2004

Asiatic Herpetological ResearchVol. 10, pp. 28-37

Molecular Systematics of Old World Stripe-Necked
Turtles (Testudines: Mauremys)
CHRIS R. FELDMAN1,* AND JAMES F. PARHAM2,3
1Department of Biology, Utah State University, Logan, UT, 84322-5305, USA,

* Corresponding Author E-mail: elgaria@biology.usu.edu.
2Evolutionary Genomics Department, Joint Genome Institute, 2800 Mitchell Drive,

Walnut Creek, CA, 94598, USA.
3University of California Museum of Paleontology, University of California, Berkeley, CA, 94720-3140, USA,

E-mail: parham@socrates.berkeley.edu
Abstract. - Nine extant species of Mauremys (including Ocadia and Chinemys) represent a geographically widespreadyet morphologically and ecologically conservative group of batagurid turtles. Here we examine the evolutionary relationships of Mauremys using 1539 base pairs of mitochondrial DNA encoding portions of COI, ND4, and three adjacent tRNA genes. These data contain 246 parsimony informative characters that we use to erect hypotheses of relationships for Mauremys. Both maximum parsimony and Bayesian methods suggest that Mauremys japonica, M.
sinensis, M. nigricans, and M. reevesii form a well-supported monophyletic clade, as do M. mutica and M. annamensis. Furthermore, our analyses show that M. mutica is paraphyletic with respect to M. annamensis. The western taxa
M. leprosa, M. caspica, and M. rivulata remain problematic and do not form a monophyletic group sister to the Asiantaxa. Nevertheless, an east-west biogeographic hypothesis cannot be discounted with our molecular genetic data.
Key words. - Turtles, Bataguridae, Mauremys, molecular phylogenetics, mitochondrial DNA

Introduction
The Old World turtle genus Mauremys is represented bymorphologically and ecologically conservative species
that are diagnosed by a rigid plastron and a striped headand neck. These semi-aquatic, batagurid (= geoemydid,
see Joyce et al., in press) turtles occupy lotic and lentic
environments in both forested and arid habitats throughout Asia and the Mediterranean.
The genus contains some of the most commercially
important freshwater turtles in Asia. For example, M.
mutica is one of the most commonly reared and highly
traded chelonians in Asia (Lau and Shi, 2000). Other
Mauremys species have been at the center of a conservation and systematics controversy. In fact, two newly
described Mauremys may be polyphyletic hybrids(Parham et al., 2001; Wink et al., 2001; Spinks et al.,2004).
Given the mounting conservation interest in the turtle fauna of Asia (van Dijk et al., 2000), understanding
the extant diversity and phylogenetic relationships
among the Bataguridae are areas of active research (Wuet al., 1999; Honda et al., 2002b; Barth et al., 2004;
Spinks et al., 2004). The genus Mauremys has received
particular attention because of this recent conservation
crisis and taxonomic confusion. The first examination ofevolutionary relationships within Mauremys was a morphological treatment of the genus based on shell and© 2004 by Asiatic Herpetological Research

scute measurements (Iverson and McCord, 1994).
Consistent with the disjunct distribution of Mauremys,
Iverson and McCord (1994) suggested that East Asiantaxa form a monophyletic group, sister to a
Mediterranean and Middle Eastern clade. A subsequent
study used 12S and 16S ribosomal genes to resolve thephylogenetic relationships among species of Mauremys(Honda et al., 2002a). In contrast to the east-west
hypothesis of Iverson and McCord (1994), Honda et al.
(2002a) suggested that the deepest phylogenetic splits
within Mauremys occur between Asian taxa. The ribosomal mtDNA data also cast doubt on the monophyly of
traditional Mauremys by including the east Asian
species, Chinemys reevesii, as the sister taxon to M.
japonica. Two recent studies examined more extensive
sequence data, predominantly cyt b mtDNA, as well asa more comprehensive sampling of batagurids (Barth etal., 2004; Spinks et al., 2004). Both studies firmly established the placement of Mauremys within the
Bataguridae and show that the Chinemys and Ocadia are
phylogenetically nested within Mauremys (Barth et al.,
2004; Spinks et al., 2004). Barth et al. (2004) offer two
possible solutions to reconcile the paraphyly ofMauremys: 1) split the species of Mauremys into four
genera; 2) lump Chinemys and Ocadia into an expanded
Mauremys. While Barth et al. (2004) refrain from a taxonomic decision, Spinks et al. (2004) adopt an expanded Mauremys. We also endorse an inclusive Mauremys

