Acta Herpetologica 10(1): 7-15, 2015 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.13128/Acta_Herpetol-15149 Haplotype variation in founders of the Mauremys annamensis population kept in European Zoos Barbora Somerová1, Ivan Rehák2,*, Petr Velenský2, Klára Palupčíková1, Tomáš Protiva1, Daniel Frynta1 1 Department of Zoology, Faculty of Science, Charles University in Prague, Viničná 7, CZ-12844, Prague 2, Czech Republic 2 Prague ZOO, U Trojského Zámku 3, CZ-171 00 Prague 7, Czech Republic. * Corresponding author. E-mail: ophis@tiscali.cz Submitted on 2014, 15th August; revised on 2014, 11th November; accepted on 2014, 12th November Editor: Uwe Fritz Abstract. The critically endangered Annam leaf turtle Mauremys annamensis faces extinction in nature. Because of that, the conservation value of the population kept in European zoos becomes substantial for reintroduction programmes. We sampled 39 specimens of M. annamensis from European zoos and other collections (mainly founders, imports and putatively unrelated individuals), and also four specimens of Mauremys mutica for comparison. In each animal, we sequenced 817 bp of the mitochondrial ND4 gene and 940 bp of the nuclear R35 intron that were used as phylogenetic markers for Mauremys mutica-annamensis group by previous authors. The sequences of the R35 intron, which are char- acteristic for M. annamensis and which clearly differ from those characteristic for M. mutica and/or other Mauremys species, were mutually shared by all of the examined M. annamensis. They also possessed mitochondrial haplotypes belonging to the annamensis subclades I and II, distinctness of which was clearly confirmed by phylogenetic analyses. Thus, both nuclear and mitochondrial markers agreed in the unequivocal assignment of the examined individuals to M. annamensis. Although no obvious hybrids were detected within the founders of the captive population, further careful genetic evaluation using genom-wide markers is required to unequivocally confirm this result. Keywords. Mauremys, Geoemydidae, conservation, mt gene ND4, nuclear intron R35, Vietnam, hybridization. INTRODUCTION Asian turtles face an extinction crisis due to habitat destruction and high demands from the Chinese markets (van Dijk et al., 2000; Le et al., 2004; Cheung and Dudg- eon, 2006; Turtle Conservation Coalition, 2011). One of the heavily exploited species is Mauremys annamensis, the Annam leaf turtle. (Siebenrock, 1903). This species of the family Geoemydidae has a very limited and fragment- ed distribution and is restricted only to central Vietnam (Le et al., 2004; Parham et al. 2006). Mauremys annamen- sis is almost extinct in the wild, with limited numbers in ex-situ populations in Vietnam, Europe and the USA. It is listed in the Appendix II of CITES and is globally red- listed as critically endangered by the IUCN (2013). Cap- tive breeding seems to be one of the long-term solutions for the survival of Asian turtles (Hudson and Buhlmann, 2002; Turtle Conservation Coalition, 2011). Mauremys annamensis has been repeatedly bred in some European zoos, including Prague Zoo (Velenský, 2006; Raffel and Meier, 2013). Currently, these zoos have started co-ordi- nated ex situ conservation breeding of the species associ- ated with a repatriation project. Among the programmes’ top priorities at present is the repatriation of the best cap- tive-bred specimens. The situation of conservation breeding is compli- cated by hybridization among distinct species and even genera of the geoemydids (Galgon and Fritz, 2002; Fritz and Mendau, 2002; Fritz et al., 2004; Schilde et al., 2004; Spinks et al., 2004; Buskirk et al., 2005; Stuart and Par- 8 B. Somerová et alii ham, 2006; Shi et al., 2008). Hybridization among Mau- remys annamensis, M. mutica, M. sinensis, M. nigricans, Cuora amboinensis and C. trifasciata was reported both in captivity and in the wild (Parham et al., 2001; Shi and Parham, 2001; Fong and Chen, 2010). The current events of natural hybridization between M. mutica and M. sinen- sis on Taiwan Island (Fong and Chen, 2010) represent an especially interesting case. The phylogenetically closest species of M. annamen- sis is M. mutica (Barth et al., 2004; Feldman and Parham, 2004; Spinks et al., 2004) and these species may inter- breed (Fong et al., 2007). This represents a serious prob- lem for the efforts to build sustainable ex-situ breeding programs enabling the reintroduction and establishment of sustainable