atripons and C. pulchristriata, to be cryptic,

© 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 94, 131–141 TYPE SPECIMEN DNA OF SOUTH-EAST ASIAN TURTLES 133 undescribed species. Spinks et al. (2004) included one a small piece of skin, muscle, or femur bone was sample of each of three species, and found C. atripons removed from the leg of seven alcoholic type speci- to be the sister taxon to a clade containing C. dentata mens of C. dentata and C. pulchristriata (Table 1). and C. tcheponensis, with C. dentata and C. tchepon- DNA was extracted following the method described by ensis separated by a very short branch. This hypoth- Kearney & Stuart (2004). Three fragments of 144, esis implies that species with yellow plastra do not 179, and 168 nucleotide basepairs (bp; after primer represent a monophyletic group. However, these sequences were trimmed) of mitochondrial DNA studies relied entirely (Spinks et al., 2004) or mostly encoding part of the cyt b gene were amplified by (Guicking et al., 2002) on pet-trade and market polymerase chain reaction (PCR; 94 °C 45 s, 48 °C samples of uncertain provenance. For example, 30 s, 72 °C 1 min) for 40 cycles using the primer Guicking et al. (2002) assigned the names C. atripons pairs L-350cytb/H-500cytb, L-430cytb/H-615cytb, and and C. pulchristriata to samples depending on L-560cytb/H-730cytb, respectively (Table 2). Primers whether they were obtained from Cambodian markets were designed so that the three fragments overlapped (C. atripons) or Vietnamese markets (C. pulchris- by 64 and 51 bp after primer sequences were triata), despite the fact that turtles collected in Cam- trimmed. This measure ensured that fragments of bodia are mass exported to Vietnam (Stuart et al., contaminant DNA were not concatenated into chi- 2000). Pet-trade and market samples do have utility meric sequences that might be erroneously judged to in phylogenetic studies, but with serious limitations be authentic (Olson & Hassanin, 2003). The 25-mL (Parham et al., 2001, 2004; Stuart & Parham, 2004). PCR reactions utilized 4 mL of bovine serum albumin The uncertainties of identifying Cyclemys species (BSA; New England BioLabs), to prevent PCR based on morphology makes the limitations of using inhibitors, a relatively large volume (4 mL) of DNA market and pet-trade samples even more severe, and template to overcome low extraction yield, the high- the hypotheses of species boundaries and relation- quality Taq polymerase AmpliTaq Gold (Roche), and ships of Cyclemys proposed in both studies need to be extra cycles (40 total) of the PCR reaction. All PCR interpreted with caution. Clearly, DNA sequences reactions contained a positive control (described from type specimens of Cyclemys would help resolve below) and a negative control (all reagents except the taxonomic uncertainty. DNA template). PCR products were electrophoresed In an effort to elucidate species boundaries in in a 1% low-melt agarose TALE gel stained with Cyclemys, we used historical (or ‘ancient’) DNA ethidium bromide and visualized under ultraviolet methods to sequence fragments of the mitochondrial light. The bands containing DNA were excised, and cyt b gene from museum type specimens, and phylo- agarose was digested from the bands using GELase genetically placed these type sequences into the (Epicentre Technologies). PCR products were context of published cyt b variation. Although mito- sequenced in both directions by direct double-strand chondrial gene trees may not always represent cycle sequencing using Big Dye version 3 (Perkin species trees owing to introgression or lineage sorting Elmer). The amplifying primers were used in the of ancestral polymorphism (Moore, 1995; Maddison, sequencing reactions. Cycle-sequencing products 1997), an accurate matching of Linnaean names to were precipitated with ethanol, 3 M sodium acetate, major mitochondrial lineages provides an evidence- and 125 mM EDTA, and were sequenced with a based hypothesis of species boundaries that is supe- Prism 3100 Genetic Analyser (ABI). Sequences were rior to the current state of taxonomic upheaval in edited using SEQUENCHER v.4.1 (Genecodes). The