Vol. 10, p. 29

Asiatic Herpetological Researchbecause we consider expanding genera to well-supported clades of species functionally preferable to proliferating monotypic genera based on subjective, typologicalideas of uniqueness (Feldman and Parham, 2002;
Parham and Feldman, 2002; Spinks et al., 2004).
Our objective here is to provide an independent estimate of Mauremys phylogeny using different molecularmarkers from other recent systematic investigations andseparate museum voucher specimens (Barth et al., 2004;
Spinks et al., 2004). We hope that our data help resolve
areas of uncertainty in the emerging consensus on
Mauremys systematics. In addition, our study will add tothe growing body of information on the evolutionary
history and diversity of Asia’s threatened batagurid
fauna (Wu et al., 1999; Honda et al., 2002b; Barth et al.,
2004; Spinks et al., 2004).

Materials and methods
Taxon sampling and laboratory protocols. - We
obtained liver tissue from 17 museum specimens representing nine currently recognized species of Mauremys
and three species of Cuora (Appendix 1). The ninespecies of Mauremys used in our study include: M.
annamensis, M. caspica, M. japonica, M. leprosa, M.
mutica, M. nigricans, M. reevesii, M. rivulata, and M.
sinensis. We do not consider “M. iversoni”, “M.
pritchardi”, “O. glyphistoma” or “O. philippeni” to bevalid taxa because specimens matching these species (alldescribed from the pet trade) are likely hybrids (Parham
et al., 2001; Wink et al., 2001; Spinks et al., 2004). In
addition, we also excluded “M. megalocephala”, which
is probably a diet-induced variant of M. reevesii (Iverson
et al., 1989; Barth et al., 2002). However, we do include
a “M. iversoni”-like hybrid specimen described in
Parham and Shi (2001) because mtDNA from thishybrid specimen is demonstrably Mauremys (Parham etal., 2001). All vouchers correspond to well-documented
reference material and original species descriptions.
We isolated genomic DNA from tissue samples bystandard proteinase K digestion and phenol/chloroform
purification (Maniatis et al., 1982). We amplified 700 bp
of mtDNA encoding a section of COI via PCR (Saiki etal., 1988) using primers HCO-2193 and LCO-1490(Folmer et al., 1994). We amplified an additional 900 bp
region of mtDNA encoding a portion of ND4 and flanking tRNA histidine (tRNAhis), serine (tRNAser), and partof leucine (tRNAleu) using primers ND4 and Leu(Arevalo et al., 1994). We used the following thermal
cycle parameters for 50µl amplification reactions: 35
cycles of 1min denature at 94°C, 1min anneal at 45°C
(COI) or 52°C (ND4), and 2min extension at 72°C. We
purified PCR products using the Wizard Prep Mini
Column Purification Kit (Promega, Inc.) and used puri-

