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Asiatic Herpetological ResearchVol. 10, pp. 120-125

An Ocadia sinensis x Cyclemys shanensis hybrid(Testudines: Geoemydidae)
MAIK SCHILDE1, DANA BARTH2 AND UWE FRITZ3
1Opalstr. 31, D-04319 Leipzig, Germany; E-mail: maik.schilde@ufz.de
2University of Leipzig, Institute of Zoology, Molecular Evolution & Animal Systematics,

Talstr. 33, D-04103 Leipzig, Germany; E-mail: dbarth@rz.uni-leipzig.de
3Zoological Museum (Museum für Tierkunde), Natural History State Collections Dresden, A. B. Meyer Building,Königsbrücker Landstr. 159, D-01109 Dresden, Germany; E-mail: uwe.fritz@snsd.smwk.sachsen.deAbstract. - A captive bred Ocadia sinensis x Cyclemys shanensis hybrid is described. Its hybrid status was confirmed
by a comparison of a 1036 bp fragment of the mitochondrial cytochrome b gene with the putative mother (C. shanensis) and genomic ISSR fingerprinting. This is the first report of an intergeneric hybrid between very distantly related
geoemydid turtles. All previous geoemydid intergeneric hybrids have been crossings within or between two sisterclades containing the currently accepted genera (Chinemys, Mauremys, Ocadia) and (Cuora, Pyxidea).
Key words. - Cyclemys, Ocadia, testudines, intergeneric hybrid.

Introduction
Recently several new cases of intergeneric chelonian
hybrids became known to science (reviewed in Galgonand Fritz, 2002). Most of them belong to the Southeast
Asian family Geoemydidae, long known under its juniorsynonym Bataguridae. However, current research on themolecular phylogeny of geoemydids has shown thatsome species traditionally attributed to different genera
are more closely related than previously thought (Wu etal., 1999; McCord et al., 2000; Honda et al., 2002a, b;
Barth et al., in press; Stuart and Parham, in press), suggesting that they should be better lumped in the samegenus. Thus, some of the hybrids may be in fact notintergeneric. In this paper we report a captive bredhybrid between two distantly related Southeast Asian
geoemydids, representing an undoubtedly intergenericcross.
According to the cited studies, there are several
major clades among geoemydids. One clade contains thecurrently recognized genera Chinemys, Mauremys,Ocadia, Cuora, and Pyxidea (McCord et al., 2000;Honda et al., 2002a, b; Barth et al., in press), and another one, being the sister clade, Cyclemys, Sacalia,Heosemys, Hieremys, Notochelys, and Leucocephalon(McCord et al., 2000; Honda et al., 2002b).
The turtle described herein is the result of a
hybridization of an Ocadia sinensis male and a
Cyclemys shanensis female, representatives of two of
the major clades of the Geoemydidae. This hybrid
demonstrates that very distantly related geoemydids arecapable of hybridizing successfully. It underlines thepossibility that some recently described Southeast Asian
chelonians (Ocadia glyphistoma, O. philippeni), which© 2004 by Asiatic Herpetological Research

are only known from few pet trade specimens, mightalso be hybrids.
The specimen. - The turtle described below hatched in
the live collection of M. Schilde from an egg of a
Cyclemys shanensis, laid August 13, 2002. The second
egg of the same clutch did not develop. The mother wasa long term captive, and kept with a Cyclemys shanensis
male and two Ocadia sinensis males. The elongatedeggs measured 56.5 x 20.0 mm. One quickly developed
a white band as typical for fertile eggs. It was incubatedconstantly at 28°C on Vermiculite. On October 26, 2002
a healthy turtle with a straight line shell length of 33 mm
hatched (Figs. 1-4). Its color pattern resembled Ocadia
sinensis but the general form was more similar toCyclemys (roofed, distinctly tricarinate shell, serrated
posterior marginal scutes), suggesting that it might be ahybrid. We decided to use two molecular methods to test
this hypothesis.

Materials and Methods
We sequenced a 1036 bp portion of the mitochondrial
cytochrome b gene (cyt b) of the captive bred turtle forcomparison with the putative mother. Because mitochondrial DNA is inherited in the maternal line, thesequence of the putative hybrid should be identical withthe mother (Cyclemys shanensis). Species identificationof the Cyclemys was done by morphological means andcomparison with the mitochondrial cyt b sequences of
Guicking et al. (2002); the Ocadia sinensis males weredetermined morphologically.
Blood samples were obtained by coccygeal veinpuncture. Samples were stored as described in Haskell

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Figure 1.