Cyclemys. Furthermore, the hypothesis that major type sequences were deposited in GenBank under mitochondrial DNA lineages of Cyclemys represent accession numbers DQ444270–DQ444275 and species is more likely to overestimate diversity than EF183501–EF183502. to underestimate diversity, a source of error that we are willing to assume in this group of threatened turtles. EXTRACTION OF DNA FROM FRESH TISSUE Total genomic DNA was extracted from fresh tissue of a C. pulchristriata that was field-collected in eastern MATERIAL AND METHODS Cambodia by B. L. Stuart (Table 1) using the Pure- Gene Animal Tissue DNA Isolation Protocol (Gentra EXTRACTION, AMPLIFICATION, AND SEQUENCING OF Systems, Inc.). This sample represented the only HISTORICAL DNA FROM MUSEUM SPECIMENS fresh tissue of Cyclemys that had previously been Small pieces of bone, dried muscle, or connective extracted in the molecular laboratory where the his- tissue were removed from the inside of seven dried torical DNA extractions and amplifications were per- (stuffed or shell) type specimens of C. atripons, formed. The extraction was amplified as a positive C. bellii, C. oldhamii, C. orbiculata, and C. ovata, and control in PCR reactions of historical DNA, and was

Table 1. Sources of mitochondrial (mt) DNA of Cyclemys turtles used in this study

Species Museum voucher Type status Year collected Condition Locality mtDNA sequence 08TeLnenSceyo London, of Society Linnean The 2008 ©

atripons USNM 81865 Holotype 1929 Dried stuffed specimen Kao Kuap, Trat, Thailand GenBank DQ444271 atripons USNM 53423 Paratype 1914 Dried shell Koh Chang, Trat, Thailand GenBank DQ444270 bellii OUMNH 8513 Holotype* By 1834† Dried shell Unknown GenBank EF183501 dentata BMNH 1946.1.22.62 Lectotype By 1828‡ Alcoholic specimen Java, Indonesia GenBank DQ444272 oldhamii BMNH 1947.3.5.63 Lectotype By 1856§ Dried shell Mergui, Myanmar GenBank DQ444274 oldhamii BMNH 1947.3.4.26 Paralectotype By 1862¶ Dried stuffed specimen Lao Mountains, Thailand GenBank DQ444273 orbiculata OUMNH 8512 Syntype By 1834** Dried shell Unknown GenBank EF183502 ovata BMNH 1863.6.21.1 Holotype By 1863** Dried shell Sarawak, Malaysia†† GenBank DQ444275 pulchristriata NMW 29525:4 Holotype By 1901‡‡ Alcoholic specimen Phuc-Son, Annam, Vietnam Failed to amplify pulchristriata NMW 29525:2 Paratype By 1901‡‡ Alcoholic specimen Phuc-Son, Annam, Vietnam Failed to amplify pulchristriata NMW 29525:3 Paratype By 1901‡‡ Alcoholic specimen Phuc-Son, Annam, Vietnam Failed to amplify pulchristriata NMW 29525:5 Paratype By 1901‡‡ Alcoholic specimen Phuc-Son, Annam, Vietnam Failed to amplify pulchristriata SMF 7667 Paratype By 1901‡‡ Alcoholic specimen Annam, Vietnam Failed to amplify ilgclJunlo h ina Society Linnean the of Journal Biological pulchristriata ZMH R00292 Paratype By 1901‡‡ Alcoholic specimen Annam, Vietnam Failed to amplify pulchristriata FMNH 259050 None 2000 Fresh tissue Mondolkiri Prov., Cambodia GenBank DQ444276

Museum abbreviations: BMNH, The Natural History Museum, London; FMNH, The Field Museum; NMW, Naturhistorisches Museum Wien; OUMNH, Oxford University Museum of Natural History; SMF, Forschungsinstitut und Naturmuseum Senckenberg, Frankfurt; USNM, National Museum of Natural History, Smithsonian Institution; ZMH, Zoologisches Museum Hamburg. *Das (2005) erroneously reported that OUMNH 8867 is the holotype of C. bellii. The second author examined OUMNH 8513 and it bears an old label stating that it is the holotype of C. bellii. OUMNH 8513 also matches the upper ﬁgure in plate 3, part 8 of Bell (1832–1836), on which Gray (1863) based his original description of C. bellii. †This specimen is simultaneously a syntype of C. orbiculata Bell, 1834 and so must have been collected by that year. ‡The original BMNH catalogue number 1828.5.12.1 implies that this specimen has been in the museum since at least 1828. §The original BMNH catalogue number 1856.5.6.1 implies that this specimen has been in the museum since at least 1856. ¶The original BMNH catalogue number 1862.8.18.20 implies that this specimen has been in the museum since at least 1862. **Based on the year of the original species description.