2004

fied template in 10µl dideoxy chain-termination reactions (Sanger et al., 1977) using ABI Big Dye chemistry(Applied Biosystems, Inc.) and the primers listed above.
Following an isopropanol/ethanol precipitation, we ran
cycle-sequenced products on a 4.8% Page Plus
(Ameresco) acrylamide gel using an ABI 377 automatedsequencer (Applied Biosystems, Inc.). We sequenced all
samples in both directions.
Sequence analyses. - We aligned DNA sequences withthe program SequencherTM 4.1 (Gene Codes Corp.), andtranslated protein coding nucleotide sequences intoamino acid sequences using MacClade 4.0 (Maddisonand Maddison, 2000). We identified tRNA genes bymanually reconstructing their secondary structures using
the criteria of Kumazawa and Nishida (1993). We
deposited all mitochondrial DNA sequences in GenBank(Appendix 1).
We performed a partition homogeneity test (PH),similar to the incongruence length differences test (ILD;
Farris et al., 1994), to determine whether the ND4 and
COI data could be combined. We used PAUP* 4.0b10(Swofford, 2002) to generate a null distribution of length
differences using 1000 same-sized, randomly generated
partitions from the original data with replacement.
To evaluate base substitution saturation at first, second, and third codon positions, we plotted the uncorrected percent sequence divergence of transitions and transversions versus the corrected maximum likelihood estimates of divergence for each codon position.
Phylogenetic analyses. - We used maximum parsimony(MP; Farris, 1983) and maximum likelihood-based
Bayesian (Larget and Simon, 1999) phylogenetic methods to infer evolutionary relationships among batagurid
species. We conducted MP analyses in PAUP* andBayesian analyses with MrBayes 3.0b4 (Huelsenbeck
and Ronquist, 2001). We polarized the phylogeny via
outgroup comparison (Maddison et al., 1984) using the
Asian box turtles Cuora mouhotii, Cuora picturata, andCuora trifasciata. Other molecular phylogenetic studies
suggest these turtles are appropriate outgroup taxa (Wuet al., 1999; Honda et al., 2002b; Barth et al., 2004;
Spinks et al., 2004).
We executed MP analyses with the branch-andbound search algorithm (Hendy and Penny, 1982) using
equally weighted, unordered characters. To assess nodal
support, we used the bootstrap resampling method (BP;Felsenstein, 1985) employing 1000 pseudoreplicates ofbranch-and-bound searches in PAUP*. Additionally, we
calculated branch support (DI; Bremer, 1994) for all
nodes using the program Tree Rot 2c (Sorenson, 1999).
We performed Bayesian analyses to estimate branch
lengths and search for additional tree topologies. To

2004

Asiatic Herpetological Research

determine the most appropriate model of DNA substitution for reconstructing Mauremys relationships underthe Bayesian method, we executed hierarchical likelihood ratio tests (LRT; Felsenstein, 1993; Goldman,
1993; Yang, 1996) in the program Modeltest 3.06
(Posada and Crandall, 1998). Because MrBayes 3.0b4
can perform singular phylogenetic analyses using different models of evolution, we performed two separate
LRTs on the two mtDNA regions. The model of
nucleotide substitution that best fit the COI data was theHKY model (Hasegawa et al., 1985) in conjunction with
(Yang, 1994a; 1994b), and I (Gu et al., 1995), while
the slightly less complex HKY + model of DNA evolution best fit the ND4 data. We then performed
Bayesian tree searches, allowing separate parameter
estimates under the two models of DNA substitution for
the COI and ND4 data partitions. We did not specify atopology or nucleotide substitution model parameters apriori. We ran Bayesian analyses for 3 x 106 generations using the Metropolis-coupled Markov chain Monte
Carlo (MCMCMC) algorithm with four heated Markov
chains per generation, sampling trees every 100 generations. To determine when the Markov chains had converged on stable likelihood values, we plotted the -lnl
scores against the number of generations (Huelsenbeck
and Ronquist, 2001). We then computed a 50 % majority rule consensus tree after excluding those trees sampled prior to the stable equilibrium. Nodal support isgiven by the frequency of the recovered clade, whichcorresponds to the posterior probability of that clade
under the given models of sequence evolution (PP;
Rannala and Yang, 1996; Huelsenbeck and Ronquist,
2001). Lastly, we performed three Bayesian runs to be
sure that independent analyses converged on similar loglikelihood scores (Leaché and Reeder, 2002).