populations of M. annamensis in the wild. Hybridization events in the annamensis-mutica complex were demonstrated by striking incongruence among phy- logenies of the individual genes, i.e., the mitochondrial and nuclear markers. Some of these incongruences may result from recent translocation and consequent hybridi- zation; however, hybridization events that took place in the past are even more likely. Fong et al. (2007) clearly demonstrated such incongruence in the Hainan popula- tion of M. mutica, which differs from the “true mutica” of the Eastern continental China by the presence of mito- chondrial haplotypes forming a clade branching with- in those belonging to the M. annamensis. In contrast, sequences of R35 intron of Hainan M. mutica are even less related to the corresponding sequences of the M. annamensis than those of the “true mutica”. Moreover, it was clearly demonstrated that mitochondrial haplotypes of the M. annamensis was split into two deeply diver- gent haplogroups, which are referred to as the annamen- sis subclade I and II (Fong et al., 2007; Fong, 2008). The phylogeographic pattern of these subclades is, however, unclear due to the extinction of most of the original pop- ulations in the nature. To organize proper ex-situ captive breeding and to remove potential hybrids from the rescue population, it is necessary to examine the genetic variation of the found- ers of the M. annamensis population. In this study, we focused on the founders, imported and putatively unre- lated individuals of the M. annamensis kept in European zoos and other collections. We sequenced mitochondrial (ND4 gene) and nuclear (R35 intron) parts of DNA to (1) verify the species determination of the founders, (2) assess sequence variation of the captive population, (3) assign captive specimens into the main haplogroups (sub- clades I and II) and to (4) exclude the discovered inter- specific species hybrids from the breeding pool. For com- parison, we also included a few specimens of M. mutica into the analyses. MATERIAL AND METHODS In this paper, we examined 39 specimens of Mauremys annamensis from European zoos and other collections (found- ers, imported and putatively unrelated individuals, i.e., captive born specimens having no shared maternal ancestors in their pedigree), and also four specimens of M. mutica were includ- ed for comparison (Table  1). For all individuals, we sequenced a combination of mitochondrial (mtDNA) and nuclear DNA (nuDNA). For sampling of individuals, we used a non-invasive method: we took the tip of the claw from each sampled ani- mal and stored in Eppendorf tubes with 96% ethanol at -20°C prior DNA extraction. We isolated the total genomic DNA with DNAeasy Tissue Kit (Qiagen, Hilden, Germany), following the manufacturer’s recommendations. Using standard conditions and the primers L-ND4 and H-Leu, we amplified an 892 bp fragment of mtDNA containing the NADH dehydrogenase subunit 4 (ND4) gene and parts of tRNA (Stuart and Parham, 2004). Following the conditions in Fujita et al. (2004), and using the primers R35Ex1 and R35Ex2, we amplified the fragment of nuDNA containing 1133 bp of the RNA fingerprint protein 35 (R35) gene intron 1. Patterns from the sequencing chromatograms indicated that at the R35 locus, some individuals were heterozygous for a length polymorphism, which usually corrupts the sequence reads downstream of the indel site (see Bhangale et al., 2005, Fig. 1B). For sequencing the R35 intron, we used internal for- ward and reverse primers (Spinks and Shaffer, 2007) in combi- nation with external primers (Fujita et al., 2004) for the putative length-polymorphic individuals (Spinks and Shaffer, 2007). Sequences of both mtDNA and nuDNA fragments were aligned and manually checked using Chromas Lite 2.01 (Tech- nelysium Pty Ltd), BioEdit (Hall, 1999) and Clustal X 1.81 (Thompson et al., 1997). Analyses of the estimates of evolutionary divergence between the sequences of ND4 gene and R35 intron were conducted using the Maximum Composite Likelihood mod- el (Tamura et al., 2004). The included codon positions were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. Evolutionary analyses were con- ducted in MEGA6 (Tamura et al., 2013). Bayesian analysis (BA) was conducted with MrBayes 3.1 (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003). The best-fit model (HKY+G) was selected by hLRT in Modeltest 3.7 (Posada and Crandall, 1998). Two independ- ent runs of Bayesian analyses were conducted with a random starting tree and run for 30x106 generations, with trees sampled every 100 generations. The burn-in command was used to dis- card the first 10% of trees (3,000,000 generations), which were generated before the chain reached equilibrium in the distribu- tion of trees. For these phylogenetic analyses, we also included some mtDNA and nuDNA sequence data used in intrageneric studies about the Mauremys mutica-annamensis complex (Fong et al., 2007; Fong and Chen, 2010) and some species from the family Geoemydidae, which were used as outgroups (GenBank num- bers are listed in Appendix 1). 