2008, , ††Collected by Alfred Russel Wallace. ‡‡Collected by Hans Fruhstorfer, a German-Swiss naturalist who traveled to Annam during a south-east Asian expedition from 1899 to 1901 (Lamas, 2005). 94 131–141 , TYPE SPECIMEN DNA OF SOUTH-EAST ASIAN TURTLES 135

Table 2. Oligonucleotide primers used to amplify and rate matrix A–C = 1.04241, A–G = 8.18342, sequence part of the mitochondrial cytochrome b gene A–T = 0.4883, C–G = 0.00549, C–T = 12.0303, propor- from Cyclemys turtles tion of invariable sites = 0.0458648, and gamma- distribution shape parameter = 0.283551. Nodal Primer Sequence support was evaluated with 500 nonparametric bootstrap pseudoreplications. ′ ′ L-350cytb 5 -TTTGAGGTGCCACCGTCATC-3 Mixed-model Bayesian analysis was performed ′ ′ H-500cytb 5 -TCGTGGAGGAAGATTAAGTGTAC-3 using MrBayes 3.1 (Ronquist & Huelsenbeck, 2003). ′ ′ L-430cytb 5 -GGGATTCTCAGTAGACAATGC-3 The data were partitioned into first-codon, second- H-615cytb 5′-TTAGTAGGAGGGTGAGTATTAGGA-3′ codon, and third-codon positions. The model of L-560cytb 5′-CAGGATTAAACTCAAACGCTG-3′ sequence evolution that best described the three data H-730cytb 5′-GCATAGGCGAATAGGAAGTATC-3′ partitions was inferred using the Akaike Information Criterion, as implemented in MODELTEST 3.7 ‘L’ and ‘H’ refer to light and heavy strands, respectively. (Posada & Crandall, 1998). The selected models were K81uf + G for the first-codon position partition, sequenced as described above. This sequence was TrN + I for the second-codon position partition, and deposited in GenBank under accession number GTR + G for the third-codon position partition. The DQ444276. K81uf and TrN models are not implemented in MrBayes 3.1, and so the next more complex model available in the program (GTR) was used for those ALIGNMENT partitions. Four independent Bayesian analyses were In addition to the eight type specimens and one performed. In each analysis, four chains were run for positive control tissue sequenced in this study, 10 000 000 generations using the default priors, trees Cyclemys cyt b sequences were also obtained from were sampled every 2000 generations, and the first three published sources. Thirty-nine sequences with a 25% of trees were discarded as ‘burn-in’. A 50% major- maximum length of 984 bp were obtained from Guick- ity rule consensus of the sampled trees was con- ing et al. (2002), three sequences of 1140 bp (GenBank structed to calculate the posterior probabilities of the AY434577, AY434579, and AY434617) were obtained tree nodes. Trace plots of clade probabilities were from Spinks et al. (2004), and one sequence of 1036 bp viewed using AWTY (Wilgenbusch, Warren & Swof- (GenBank AJ604513) was obtained from Schilde, ford, 2004). Barth & Fritz (2004). Sequences from Leucocephalon yuwonoi (GenBank AY434608) and Heosemys spinosa RESULTS (GenBank AY434578) were used as outgroups, based HISTORICAL DNA SEQUENCES on the findings of Spinks et al. (2004). Sequences were aligned using SEQUENCHER v.4.1 (Genecodes). All three overlapping cyt b fragments, consisting of a total of 376 bp, were obtained from the seven dried type specimens. Two overlapping cyt b fragments PHYLOGENETIC ANALYSES (from the primer pairs L-430cytb/H-615cytb and Phylogenies were reconstructed using maximum- L-560cytb/H-730cytb), consisting of a total of 296 bp, parsimony and maximum-likelihood optimality crite- were obtained from the alcoholic lectotype of C. ria, and mixed-model Bayesian inference. dentata. None of the fragments were successfully Maximum-parsimony analysis was performed using amplified from the alcoholic type specimens of C. pul- PAUP* 4.0b10 (Swofford, 2002). A heuristic search christriata (Table 1). was performed with equal weighting of nucleotide The electropherograms of the obtained sequences substitutions, stepwise addition with 100 random- had strong signals with very little or no background addition replicates, and TBR branch swapping was noise. Identical sequences were obtained in all over- limited to 1000 trees per replicate. Nodal support