Figure 2.

Figure 3.

Figure 4.

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Fig. 1-4. The captive bred Ocadia sinensis x Cyclemys shanensis hybrid, September 2003 (11 months old). The roofed,
distinctly tricarinate shell and the serrated posterior marginals resemble Cyclemys spp. The neck and facial stripes aswell as the plastral pattern are similar to O. sinensis. The plastral pattern was more contrasting as a hatchling and has
faded during growth. Photos: F. Höhler.

and Pokras (1994) and Arctander (1988). Additional
blood samples and photographs (dorsal and ventralaspects) of the captive bred turtle (MTD T1262), theCyclemys shanensis female (MTD T816), and the twoOcadia sinensis males (MTD T817-818) are in the tissue
collection of the Zoological Museum Dresden. DNA
extraction was carried out using the QIAamp Blood
Mini Kit (Qiagen). Primers mt-A (Lenk and Wink, 1997)
and H15909 (Lenk et al., 1999) were used to amplify aDNA fragment containing 1036 bp of cyt b. PCR andsequencing conditions were as described in Barth et al.
(in press). Sequencing reactions were performed on an
ABI 3100 Genetic Analyzer. Alignment was carried out
with CLUSTAL X, v. 1.8 (Thompson et al., 1997) withdefault parameters. To demonstrate the considerable
genetic difference between Cyclemys and Ocadia compared to other hybridizing geoemydids, Mega 2.1(Kumar et al., 2001) was used for estimation of genetic
distances. Cyt b sequence data for calculating genetic
distances are from Barth et al. (in press) and Guicking etal. (2002).
To exclude the possibility of a gynogenetic orparthenogenetic origin of the specimen and to identify

the putative father, we conducted genomic fingerprinting with Inter Simple Sequence Repeats (ISSR) for thecaptive bred specimen, the Cyclemys female and both
Ocadia males. ISSR PCR produces species-specificgenomic fingerprints (Gupta et al., 1994; Zietkiewics etal., 1994; Wink et al., 1998, 2001; Nagy et al., 2003)
useful in detecting turtle hybrids (Wink et al., 2001).
Hybrid specimens share about 50% of their ISSR PCR
products with the respective paternal and maternalspecies (Wolfe et al., 1998; Wink et al., 2001; Storch etal., 2001). ISSR PCR is a simple and cheap method, andthe results are easily reproducible (Bornet andBranchard, 2001). Gynogenesis or pseudogamy, thedevelopment of unfertilized eggs by activation throughsperm of another species, as well as parthenogenesis is
not known in turtles. However, if the captive bred specimen should be of such origin, the ISSR profiles should
be identical with its biological mother.
The primer 5´-GACAGACAGACAGACA-3` wasused to generate ISSR fingerprints for the captive bredspecimen, the putative mother (Cyclemys shanensis),and both Ocadia sinensis males. Each reaction mix contained 100 ng of genomic DNA, 20 pmol primer, 1 U

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Asiatic Herpetological ResearchVol. 10, p. 122

Table 1. ISSR fingerprints of the Ocadia sinensis x Cyclemys shanensis hybrid and parental species (biologicalmother and father plus the second O. sinensis male kept with the mother). Only polymorphic and diagnostic PCR
products shown.
MTD T = Museum für Tierkunde Dresden Tissue Collection; + = PCR product present, - = PCR product lacking.
Fragment Length

Cyclemys
shanensis
MTD T816

HybridMTD T1262

Ocadiasinensis
MTD T817

Ocadiasinensis
MTD T818

100 bp
480 bp590 bp810 bp940 bp1100 bp1450 bp
1500 bp1550 bp1700 bp2400 bp2700 bp2900 bp++++++++++++++++++++++++++-+++++-

Taq-polymerase (SIGMA), 2.5 µl 10x PCR buffer(SIGMA) and 2.5 µl of 200 µM dNTPs in a total volumeof 25 µl. Amplification conditions were 4 min initialdenaturation (94°C), followed by 31 cycles of 1 min at94°C, 1 min at 54°C, and 2 min at 72°C, final extensionof 7 min (72°C). PCR reactions were performed on an
Eppendorf thermocycler.
15 µl of each PCR reaction was separated on 2%
agarose gels (25 cm), stained in ethidium bromide solution (0.5 µg/ml) and visualized under UV light. The 100
bp DNA ladder Plus (MBI Fermentas) was used as a size
standard. PCR was repeated under identical conditions
to test reproducibility of results. DNA fragments werescored manually. Band sharing coefficients were calculated according to Storch et al. (2001).