Results
Genetic variation. - Sequences from the protein coding
regions appear functional and there are no generearrangements in the data (Kumazawa and Nishida,
1995; Kumazawa et al., 1996; Macey and Verma, 1997;
Macey et al., 1997). However, ND4 in the batagurids
studied here appears truncated relative to that of emydid
turtles, which have three additional residues:
Phenylalanine, Tyrosine, and Cysteine (Feldman andParham, 2002). Instead, these batagurids possess a stopcodon, followed by a 12 bp stretch of highly polymorphic DNA between ND4 and tRNAhis. Additionally,tRNAser has a short D-stem, instead of a D-arm replacement loop like that of most metazoan taxa (Kumazawa
and Nishida, 1993). This unusual tRNA condition is also
seen in emydid turtles (Feldman and Parham, 2002).
The PH test shows that length difference between

Vol. 10, p. 30

the sum of the COI and ND4 trees and the combined
COI and ND4 trees is not significantly different from therandomly generated test statistic (P = 0.93). Therefore,
we combined the aligned DNA sequences for subsequent phylogenetic analyses.
Of the 1539 aligned nucleotides, 369 are variable
and 246 are parsimony informative. Among ingrouptaxa, 289 sites are variable and 205 parsimony informative. Of the 369 variable characters, 60 occur at 1stcodon positions, 15 at 2nd positions, 261 at 3rd positions,and 33 in tRNAs. The scatter diagrams are linear andshow no evidence of multiple hit problems for transitions or transversions (data not shown).
Phylogenetic relationships. - The branch-and-boundequally weighted MP analysis produces a single mostparsimonious tree (L = 661; CI = 0.626; RI = 0.683) thatis consistent with the model-based Bayesian analyses(Fig. 1). All three Bayesian analyses converge on thesame topology and nearly identical mean log-likelihood
values, parameter estimates, and nodal support. Thus we
simply present results from the final search. The partitioned HKY + + I and HKY + Bayesian analysis (3 x
106 generations) attains stable log-likelihood values
within the first 15,000 generations, but we were conservative and discarded the first 20,000 generations.
Because we sampled trees every 100 generations, we
discarded the first 200 trees and retained 29,800
Bayesian trees, which we used to generate a 50% majority rule tree, and for which consensus values represent agroup’s posterior probability (Huelsenbeck andRonquist, 2001). The summary topology of the nearly
30,000 Bayesian trees (mean -lnl = 5205.5110, 2 =24.5038; mean ti/tv (COI) = 10.8360; 2 = 10.6335;
mean (COI) = 0.5479, 2 = 0.8874; mean Pinvar (COI)= 0.4163, 2 = 0.0291; mean ti/tv (ND4) = 12.3499; 2= 9.0505; mean (ND4) = 0.2431, 2 = 0.0009) differsfrom the MP tree in the placement of only one taxon(Fig. 1).
In both analyses, species of Cuora unambiguouslygroup to the exclusion of Mauremys, (BP = 100%; DI =19; PP = 100%). Mauremys japonica is a member of aclade containing M. nigricans, M. reevesii and M. sinensis (BP = 100%; DI = 13; PP = 100%), yet relationships
among these taxa are not well resolved, as indicated by
the low nodal support and conflict between MP and
Bayesian reconstructions. The MP tree places M. sinensis sister to a group linking M. japonica, M. nigricans,and M. reevesii (DI = 1), wherein M. nigricans and M.
reevesii form an additional clade (BP = 86%; DI = 5).
Alternatively, the Bayesian tree connects M. japonica to
M. sinensis (PP = 59%), sister to the M. nigricans the M.
reevesii clade (PP = 99%). The M. japonica, M. nigricans, M. reevesii, and M. sinensis clade is sister to a

Asiatic Herpetological Research

Figure 1. Phylogenetic trees for Mauremys. Country of origin given for species with multiple samples; hybrid taxon is in quotes. A) Single most parsimonious tree
based on equally weighted characters (L = 661; CI = 0.626; RI = 0.683). Numbers above nodes indicate bootsrap support, those below nodes represent decay indices.
B) Bayesian estimate of Mauremys phylogeny based on 29,800 trees built under partitioned HKY + + I and HKY + models of DNA evolution (mean -lnl =5205.5110, 2 = 24.5038; mean ti/tv (COI) = 10.8360; 2 = 10.6335; mean (COI) = 0.5479, 2 = 0.8874; mean Pinvar (COI) = 0.4163, 2 = 0.0291; mean ti/tv(ND4) = 12.3499; 2 = 9.0505; mean (ND4) = 0.2431, 2 = 0.0009). Numbers along nodes represent posterior probability values. Branch lengths drawn proportional to Bayesian estimates of genetic divergence.