was lapping fragments. The eight type sequences were evaluated with 500 nonparametric bootstrap pseu- genetically distinct from the positive control that rep- doreplications (Felsenstein, 1985) using the heuristic resented the only fresh tissue of Cyclemys previously search option, with TBR branch swapping limited to extracted in the same laboratory. Consequently, the 10 000 000 rearrangements per replicate. sequences of the eight type specimens used in the Maximum-likelihood analysis was performed using analyses here appear to be authentic. GARLI 0.95 (Zwickl, 2006). Five independent analyses were performed using the default settings, the PHYLOGENETIC RELATIONSHIPS GTR + I + G model, and estimated base frequencies The alignment contained no insertion-deletions and A = 0.31052, C = 0.30726, G = 0.11636, T = 0.26586, was unambiguous. Of the 1140 aligned characters,

297 were variable and 187 were parsimony informa- hypothesis supports the recognition of four named tive. Strict consensus of the saved 100 000 equally species (C. atripons, C. dentata, C. oldhamii, and most parsimonious trees (tree length, L = 435; consis- C. pulchristriata), and a fifth species of unknown geo- tency index, CI = 0.766; retention index, RI = 0.944) graphical origin. The substantial differences in iden- showed that these trees differed only by the arrange- tifications among Guicking et al. (2002), Spinks et al. ment of individuals on short-terminal branches. The (2004), and this study incorporating type sequences, score of the best-likelihood tree (–ln L 3565.994676) support the notion that Cyclemys of unknown prov- was within 0.043067 likelihood units of the best tree enance are not reliably identified to species solely on recovered in each of the other four runs, suggesting the basis of morphology. The corrected taxonomy that the five runs had converged. The standard devia- based on the type sequences (Fig. 1) shows that many tion of split frequencies among the four Bayesian runs samples were misidentified in previous studies was 0.004991, and trace plots of clade probabilities (Guicking et al., 2002; Spinks et al., 2004). Unfortu- viewed using AWTY were relatively stationary. These nately, the lack of voucher specimens associated with two measures suggest that the four runs had con- some (Guicking et al., 2002) or all (Spinks et al., 2004) verged, and that topologies were sampled in propor- of the genetic material analysed in those studies tion to their true posterior probability distribution. prevents re-evaluating identifications in the light of The maximum-parsimony trees, maximum-likelihood our new findings with type sequences. tree, and Bayesian-consensus tree (Fig. 1) were The absence of substantial genetic variation among highly consistent, and differed only by the arrange- the types of C. bellii, C. dentata, C. orbiculata, and ment of individuals on short-terminal branches. C. ovata corroborates the hypothesis based on mor- Five major, well-supported clades were recovered phology (Fritz et al., 1997) that C. bellii, C. orbicu- (Clades A–E, Fig. 1). Clade A (Fig. 1) was sister to all lata, and C. ovata are junior synonyms of C. dentata, other Cyclemys, and contained samples obtained from the oldest available name. Likewise, the absence the Hong Kong pet trade (Guicking et al., 2002). of substantial genetic variation among the types These were reported to have originated from Kachin, of C. oldhamii and samples identified as C. shanensis Myanmar, and were identified as an undescribed (including topotypes of C. s. shanensis from Lake Inlé, species morphologically resembling C. oldhamii Myanmar) and C. tcheponensis suggests that C. shan- (Guicking et al., 2002). None of the type sequences ensis and C. tcheponensis are junior synonyms of that we obtained belong to this clade. Clade B (Fig. 1) C. oldhamii, the oldest available name. Cyclemys old- contained samples identified as two subspecies of hamii, C. shanensis, and C. tcheponensis differ mor- C. shanensis by Guicking et al. (2002), as