9Haplotype variation in Mauremys annamensis RESULTS In an alignment of the mitochondrial ND4 gene (817 bp), we detected 16 haplotypes, 25 variable sites and 17 parsimony-informative sites. All individuals of M. anna- mensis examined in this study possessed the mitochon- drial ND4 gene (p-distaces ranging from 0.127% to 1.826%) typical for this species (Fong et al., 2007). Phylogenetic analyses containing our sequences in the context of those available in the GenBank confirmed haplogroups and the general topology of previously pub- lished trees (Fong et al., 2007). The BA tree (Fig. 1) sug- gests a principal split between the “true mutica clade” (BA posterior probability  =  1.00) and a clade (BA  =  1.00) containing both the M. annamensis and Hainan M. muti- ca. The latter clade further splits into three distinct clades (all BA probabilities  =  1.00). These are an “annamensis subclade I”, “annamensis subclade II” and the “Hainan mutica clade”. Average uncorrected p-distance between the “annamensis subclade I” and “annamensis subclade II was 1.968%”. The sister relationship between the “anna- mensis subclade I” and the “Hainan mutica clade” is moderately supported (BA = 0.82). ND4 sequences of our M. annamensis samples belong to the haplogroups previously described as the “anna- mensis subclade I” and “annamensis subclade II” (13 and 26 cases, respectively). Out of four examined samples of the putative M. mutica, ND4 sequences branch within the “true mutica clade” and one within the “annamensis sub- clade I”. P-distances among these four clades computed from all available sequences (including GenBank sources) suggest low mutual divergence among both the “anna- mensis” and “Hainan mutica” clades (Table 1). In an alignment of nuclear R35 intron (918 bp), we detected 25 haplotypes, 20 variable sites and 7 par- simony-informative sites. All 39 specimens putatively belonging to the M. annamensis shared mutually simi- lar sequences of R35 intron (p-distaces from 0.132% to 0.932%). The R35 sequences in three of four M. mutica samples clearly differed from those of the M. annamensis. Phylogenetic analysis of these sequences and those available in the GenBank (alignment of 940 bp, see Fig. 2) confirmed the presence of the three previously described clades (Fong et al., 2007) within the annamen- sis-mutica complex: the “Hainan mutica clade” (BA pos- terior probability  =  0.98) is the sister group of the true mutica-annamensis clade (BA  =  1.00), which contains a group of mutica sequences corresponding to the “true mutica clade” (BA  =  0.53) and a well-supported “anna- mensis clade” (BA = 1.00). In BA tree, the “true mutica” is paraphyletic with respect to the “annamensis clade”, how- ever, most of the sequences of this group form a single branch with low support (BA = 0.53). The BA analysis placed all 39 examined sequences of the M. annamensis into the “annamensis clade”. Out of four of the M. mutica sequences, one belongs to the “Hainan mutica clade”, one into the “annamensis clade” and the remaining two into the “true mutica” (Table 2). DISCUSSION We have no evidence suggesting the presence of the interspecific hybrids among the examined founders of the M. annamensis kept in European collections. Of course, without an application of expensive genome-wide mark- ers (like SNPs, extensive number of microsatellites), it is impossible to entirely rule out partial introgression of the genomes of other related geoemydids into some found- ers of the European population of the M. annamensis (i.e., presence of hybrids of a higher order - F2 and higher generations and backcrosses). Also, without cloning, we are unable to evaluate the affinity of potential heterozy- gots of the R35 intron to individual mitochondrial sub- clades. Nevertheless, when considering other supportive evidence (age, origin), the presence of hybrids seems to be fairly unlikely. The original geographic distribution of the Maure- mys annamensis is unknown, only few records document it. That is why it is hard to understand the significance of the two distinct mitochondrial clades, which we, as well as previous authors, detected in the M. annamensis. It is unclear whether these clades occur or occurred in the wild in syntopy or allopatrically. The sequence divergence Table 1. Average values of estimates of evolutionary divergence between sequences (the p-values are expressed in per cents).   