Results and Discussion
As expected, the cyt b sequence of the captive bred turtle and the putative mother (Cyclemys shanensis; EMBL
acc. no. AJ604513) proved to be identical. ISSR fingerprinting produced highly variable profiles which werespecies-specific and permitted individual identificationof both Ocadia sinensis males (Table 1). The captive
bred turtle shared 6 of its 13 bands with the mother(band sharing coefficient 0.5) and 7 bands with one ofthe O. sinensis males (band sharing coefficient 0.52).
Because the captive bred turtle and this O. sinensis maleexclusively share some fragments, we identified this turtle as the biological father. Thus, both methods confirmed the hybrid origin of the captive bred turtle.
Except for an unconfirmed, anecdotal newspaper
record of natural hybrids between Cuora flavomargina-ta and Geoemyda japonica in Japan (Anonymous,1995), all previous geoemydid intergeneric hybrids havebeen crossings within or between two sister clades containing the currently accepted genera (Chinemys,Mauremys, Ocadia) and (Cuora, Pyxidea): Chinemys
reevesii x Cuora amboinensis kamaroma (Galgon andFritz, 2002), Chinemys reevesii x Mauremys japonica(Yasukawa et al., 1992), Chinemys reevesii x Mauremys
mutica (= "Mauremys pritchardi", Wink et al., 2001),
Cuora amboinensis kamaroma x Mauremys annamensis(Fritz and Mendau, 2002), Cuora bourreti x Pyxidea
mouhotii (= "Cuora serrata", Parham et al., 2001; Stuart
and Parham, in press), Cuora galbinifrons x Pyxidea
mouhotii (= "Cuora serrata", Parham et al., 2001; Stuart
and Parham, in press), and Cuora trifasciata x
Mauremys mutica (= "Mauremys iversoni", Parham etal., 2001; Wink et al., 2001). Cyclemys belongs to another major clade, comprising the genera Cyclemys,Sacalia, Heosemys, Hieremys, Notochelys, andLeucocephalon (McCord et al., 2000). Cyclemys is separated by a considerable genetic distance from Ocadia(Table 2), surpassing the genetic distances of the otherhybridizing geoemydids.
Superficially our hybrid Ocadia sinensis x
Cyclemys shanensis resembles O. sinensis due to its
striped head and neck and the spotted plastral pattern.
This leads to the speculation that the morphologicallysimilar Ocadia philippeni McCord and Iverson, 1992and O. glyphistoma McCord and Iverson, 1994 might bealso intergeneric hybrids, as earlier suggested by van
Dijk (2000), Lau and Shi (2000), Parham and Shi(2001), and Galgon and Fritz (2002). Both species were
described on the basis of only a few pet trade turtles(McCord and Iverson, 1992, 1994), and until now noVol. 10, p. 123

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Table 2. Pairwise genetic distances (cyt b) between
hybridizing geoemydid species.

Literature Cited

Cyt b
pairwisedistances
Chinemys reevesii - Cuora amboinensis
0.104
Chinemys reevesii - Mauremys japonica
0.050
Chinemys reevesii - Mauremys mutica
0.070
Cuora amboinensis - Mauremys annamensis
0.098
Cuora galbinifrons - Pyxidea mouhotii
0.059
Cuora trifasciata - Mauremys mutica
0.104
Ocadia sinensis - Cyclemys shanensis
0.118

Allendorf, F. W., R. F. Leary, P. Spruell and J. K.
Wenburg. 2001. The problems with hybrids: setting
conservation guidelines. TREE 16:613-622.