C. shanensis phologically only by the retention or loss of head by Schilde et al. (2004), as C. dentata and C. tchepon- stripes with maturity (Fritz et al., 1997; Guicking ensis by Spinks et al. (2004), and the lectotype and et al., 2002), and adopting this taxonomy implies that paralectotype of C. oldhamii. Clade C (Fig. 1) con- the retention or loss of head stripes with maturity tained samples identified as C. atripons and C. pul- represents only geographical variation within a christriata by Guicking et al. (2002), and the positive species. control sample of C. pulchristriata from eastern Cam- Cyclemys atripons and C. pulchristriata are mor- bodia. Clade D (Fig. 1) contained samples identified phologically indistinguishable, but are paraphyletic as C. dentata and C. oldhamii by Guicking et al. with respect to C. dentata. Because we were unsuc- (2002), the lectotype of C. dentata, the syntype of cessful in obtaining DNA from the type series of C. orbiculata, the holotype of C. bellii, and the holo- C. pulchristriata (Table 1), our identification of the type of C. ovata. Clade E (Fig. 1) contained samples C. pulchristriata clade is tentatively based on the identified by Guicking et al. (2002) as an undescribed assumption that the positive control sample belongs species morphologically resembling C. atripons and to this species. This sample was field-collected in hilly C. pulchristriata, and the holotype and paratype of eastern Cambodia by B. L. Stuart, in the same Anna- C. atripons. Species with yellow plastra (Clades C–E, mite (Truong Son) Mountain range that contains the Fig. 1) form a well-supported monophyletic group, but type locality of C. pulchristriata in central Vietnam. species with dark plastra (Clades A–B, Fig. 1) are Stuart & Platt (2004) tentatively referred this speci- paraphyletic (Fig. 1). men to C. atripons, on the basis that most of the Cambodian Cyclemys specimens with yellow plastra they examined originated from the mountains of DISCUSSION south-western Cambodia, close to the type locality of Our phylogenetic analysis of mitochondrial DNA C. atripons, and that no morphological differences sequences obtained from museum type specimens of among the specimens were apparent. Stuart, Sok & Cyclemys greatly aids the resolution of species bound- Neang (2006b) identified additional specimens from aries in the genus. Specifically, our phylogenetic eastern Cambodia as C. atripons to maintain consis-

atripons paratype 1.00/100/99 atripons holotype atripons Cyclemys .63/-/58 ' n. sp. 2 Cambodia' E .70/97/91 'Cyclemys n. sp. 2 Vietnam, Saigon' 1.00/89/81 ovata holotype 1.00/ bellii holotype 94/94 oldhamii .94/ ' Malaysia, Penang 1' 96/94 'oldhamii Malaysia, Penang 2' 1.00/ 'dentata Indonesia 3' dentata .97/79/60 90/99 'dentata Indonesia 1' .96/ D 84/64 'dentata Indonesia 2' 'dentata' 1.00/96/99 dentata lectotype .99/93/67 orbiculata syntype pulchristriata Mondolkiri, Cambodia 'atripons Cambodia 2' 'atripons Cambodia 3' 1.00/99/97 'atripons Cambodia 4' 'atripons Cambodia 5' 1.00/100/100 'atripons Cambodia 6' pulchristriata 'atripons Cambodia 7' 'atripons Cambodia 1' C 'atripons'* 1.00/95/98 1.00/97/99 'pulchristriata Vietnam, Saigon' .95/73/71 'pulchristriata Vietnam, Saigon' .99/67/61 'pulchristriata Vietnam, Saigon' 'dentata'* oldhamii paralectotype oldhamii lectotype 'shanensis'** 'shanensis shanensis Myanmar, Lake Inlé 7' 'shanensis shanensis Cambodia, Ratanakiri' 'shanensis shanensis Thailand 1' 'shanensis shanensis Thailand 2' 'shanensis shanensis Thailand 3' 1.00/100/100 'shanensis shanensis Thailand 4' oldhamii 1.00/93/97 'shanensis shanensis Myanmar, Lake Inlé 1' .64/56/58 'shanensis shanensis Myanmar, Lake Inlé 2' B 'shanensis shanensis Myanmar, Lake Inlé 3' 'shanensis shanensis Myanmar, Lake Inlé 4' .76/66/- 'shanensis shanensis Myanmar, Lake Inlé 5' 'shanensis shanensis Myanmar, Lake Inlé 6' 'tcheponensis'* 'shanensis tcheponensis Vietnam, Lao Bao 1' 'shanensis tcheponensis Vietnam, Lao Bao 2' 1.00/93/93 'shanensis tcheponensis Vietnam, Lao Bao 3' 'shanensis tcheponensis Vietnam, Lao Bao 4' 'Cyclemys n. sp. 1 Myanmar, Kachin (?) 1' 'Cyclemys n. sp. 1 Myanmar, Kachin (?) 2' 1.00/100/100 'Cyclemys n. sp. 1 Myanmar, Kachin (?) 3' unknown species 'Cyclemys n. sp. 1 Myanmar, Kachin (?) 4' A 'Cyclemys n. sp. 1 Myanmar, Kachin (?) 5' 0.1 substitutions/site