Vol. 10, p. 31
2004

8.58
8.32
9.62
8.52
8.52
9.49
9.36
8.13
8.13
9.49
9.49
9.49
8.32
8.26
7.35
6.63
8.71
8.39
9.30
8.52
8.52
9.17
9.75
8.13
8.13
9.49
9.17
9.17
8.00
8.39
6.57
9.72
8.65
7.61
6.18
4.16
8.65
7.61
6.18
4.42
8.97
0.13
6.83
8.32
8.71
6.76
5.79
6.24
8.71
6.76
5.79
6.24
8.84
0.26
6.96
8.32
8.52
7.48
4.94 10.14
8.45
7.54
6.96
6.83
8.45
7.54
6.96
6.83
9.69
6.18
7.54
7.41
8.84
0
6.70
8.19
8.84
6.70
8.19
8.32
9.61
6.05
9.04
12.94 8.49
14.50 12.99 15.16
15.54 12.48 13.81 9.26
16.31 13.51 13.85 11.02
7.61
7.61
0.13
6.76
6.76
0.26
8.52
7.54
7.54
6.18
0
9.61
12.94
14.50
15.54
16.31
8.26
8.06
6.31
7.09
7.09
6.31
8.58
7.80
7.80
8.72
8.72
11.30
11.25
16.55
16.33
16.55
6.31
6.50
7.67
5.92
5.92
7.67
8.00
0
12.13
11.32
11.32
10.15
10.09
13.64
13.10
13.42
5.66
5.33
6.89
0
10.00
9.78
7.98
7.98
10.34
9.51
9.51
7.75
8.74
13.80
13.56
13.90
1 M. nigricans
2 M. reevesii
3 M. annamensis
4 M. caspica (Iran)
5 M. caspica (Bahrain)
6 "M. iversoni"
7 M. japonica
8 M. leprosa (Spain)
9 M. leprosa (Morocco)
10 M. mutica (China)
11 M. mutica (Vietnam)
12 M. mutica (Vietnam)
13 M. rivulata
14 M. sinensis
15 Cuora picturata
16 Cuora trifasciata
17 Cuora mouhotii

3.68
11.72
7.56
7.56
11.71
6.63
8.92
8.92
12.83
11.45
11.45
8.57
5.17
13.92
14.29
14.33

3.12
11.72
7.01
7.01
11.71
6.16
9.28
9.28
12.68
11.44
11.44
8.65
5.62
14.08
13.67
13.85

7.74
7.74
9.75
9.75
0.26
13.95
11.59
11.59
8.96
0.13
0.13
9.86
13.24
14.82
15.88
16.64

5.66
5.33
6.89
0
10.00
9.78
7.98
7.98
10.34
9.51
9.51
7.75
8.74
13.80
13.56
13.90

7.74
7.74
0.26
7.02
7.02
13.94
11.59
11.59
8.96
0.26
0.26
10.11
13.22
14.50
15.54
16.31

5.07
4.75
8.65
6.76
6.76
8.65
12.66
12.66
13.95
13.64
13.64
11.37
6.52
18.11
17.35
16.55

6.31
6.50
7.67
5.92
5.92
7.67
8.00
0
12.13
11.32
11.32
10.15
10.09
13.64
13.10
13.42

11
10
4
2

3

5

6

7

8

9

12

13

14

15

16

17

Asiatic Herpetological Research

1

Table 1. Pairwise comparisons of mtDNA sequences among Mauremys and related taxa. Note: values above the diagonal indicate uncorrected pairwise differences (%) while
those below the diagonal denote HKY + + I sequence divergences (%).