Geomydid species

additional specimens became known to science. For
some individuals of three other pet trade taxa a hybrid
status has been unambiguously demonstrated: Two turtles identified as Mauremys pritchardi McCord, 1997
proved to be hybrids of Chinemys reevesii andMauremys mutica (Wink et al., 2001). Three Mauremys
iversoni Pritchard and McCord, 1991 originated fromcrossing Cuora trifasciata and Mauremys mutica(Parham et al., 2001; Wink et al., 2001), and several
Cuora serrata Iverson and McCord, 1992, have been
demonstrated to be hybrids between Cuora galbinifrons
and Pyxidea mouhotii and of Cuora bourreti andPyxidea mouhotii (Parham et al., 2001; Stuart andParham, in press).
Until now it is unknown whether all specimens ofthese taxa are of hybrid origin, and if so, whether thecrosses occurred in the wild, in captivity, or whether one
or the other form might represent a natural, stable hybridtaxon (Parham et al., 2001; Wink et al., 2001). Many
Southeast Asian chelonians are facing extinction due tooverexploitation (van Dijk et al., 2000). Therefore,many conservation efforts are established around theglobe, including CITES listing and captive breeding
programs for several species. A correct taxonomy is theprerequisite for any conservation measure. Hence, it is
crucial to determine whether the mentioned taxa represent real evolutionary entities and deserve high priority
in conservation, this includes also natural hybrid taxa
(Allendorf et al., 2001), or whether they are only incidentally occurring hybrids, without any conservation
relevance.

Acknowledgments
Special thanks go to Michael Wink and Daniela
Guicking, Heidelberg, for sharing unpublished geoemydid sequences with us and to Thomas U. Berendonk,Leipzig, for technical advice. James R. Buskirk,Oakland, provided the newspaper report on Cuora flavomarginata x Geoemyda japonica hybrids.

Anonymous. 1995. Hybrids between two protected turtles increase. Okinawa Times 15 August 1995.
Arctander, P. 1988. Comparative studies of Avian DNA
by restriction fragment polymorphism analysis.
Journal of Ornithology 129:205-216.
Barth, D., D. Bernhard, G. Fritzsch and U. Fritz. in press.
The freshwater turtle genus Mauremys - a textbook
example of an east-west disjunction or a taxonomic
misconcept? Zoologica Scripta.
Bornet, B. and M. Branchard. 2001. Nonanchored intersimple sequence repeat (ISSR) markers: reproducible and specific tools for genome fingerprinting. Plant Molecular Biology Reporter 19:209-215.
Fritz, U. and D. Mendau. 2002. Ein Gattungsbastard
zweier südostasiatischer Schildkröten: Cuora
amboinensis kamaroma Rummler and Fritz, 1991 x
Mauremys annamensis (Siebenrock, 1903).
Salamandra 38:129-134.
Galgon, F. and U. Fritz. 2002. Captive bred hybridsbetween Chinemys reevesii (Gray, 1831) and Cuora
amboinensis kamaroma Rummler and Fritz, 1991.
Herpetozoa 15:137-148.
Guicking, D., U. Fritz, M. Wink and E. Lehr. 2002. New
data on the diversity of the Southeast Asian leaf turtle genus Cyclemys Bell, 1834. Molecular results.
Faunistische Abhandlungen, Museum für Tierkunde
Dresden 23:75-86.
Gupta, M., Y.-S. Chyi, J. Romero-Severson and J. L.
Owen. 1994. Amplification of DNA markers fromevolutionary diverse genomes using single primersof simple-sequence repeats. Theoretical AppliedGenetics 89:998-1006.
Haskell, A. and M. A. Pokras. 1994. Nonlethal bloodand muscle tissue collection from redbelly turtles
for genetic studies. Herpetological Review 25:1112.
Honda, M., Y. Yasukawa and H. Ota. 2002a. Phylogenyof the Eurasian freshwater turtles of the genus
Mauremys Gray, 1869 (Testudines), with special