Figure 1. Fifty-percent majority-rule consensus phylogram resulting from mixed-model Bayesian analysis of the mitochondrial cytochrome b gene from Asian leaf turtles (Cyclemys). Maximum-likelihood and maximum-parsimony analysis recovered the same topology, and differed only by the arrangement of individuals on short-terminal branches. Numbers next to nodes are Bayesian posterior probabilities/maximum-likelihood bootstrap values/parsimony bootstrap values. Haplotypes in boldface were sequenced in this study, haplotypes marked with an asterisk (*) were obtained from Spinks et al. (2004), the haplotype marked with two asterisks (**) was obtained from Schilde et al. (2004), and the remaining haplotypes were obtained from Guicking et al. (2002). Species identiﬁcations of haplotypes obtained from Guicking et al. (2002), Schilde et al. (2004), and Spinks et al. (2004) are enclosed in single quotations. Our recommended taxonomy is on the right. tency with Stuart & Platt (2004). Sequences from et al. (2002) to be a cryptic, undescribed species mor- type material of C. pulchristriata or from fresh topo- phologically resembling C. atripons and C. pulchris- typic samples are needed to identify the C. pulchristriata (‘Cyclemys n. sp. 2’) is C. atripons. triata lineage with certainty. It is clear, however, from The lineage interpreted by Guicking et al. (2002) to our results that the lineage interpreted by Guicking be a cryptic, undescribed species morphologically

© 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 94, 131–141 138 B. L. STUART and U. FRITZ resembling C. oldhamii (‘Cyclemys n. sp. 1’) does not dentata, supporting the hypothesis that the Penang match any of the type sequences obtained in this samples are an example of mitochondrial DNA intro- study, and may indeed represent an undescribed gression. Cyclemys oldhamii and C. dentata occur in species. We did not attempt to sequence the type sympatry in Peninsular Malaysia (Fritz et al., 1997), material of three described taxa of Cyclemys that are but owing to the trade origin of the Penang samples, currently considered to be junior synonyms of other the introgression may alternatively have occurred in species, and which exhibit a dark plastron like that of captivity. Field sampling in Peninsular Malaysia ‘Cyclemys n. sp. 1’: C. tcheponensis, C. dhor shanensis, should offer insight into this topic. A nuclear DNA and C. tiannanensis. The sequences obtained from dataset of the type sequences would also provide an Guicking et al. (2002) include samples from Lake Inlé, independent estimate of relationships and species Myanmar, the type locality of C. dhor shanensis boundaries in Cyclemys. However, obtaining nuclear (referred to as C. s. shanensis in Guicking et al., 2002) DNA sequences from degraded samples is more chal- and from Lao Bao, Vietnam, near to the type locality lenging than obtaining mitochondrial DNA sequences, of C. tcheponensis (referred to as C. s. tcheponensis in probably because mitochondrial DNA occurs in higher Guicking et al., 2002), and so it is unlikely that copy number in the cell, and is therefore more likely ‘Cyclemys n. sp. 1’ represents either of these two taxa. to be retrieved (Hofreiter et al., 2001; Huynen et al., Cyclemys tiannanensis has not yet been characterized 2003; Isenberg, 2005). molecularly, and its relationship to ‘Cyclemys n. sp. 1’ Sampling genetic material from a museum speci- remains unevaluated. Unfortunately, the provenance men necessarily requires removing part of the speci- of ‘Cyclemys n. sp. 1’ is unknown. These samples were men, and there is justifiably great concern that obtained from a Hong Kong pet dealer who reported damage to the specimen be kept to a