annamensis subclade II annamensis subclade I Hainan mutica clade true mutica clade annamensis subclade II 0.083-0.167     annamensis subclade I 0.844-1.193 0.167-0.420   Hainan mutica clade 1.020-1.281 0.589-0.934 0.083-0.167 true mutica clade 3.952-4.895 3.549-4.782 3.952-4.782 0.083-1.554 10 B. Somerová et alii between the two clades is only about 0.84-1.19 %. Thus, we cannot reject the possibility that retention of ancestral polymorphism is a cause of a simultaneous occurrence of these related, but still distinct clades, in the sampled pop- ulation of the M. annamensis. Ancestral polymorphism may be irrelevant to an original population structure of the species prior to its recent decline leading to near extinction in the wild. The distinction between the mitochondrial haplo- type groups I and II has been recognized only recently and thus, the species has been treated as a single con- servation unit in most zoos and collections. However, it is possible to keep the animals of the two groups apart. This would be recommended especially in the case of animals producing offspring suitable for repatriation projects. Nevertheless, such a precaution cannot sub- Fig. 1. Bayesian tree of mitochondrial DNA (ND4) of the genus Mauremys. Numbers at branches are support values, only values >  0.95 are shown. Samples sequenced in this study, which correspond to Table 1, in bold, remaining samples were sequenced by previous authors (Fong et al., 2007; Fong and Chen, 2010), Mm  =  Mauremys mutica, Ma  =  Mauremys annamensis. Countries of the origins of samples are shown at individuals with reliable locality. 11Haplotype variation in Mauremys annamensis Table 2. List of samples used in this study containing information about species, breeder, nuclear and mitochondrial haplotype subclades. Nr.   Species Breeder ND4 R35 Mauremys annamensis mtDNA subclade II 1 Mauremys annamensis H. Becker annamensis subclade II annamensis clade 2 Mauremys annamensis H. Becker annamensis subclade II annamensis clade 3 Mauremys annamensis H. Becker annamensis subclade II annamensis clade 3D Mauremys annamensis D. Frynta annamensis subclade II annamensis clade 5D Mauremys annamensis D. Frynta annamensis subclade II annamensis clade 6 704774 Mauremys annamensis Rotterdam annamensis subclade II annamensis clade 8 704525 Mauremys annamensis Rotterdam annamensis subclade II annamensis clade 10 704524 Mauremys annamensis Rotterdam annamensis subclade II annamensis clade 11 705067 Mauremys annamensis Rotterdam annamensis subclade II annamensis clade 12 3 Mauremys annamensis Münster annamensis subclade II annamensis clade 13 4 Mauremys annamensis Münster annamensis subclade II annamensis clade 15 9 Mauremys annamensis Münster annamensis subclade II annamensis clade 16 1 Mauremys annamensis Münster annamensis subclade II annamensis clade 17 2 Mauremys annamensis Münster annamensis subclade II annamensis clade 19 6 Mauremys annamensis Münster annamensis subclade II annamensis clade 20 Mauremys annamensis M. Schilde annamensis subclade II annamensis clade 23 M53 Mauremys annamensis Praha annamensis subclade II annamensis clade 25 F21 Mauremys annamensis Praha annamensis subclade II annamensis clade 26 F9 Mauremys annamensis Praha annamensis subclade II annamensis clade 32 ROO718 Mauremys annamensis Leipzig annamensis subclade II annamensis clade 35 32 Mauremys annamensis Panuška annamensis subclade II annamensis clade 36 33 Mauremys annamensis Panuška annamensis subclade II annamensis clade 37 34 Mauremys annamensis Panuška annamensis subclade II annamensis clade 126 COS679 Mauremys annamensis Chester annamensis subclade II annamensis clade 127 COS678 Mauremys annamensis Chester annamensis subclade II annamensis clade 130 COS349 Mauremys annamensis Chester annamensis subclade II annamensis clade Mauremys annamensis mtDNA subclade I 1D Mauremys annamensis D. Frynta annamensis subclade I annamensis