2004

Asiatic Herpetological Researchreference to a close affinity of Mauremys japonica
with Chinemys reevesii. Journal of Zoological
Systematics and Evolutionary Research 40:195200.
Honda, M., Y. Yasukawa, R. Hirayama and H. Ota.
2002b. Phylogenetic relationships of the Asian Boxturtles of the genus Cuora sensu lato (Reptilia:Bataguridae) inferred from mitochondrial DNAsequences. Zoological Science 19:1305-1312.
Kumar, S., K. Tamura, I. B. Jakobsen and M. Nei. 2001.
MEGA2: Molecular Evolutionary Genetics
Analysis Software. Bioinformatics 17:1244-1245.
Lau, M. and H. Shi. 2000. Conservation and trade of terrestrial and freshwater turtles and tortoises in the
People's Republic of China. Chelonian ResearchMonographs 2:30-38.
Lenk, P., U. Fritz, U. Joger and M. Wink. 1999.
Mitochondrial phylogeography of the European
pond turtle, Emys orbicularis (Linnaeus 1758).
Molecular Ecology 8:1911-1922.
Lenk, P. and M. Wink. 1997. A RNA/RNA heteroduplex
cleavage analysis to detect rare mutations in populations. Molecular Ecology 6:687-690.
McCord, W. P., J. B. Iverson, P. Q. Spinks and H. B.
Shaffer. 2000. A new genus of geoemydid turtle
from Asia. Hamadryad 25:20-24.
McCord, W. P. and J. B. Iverson. 1992. A new species ofOcadia (Testudines: Bataguridae) from Hainan
Island, China. Proceedings of the BiologicalSociety of Washington 105:13-18.
McCord, W. P. and J. B. Iverson. 1994. A new species ofOcadia (Testudines: Batagurinae) from southwestern China. Proceedings of the Biological Society ofWashington 107:52-59.
Nagy, Z. T., U. Joger, D. Guicking and M. Wink. 2003.
Phylogeography of the European whip snake
Coluber (Hierophis) viridiflavus as inferred fromnucleotide sequences of the mitochondrial
cytochrome b gene and ISSR genomic fingerprinting. Biota 3:109-118.
Parham, J. F., W. B. Simison, K. H. Kozak, C. R.
Feldman and H. Shi. 2001. New Chinese turtles:
endangered or invalid? A reassessment of twospecies using mitochondrial DNA, allozyme elec-Vol. 10, p. 124

trophoresis and known-locality specimens. Animal
Conservation 4:357-367.
Parham, J. F. and H. Shi. 2001. The discovery ofMauremys iversoni-like turtles at a turtle farm in
Hainan province, China: the counterfeit goldencoin. Asiatic Herpetological Research 9:71-76.
Storch, V., U. Welsch and M. Wink. 2001.
Evolutionsbiologie. Springer Verlag Berlin,
Heidelberg, New York, 450 pp.
Stuart, B. L and J. F. Parham. in press. Molecular phylogeny of the critically endangered Indochinese box
turtle (Cuora galbinifrons). Molecular Phylogenetics and Evolution.
Thompson, J. D., T. J. Gibson, F. Plewniak, F.
Jeanmougin and D. G. Higgins. 1997. The ClustalX
windows interface: flexible strategies for multiple
sequence alignment aided by quality analysis tools.
Nucleic Acids Research 24:4876-4882.
van Dijk, P. P. 2000. The status of turtles in Asia.
Chelonian Research Monographs 2:15-23.
van Dijk, P. P., B. L. Stuart and A. G. J. Rhodin (eds.).
2000. Asian Turtle Trade. In Proceedings of aWorkshop on Conservation and Trade of Freshwater
Turtles and Tortoises in Asia. Chelonian Research
Monographs 2:1-164.
Wink, M., H. Sauer-Gürth, F. Martinez, G. Doval, G.
Blanco and O. Hatzofe. 1998. Use of GACA-PCR
for molecular sexing of Old World vultures (Aves:Accipitridae). Molecular Ecology 7:779-782.
Wink, M., D. Guicking and U. Fritz. 2001. Molecularevidence for hybrid origin of Mauremys iversoni
Pritchard et McCord, 1991, and Mauremys
pritchardi
McCord,1997.
ZoologischeAbhandlungen, Staatliches Museum für Tierkunde
Dresden 51:41-49.
Wolfe, A. D., Q.-Y. Xiang and S. R. Kephart. 1998.
Assessing hybridization in natural populations ofPenstemon (Scrophulariaceae) using hypervariableintersimple sequence repeat (ISSR) bands.
Molecular Ecology 7:1107-1125.
Wu, P., K. Zhou and Q. Yang. 1999. Phylogeny of Asian
freshwater and terrestrial turtles based on sequence
analysis of 12S rRNA gene fragment. Acta
Zoologica Sinica 45:260-267.

Vol. 10, p. 125

Asiatic Herpetological Research

Yasukawa, Y., N. Kamezaki and N. Ichikawa. 1992. Onhybrids between Mauremys japonica and Chinemys
reevesii. Japanese Journal of Herpetology 14:206207.
Zietkiewicz, E., A. Rafalski and D. Labuda. 1994.
Genome fingerprinting by simple sequence repeat(ISSR)-anchored polymerase chain reaction amplification. Genomics 20:176-183.

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