mimimum, that they originated from Kachin Province, Myanmar particularly with those specimens that are as irre- (Guicking et al., 2002). During the past two decades, placeable as types (Graves & Braun, 1992; Mundy, many newly described turtle species were based on Unitt & Woodruff, 1997; Rohland, Siedel & Hofreiter, specimens purchased from a Hong Kong pet dealer 2004; Wisely et al., 2004; Asher & Hofreiter, 2006). (Parham et al., 2001; Dalton, 2003). Some of these New protocols for obtaining DNA from museum speci- newly described species have been shown to be mens are commendably seeking to minimize such hybrids of other, better known species (Parham et al., damage (Mundy et al., 1997; Rohland et al., 2004; 2001; Wink, Guicking & Fritz, 2001; Spinks et al., Wisely et al., 2004; Asher & Hofreiter, 2006), but still 2004; Stuart & Parham, 2007), whereas others have modify specimens in some manner. We submit that been shown to represent distinct, evolutionary lin- sampling genetic material from freshly collected eages that remain unknown in the wild (Parham specimens of turtles also requires destructive sam- et al., 2004). Because the mitochondrial DNA of pling by removing tissue (typically muscle or liver) ‘Cyclemys n. sp. 1’ does not match any other known from the carcass. As with freshly collected specimens, taxon (except potentially C. tiannanensis), this dis- in this study we aimed to sample tissues that were tinct lineage is unlikely to be another example of a subjectively deemed to have limited phylogenetic pet-trade species with a recent hybrid origin. In many utility. These included small pieces of skin, muscle, cases, the provenance supplied by the Hong Kong pet bone, or connective tissue, usually in the form of dealer for specimens have been questioned (de Bruin unrecognizable organic material adhering to the & Artner, 1999; Parham & Li, 1999; Dalton, 2003; Shi inside of dried specimens. The most drastic sampling et al., 2005), underscoring the urgent need for scien- (and ironically the least successful) entailed removing tific fieldwork. a femur bone from a leg that was retracted within the Although we hypothesize that the major mitochon- shell of some alcoholic specimens. Efforts should con- drial DNA lineages represent species of Cyclemys, one tinue to find new means to extract DNA with minimal apparent example in our dataset of introgression damage to specimens, and curators should continue between mitochondrial DNA lineages suggests that to critically review requests to destructively sample these lineages may be imperfect proxies for species. specimens in their care (Graves & Braun, 1992). Mitochondrial DNA sequences obtained by Guicking However, our ability to sample type specimens of et al. (2002) from two turtles with black plastra pur- Cyclemys in this manner enabled us to reveal new chased in local trade at Penang, Peninsular Malaysia, phylogenetic characters from critically important and identified by those authors based on morphology specimens, including one notably collected by Alfred as C. oldhamii, are deeply nested within a clade of Russel Wallace. turtles with yellow plastra referred here to C. dentata The type sequences generated in this study provide (Fig. 1). A nuclear DNA dataset (ISSR-PCR) pre- useful reference points for future phylogenetic studies sented by Guicking et al. (2002) on the same samples of Cyclemys because their species identifications are readily distinguished the Penang turtles from C. unambiguous. New efforts to resolve Cyclemys species