clade 2D Mauremys annamensis D. Frynta annamensis subclade I annamensis clade 4D Mauremys annamensis D. Frynta annamensis subclade I annamensis clade 7 704212 Mauremys annamensis Rotterdam annamensis subclade I annamensis clade 9 704523 Mauremys annamensis Rotterdam annamensis subclade I annamensis clade 18 8 Mauremys annamensis Münster annamensis subclade I annamensis clade 21 Mauremys annamensis M. Schilde annamensis subclade I annamensis clade 22 Mauremys annamensis M. Schilde annamensis subclade I annamensis clade 24 M7 Mauremys annamensis Praha annamensis subclade I annamensis clade 33 ROO720 Mauremys annamensis Leipzig annamensis subclade I annamensis clade 34 ROO719 Mauremys annamensis Leipzig annamensis subclade I annamensis clade 128 CZ/921 Mauremys annamensis Chester annamensis subclade I annamensis clade 129 CZ/922 Mauremys annamensis Chester annamensis subclade I annamensis clade Mauremys mutica 4   Mauremys mutica H. Becker true mutica clade true mutica clade 5 Mauremys mutica H. Becker true mutica clade true mutica clade Animals of hybrid origin 28 3 Mauremys mutica Praha true mutica clade annamensis clade 27 2 Mauremys mutica Praha annamensis subclade I Hainan mutica clade 12 B. Somerová et alii stantiate the elimination of the descendants of parents belonging to different clades from the studbook popu- lation. There is an urge call for further research of the genetic variation in the M. annamensis using multiple nuclear markers and/or advanced genomic methods, especially to enable a better understanding of the diver- gence of the two distinct subclades. ACKNOWLEDGEMENTS We would like to thank the following zoological gardens (and their curators): Chester, Leipzig, Münster, Prague, Rotterdam and the private breeders Herbert Becker, Jíří Panuška and Maik Schilde for the possibil- ity to access the turtles for measurement and for tak- ing DNA samples and photographs. The research was supported by Charles University in Prague (project No. 9873) and Prague Zoo. 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Name of sequence Species ND4 R35 1Ma M. annamensis EF034098 EF587934 10Ma M. annamensis AF348280 DQ386668 11Mm.Hainan M. annamensis EF034096 EF87929 12Ma M. annamensis EF034105 — 13Ma.Hainan M. annamensis EF034104 EF587915 14Mm.Hainan M. mutica EF034097 EF587925 15.Mm.Hainan M. mutica EF034101 EF587917 16.Mm.Hainan M. mutica EF034095 EF587930 17Mm.Vietnam M. mutica AF348278 DQ386664 18Ma M. annamensis EF034104 EF587923 19Ma M. annamensis EF034106 EF587928 20Ma M. annamensis EF034107 EF587924 21Ma M. annamensis EF034112 DQ386656 22Ma M. annamensis EF587914 EF587921 23Ma M. annamensis EF034099 EF587919 24Ma M. annamensis EF034100 — 25Ma M. annamensis EF034108 — 26Mm M. mutica EF034092 — 27Mm M. mutica EF034093 EF587931 28Mm M. mutica EF034089 EF587932 29Mm M. mutica AF348278 DQ386666 2Ma M. annamensis AY337338 EF587933 30Mm M. mutica EF034090 — 31Mm M. mutica EF034092 EF587916 32Mm M. mutica EF034094 EF587927 3Ma M. annamensis EF034103 — 4Ma M. annamensis EF034105 EF587922 15Haplotype variation in Mauremys annamensis Name of sequence Species ND4 R35 6Ma M. annamensis EF034102 EF587929 7Ma M. annamensis EF034109 EF587926 8Ma M. annamensis EF034113 DQ386655 9Mm.Vietnam M. mutica AF348279 –––– Cuora amboinensis Cuora amboinensis EF011357 HQ442382 Cuora galbinifrons Cuora galbinifrons AY364617 –––– Cuora pani Cuora pani — EF011442 Cuora trifasciata Cuora trifasciata — JQ596437 Cyclemys dentata Cyclemys dentata — AM931697 Leucocephalon yuwonoi Leucocephalon yuwonoi — AM931708 M. nigricans M. nigricans EF034111 –––– M. reevesii M. reevesii EF034110 –––– M. reevesii.Taiwan4 M. reevesii GQ259438 GQ259459 M. reevesii.Taiwan7 M. reevesii GQ259441 GQ259464 M. sinensis.Hainan13 M. sinensis AY337345 DQ386678 M. sinensis.Taiwan9 M. sinensis GQ259443 GQ259465 M. caspica M. caspica AY337340 –––– Mauremys japonica Mauremys japonica — HQ442386 Mm.Taiwan19 M. mutica GQ259452 GQ259471 Mm.Taiwan20 M. mutica GQ259453 GQ259472 Mm.Taiwan21 M. mutica GQ259454 GQ259473 Mm.Taiwan25 M. mutica GQ259457 GQ259474 Mm.Taiwan26 M. mutica GQ259458 GQ259475 Ocadia glyphistoma Ocadia glyphistoma — DQ386663 Sacalia quadriocellata Sacalia quadriocellata — HQ442384 Siebenrockiella Siebenrockiella leytensis — AM931708 Siebenrockiella crassicollis Siebenrockiella crassicollis — AY954913 Acta Herpetologica Vol. 10, n. 1 - June 2015 Firenze University Press Obituary: Valery K. 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