2004

Vol. 10, p. 32

poorly supported M. mutica, M. annamensis, “M. iversoni”, M. leprosa, and M. caspica assemblage (DI = 2;
PP = 61%). Within this large group, M. mutica, M. annamensis and “M. iversoni” form a strongly supported
clade (BP = 99; DI = 15; PP = 100%). In fact, inclusionof both M. annamensis and “M. iversoni” render M.
mutica paraphyletic; two M. mutica (ROM 25613,
25614) are more closely related to M. annamensis and“M. iversoni” than they are to a Chinese M. mutica(MVZ 230476) (BP = 100%; DI = 36; PP = 100%).
Within this “mutica complex”, M. annamensis and “M.
iversoni” are weakly allied (BP = 60%; DI = 1; PP = 86).
This entire “mutica complex” is then sister to a weakly
supported M. leprosa and M. caspica clade (DI = 1).
Finally, both MP and Bayesian analyses suggest that M.
rivulata is sister to a monophyletic clade containing therest of Mauremys, but this phylogenetic arrangement
receives almost no statistical support (DI = 2; PP =60%).

Discussion
Phylogenetic relationships. - Both MP and Bayesian
phylogenetic methods show that M. japonica is a member of a clade containing M. nigricans, M. reevesii, andM. sinensis, exclusive of other Mauremys. The M.
japonica, M. nigricans, M. reevesii, and M. sinensis
clade is joined to a poorly supported M. mutica, M.
annamensis, M. leprosa, and M. caspica assemblage.
Within this grouping, M. mutica and M. annamensis
form a solid clade, congruent with shell and scute data
(Iverson and McCord, 1994), other molecular data
(Barth et al., 2004; Spinks et al., 2004) but not 12S and
16S mtDNA data (Honda et al., 2002a). Our analyses
further suggest that M. mutica is paraphyletic. Two M.
mutica (ROM 25613, 25614) purchased in Vietnam are
more closely related to M. annamensis than they are to
topotypic M. mutica (MVZ 230476) from China. We
tested the paraphyly of M. mutica by constraining the
MP searches to recover only those trees that produce amonophyletic M. mutica. The shortest two trees generated by the constraint search are 697 steps long (CI =0.594; RI = 0.636), 36 steps longer than the unconstrained MP tree. The two-tailed Wilcoxon signed-ranks
test (Templeton, 1983) fails to support (P < 0.0001) the
monophyly of M. mutica. The mutica complex is linked
to a tenuous M. leprosa and M. caspica group. Lastly,
both MP and Bayesian phylogenetic analyses tentatively
place M. rivulata sister to a monophyletic clade containing the remaining ingroup taxa.
Genetic Variation. - Our samples of M. leprosa from
Spain and Morocco, and M. caspica from Iran and
Bahrain, show no intraspecific haplotype diversity

Vol. 10, p. 33

Asiatic Herpetological Research(Table 1), yet exhibit sizeable morphological variation(Busack and Ernst, 1980). This discrepancy between
intraspecific mtDNA diversity and geographic variation
seems to be common among turtles (e.g., Lenk et al.,
1999; Starkey et al., 2003) and may be related to extensive phenotypic plasticity or the slow rate of molecular
evolution in turtles (Avise et al., 1992; Lamb et al.,1994).
In contrast, most interspecific mtDNA variation
appears extensive, with uncorrected sequence divergences higher than 8% between a number of ingrouptaxa (Table 1). Additionally, the mitochondrial sequence
divergences between M. rivulata and M. caspica (Table1), formerly considered conspecifics (Fritz and Wischuf,
1997), are equivalent to or greater than the genetic distances observed between other congeneric emydid andbatagurid turtles (e.g., Feldman and Parham, 2002;
Starkey et al., 2003; Stuart and Parham, 2004). Hence,these mtDNA data, together with the differing shell morphologies, distinct color patterns, and unique habitat
preferences of M. rivulata and M. caspica (Busack and
Ernst, 1980), support the recent elevation of M. rivulata
as a distinct evolutionary lineage independent of M.
caspica (Fritz and Wischuf, 1997).
Mauremys annamensis, a robust batagurid endemic
to central Vietnam, is characterized by extensive axillarybuttresses, a massive bridge, a slightly tricarinate andhigh-domed shell, a vividly striped head and neck, andreverse sexual size-dimorphism (McDowell, 1964;
Iverson and McCord, 1994). The taxon is so distinctive
it was once placed into its own genus, Annamemys
Bourret 1939. McDowell (1964) originally demonstrated that M. annamensis and M. mutica share a number ofderived features and Iverson and McCord (1994) subsequently confirmed a close kinship between these taxa
with shell measurements. Hence, the close relationshiprevealed by our mitochondrial genes is not novel. Whatis surprising, however, is that M. annamensis differsfrom Vietnamese M. mutica and our “M. iversoni”-likehybrid by only two transitions. Furthermore, this cladeshows a roughly 6% uncorrected sequence divergencefrom topotypic M. mutica from Zoushan Island,
Zheijung Province, eastern China. In contrast, distantly
collected samples of M. leprosa and M. caspica show nosuch intraspecific mtDNA variation (Table 1). These
data question our ideas of species limits within
Mauremys. Is M. annamensis a distinct species? Does
M. mutica represent multiple species?
Several potential hypotheses might account forthese unexpected results. M. annamensis may simply
represent a recent species, derived from M. mutica, oreven a geographical variant of M. mutica. The dramatic
morphological differences exhibited by M. annamensis
could reflect intense selection and rapid phenotypic evo-