© 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 94, 131–141 TYPE SPECIMEN DNA OF SOUTH-EAST ASIAN TURTLES 139 boundaries and relationships should be directed Asher RJ, Hofreiter M. 2006. Tenrec phylogeny and the towards obtaining mitochondrial DNA sequences from noninvasive extraction of nuclear DNA. Systematic Biology C. pulchristriata and C. tiannanensis type material, 55: 181–194. obtaining nuclear DNA sequences from all type Austin JJ, Arnold EN. 2001. Ancient mitochondrial DNA material, and especially, obtaining field-collected, and morphology elucidate an extinct island radiation of vouchered, genetic material of known provenance. Indian Ocean giant tortoises (Cylindraspis). Proceedings of The genus Cyclemys contains multiple, distinct evo- the Royal Society B 268: 2515–2523. lutionary lineages that each warrant recognition and Austin JJ, Arnold EN, Bour R. 2002. The provenance of type specimens of extinct Mascarene Island giant tortoises protection, but an effective conservation strategy (Cylindraspis) revealed by ancient mitochondrial DNA requires knowing where these lineages exist in sequences. Journal of Herpetology 36: 280–285. nature. Austin JJ, Arnold EN, Bour R. 2003. Was there a second adaptive radiation of giant tortoises in the Indian Ocean? Using mitochondrial DNA to investigate speciation and ACKNOWLEDGEMENTS biogeography of Aldabrachelys (Reptilia, Testudinidae). The following people (and institutions) made this Molecular Ecology 12: 1415–1424. study possible by sampling and loaning tissue from Austin JJ, Arnold EN, Jones CG. 2004. Reconstructing an type specimens in their care: George Zug (National island radiation using ancient and recent DNA: the extinct Museum of Natural History, Smithsonian Institu- and living day geckos (Phelsuma) of the Mascarene islands. tion), Colin McCarthy (The Natural History Museum, Molecular Phylogenetics and Evolution 31: 109–122. London), Richard Gemel and Franz Tiedemann Austin JJ, Melville J. 2006. Incorporating historical (Naturhistorisches Museum Wien), Gunther Köhler museum specimens into molecular systematic and conservation genetics research. Molecular Ecology Notes 6: 1089– (Forschungsinstitut und Naturmuseum Senckenberg, 1092. Frankfurt), Tom Kemp and Malgosia Nowak-Kemp Bell T. 1832–1836. A monograph of the Testudinata. London: (Oxford University Museum of Natural History), and Samuel Highley. Jakob Hallermann (Zoologisches Museum Hamburg). Bell T. 1834. Characters of a new genus of freshwater tortoise The specimen from Mondolkiri, Cambodia, was col- (Cyclemys). Proceedings of the Zoological Society of London lected under the auspices of the Wildlife Conserva- 1834: 17. tion Society/Ministry of Agriculture, Forestry and Bourret R. 1939. Notes herpétologiques sur l’Indochine Fisheries/Ministry of Environment Collaborative française. XVI. Tortues de la collection du Laboratoire des Program with field assistance from An Dara and Sciences naturelles de l’Université. Description d’une espèce Suon Phalla. Editha Schubert and Holger Dathe nouvelle. Annexe au Bulletin Général de l’Instruction Pub- (Deutsches Entomologisches Institut, Müncheberg) lique 1938: 5–12. and Hannes Baur (Naturhistorisches Museum Bern) de Bruin RWF, Artner HG. 1999. On the turtles of Hainan provided biographical information on Hans Fruhstor- Island, southern China. Chelonian Conservation and fer. Sequencing was performed in The Field Museum’s Biology 3: 479–486. Pritzker Laboratory for Molecular Systematics and Cooper A, Poinar HN. 2000. Ancient DNA: do it right or not Evolution operated with support from the Pritzker at all. Science 289: 1139. Foundation. Rob Vogelbacher and the DePaul Univer- Dalton R. 2003. Mock turtles. Nature 423: 219–220. sity Bioinformatics Group, in conjunction with the Das I. 2005. Asian turtles in the collection of the Oxford Illinois Bio-Grid, executed the Bayesian analysis on a University Museum, including a list of type specimens. computer cluster. The work was supported by a grant Herpinstance 3: 4–7. from The John D. and Catherine T. MacArthur Foun- van Dijk PP, Stuart BL, Rhodin AGJ, eds. 2000. Asian dation to the Field Museum. James Parham (Califor- turtle trade: proceedings of a workshop on conservation and trade of freshwater turtles and tortoises in Asia. Chelonian nia Academy of Sciences) and two anonymous Research Monographs 2. Lunenburg: Chelonian Research reviewers improved the manuscript. Foundation. Felsenstein J. 1985. 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