2004

lution while the minute mitochondrial divergences andparaphyly represent the nature of speciation and unsorted polymorphism. Alternatively, there may be historicalor ongoing introgression between M. annamensis andVietnamese M. mutica, perhaps facilitated by selection.
Two additional hypotheses involve the possibilityof hybridization. While our specimen of M. annamensis
conforms to the species description, it was acquired
from a Chinese turtle farm (Appendix 1) where M. annamensis and M. mutica are reared together in large numbers (J.F. Parham, pers. obs.). Hence, our M. annamensis could be a captive hybrid between M. annamensis
and M. mutica, though we find no morphological characters supporting this notion. Ideally, we would examine
the morphology and compare the sequences of a wildcaught M. annamensis to our sample, but to our knowledge, no tissued, field-collected vouchers of M. annamensis exist in collections; all modern museum specimens of M. annamensis have been obtained from either
animal markets or the pet trade.
Another possibility is that the Vietnamese M. mutica could be hybrid offspring of female M. annamensis
and male M. mutica, accounting for the scant mtDNA
differences between Vietnamese M. mutica and M.
annamensis and the sizeable divergences between thesesamples and topotypic M. mutica. Although the“Vietnamese M. mutica” are phenotypically similar totypical M. mutica, their darker coloration is evocative ofM. annamensis. Both Barth et al. (2004) and Spinks etal. (2004) found substantial mitochondrial variation
between M. mutica and M. annamensis, but we do notknow the provenance or morphology of their samples.
The hybridization of batagurid turtles has lead toother cases of taxonomic confusion (Parham and Shi,
2001; Parham et al., 2001; Shi and Parham, 2001; Wink
et al., 2001; Spinks et al., 2004) and cannot be discounted here. Unfortunately, our small sample size prohibits
us from effectively evaluating these hypotheses. Clearlya more detailed genetic study is needed to unravel thisproblem. With our present knowledge, any change inconservation policies for M. annamensis, one of theworld’s most poorly known turtles, would be premature.
Biogeography. - The distribution of Mauremys is characterized by a major break between the Zagros
Mountains of western Iran (easternmost M. caspica) andthe Annamite Mountains of central Vietnam (range of M.
annamensis). This disjunction includes the entire Indian
subcontinent (home to a diverse, endemic batagurid
fauna), and the inhospitable Tibetan plateau. We suggestthat the collision of India into Asia may be the vicariant
event responsible for the current distribution ofMauremys, as proposed for anguine lizards (Macey etal., 1999). Molecular data are ambiguous on this point.

2004

Asiatic Herpetological Research

Given that neither eastern nor western species assemblages appear monophyletic (though a Wilcoxon signed
ranks test topology test cannot discount this hypothesis[P = 0.35]), the current divergences between the living
species may have occurred before the development of
the Indo-Tibetan gap. The collision and subsequent
uplift of the Tibetan plateau took place in multiple stages
between 50 and 10 MYBP (Shackleton and Chang,
1988; Dewey et al., 1989; Windley, 1988). Hervet(2004) attributed some Paleogene (>50 MYBP)
European fossils to the stem of Mauremys, but did notinvestigate their relations to east Asian Mauremys. In
addition to employing additional molecular markers tovouchered museum specimens, the integration of all
extant Mauremys into analyses of morphological characters and fossil taxa will be necessary to unravel the historical biogeography of this clade of turtles.

Acknowledgments
We thank T. J. Papenfuss, C. Cicero, and D. B. Wake
(MVZ), and R. W. Murphy (ROM) for kindly contributing specimens and tissues essential to this project. We
are grateful to M. E. Pfrender and P. G. Wolf for generously providing laboratory space, and D. G. Mulcahy
and W. B. Simison for much needed lab assistance. We
appreciate helpful discussions about phylogenetic methods from E. M. O’Neill, A. D. Leaché, and F. T.
Burbrink, and useful comments on this manuscript from
M. D. Matocq, J. R. Mendelson III, E. D. Brodie Jr., H.
B. Shaffer, and two anonymous reviewers. We also
thank P. Q. Spinks and U. Fritz for sharing unpublisheddata. Finally, we thank K. Padian and T. J. Papenfuss forfunding, space, advice, and encouragement. This is
University of California Museum of Paleontology
Contribution #1826 and LBNL #54656. This research
was performed under the auspices of the U.S.
Department of Energy, Office of Biological andEnvironmental Research.

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Appendix 1.
Specimens used and GenBank Accession numbers for
DNA sequence data. Acronyms are: MVZ = Museum ofVertebrate Zoology, Berkeley, California; ROM = Royal
Ontario Museum, Toronto, Ontario; AF or AY =GenBank (http://www.ncbi.nlm.gov).
Mauremys annamensis - Purchased in turtle farm in
Hainan Province, China, no real locality data; MVZ
238937; AY337338, AY337346. Mauremys caspica Field collected on Bahrain Island, Bahrain; MVZ
230971; AY337339, AY337347. Mauremys caspica Field collected in West Azarbaijan Province, Iran; MVZ
234281; AY337340, AY337348. “Mauremys iversoni” Purchased in turtle farm in Hainan Province, China, noreal locality data; MVZ 230475; AF348275, AF34281.
Mauremys japonica - Pet trade specimen, no locality
data; MVZ 234647; AY337341, AY337349. Mauremys
leprosa - Field collected in Tetouan Province, Morocco;
MVZ 178059; AY337342, AY337350. Mauremys leprosa - Field collected in Cadiz Province, Spain; MVZ
231989; AY337343, AY337351. Mauremys mutica Field collected in Zoushan Island, Zhejiang Province,
China; MVZ 230476; AF348262, AF348278. Mauremys
mutica - Purchased from a turtle trader in Yen Bai
Province, Vietnam; ROM 25613; AF348260,
AF348279. Mauremys mutica - Purchased from a turtle
trader in Yen Bai Province, Vietnam; ROM 25614;
AF348261, AF348280. Mauremys rivulata - Field-collected in Bursa Province, Turkey; MVZ 230212;
AY337344, AY337352. Mauremys (=Ocadia) sinensis Field-collected in Hainan Province, China; MVZ
230479; AY337345, AY337353. Mauremys nigricans Pet trade specimen, no locality data, MVZ 130463;
AF348264, AF348289. Mauremys reevesii - Pet trade
specimen, no locality data, MVZ 230533; AF348263,
AF348288. Cuora picturata - Purchased from a turtle
trader in Dong Nai Province, Vietnam, ROM 37067;
AF348265, AF348292. Cuora trifasciata - Pet trade
specimen, no locality, MVZ 230636; AF348270,
AF348297. Cuora mouhotii - Purchased from a turtle
trader in Bac Thai Province, ROM 35003; AF348273,
AF348286.