越南三线闭壳龟、中国三线闭壳龟和金头闭壳龟的新亚种

Torsten Blanck， 周婷， 李艺， Tomáš Protiva， Paul Crow， Ralph Tiedemann

(1.闭壳龟保育中心-8530德意志兰茨贝格，奥地利； 2. 海南省林业科学研究所，海口571100； 3. 惠州李艺金钱龟生态发展有限公司，广东惠州516157； 4. 查尔斯大学动物学系理学部，布拉格-Vininá 7， 12844布拉格， 捷克共和国；5. 嘉道理农场&植物园-林锦公路，大浦，新界，香港特别行政区， 中国； 6. 波茨坦大学，生物和生物化学学院，进化生物学/系统动物学，卡尔-李卜克内西街24-25，豪斯26，德国波茨坦14476)



越南三线闭壳龟、中国三线闭壳龟和金头闭壳龟的新亚种

Torsten Blanck1*， 周婷2， 李艺3， Tomáš Protiva4， Paul Crow5， Ralph Tiedemann6

最近，遗传学研究证明了越南三线闭壳龟Cuorocyclornata的有效性，并表明这个种内还存在第3个尚未描述的亚种，这个亚种也能从体色和形态量度上与以前认定的2个亚种相区分。同时，也证明中国三线闭壳龟C.trifasciata有一个来自中国海南岛在遗传学上存在歧化的种群。此现象也存在于金头闭壳龟C.aurocapitata，很久以来就发现该种有一个在体色量度上明显不同的种群，并得到了微卫星标记和形态学证据的支持。以上两种方法都显示了中国三线闭壳龟、越南三线闭壳龟和尚未描述的亚种之间的区别。为此，本文将这3个种群分别描述为新亚种C.cyclornataannamitica，C.trifasciataluteocephala和C.aurocapitatadabieshani。

龟鳖目；地龟科；闭壳龟属的新亚种；分类；越南；中国；微卫星；形态测量

The Asian box turtle genusCuora(Gray， 1855) includes 13 species， with 6-7 recognized subspecies (TTWG， 2014). All but one species are listed as Critically Endangered by the IUCN， and most of them rank in the World’s 25+ Most Endangered Tortoises and Freshwater Turtles (TCC， 2011). Two species，CuoramccordiandC.zhoui， might already be extirpated in the wild， but all members of the genus are highly threatened by habitat destruction and collection for human consumption and as pets. The genusCuoraincludes species that are aquatic as well as some that are essentially terrestrial. They inhabit the rainforests of south and east Asia and range in size from 12-35 cm straight carapace length (SCL).

The taxonomy of this genus has remained controversial (Blancketal.， 2006； Spinks & Shaffer， 2007； Spinksetal.， 2009， 2012) despite numerous recent molecular studies. Whereas mitochondrial DNA (mtDNA) studies appeared to provide a sound framework for taxonomic decisions (Spinksetal.， 2004； Stuart & Parham， 2004)， recent work with a few nuclear single copy loci identified some putative “species” as hybrids (Stuart & Parham， 2007). Taxonomic uncertainty has been particularly apparent for the clade includingC.trifasciatasensu lato， which was recently suggested to consist of two species (Blancketal.， 2006)， i.e.，C.trifasciatasensu stricto (=Clade A； Fig. 1) andC.cyclornata(=Clade C) based on morphological differences and mtDNA sequence divergence. However， the species rank ofC.cyclornatahas been challenged by others (Artner， 2007， 2008； Fritz & Havas， 2007； Spinks & Shaffer，2007； Spinksetal.， 2009， 2012). Within Clade C (C.cyclornata)， Blancketal. (2006) could clearly differentiate between two divergent lineages both morphologically and by mtDNA， which they described as subspecies， namelyC.cyclornatacyclornataandC.cyclornatameieri. The validity ofC.cyclornatawas genetically reverified by the recent studies of Tiedemannetal. (2014) and Lietal. (2015a， 2015b) where microsatellite markers and mt genome analysis clearly supported its genetic distinctiveness.

Fig. 1 Individually based neighbor-joining tree of Nei’s minimum genetic distance for the microsatellite data (Tiedemann et al.， 2014)

Fig. 2 Coloration (left to right： plastron in adults， plastron in juveniles， head lateral)， distribution， and sample origins of the analyzed Cuora taxa Letters in the map correspond to the letters to the left.

Furthermore， several specimens of unknown origin， belonging to yet another genetically divergent clade ofC.trifasciatasensu lato were identified (i.e.C.trifasciatacf.trifasciataor Clade B， see Fig. 1). Mitochondrial data on this clade-however-may be obscured by the possibility of erroneously containing numts (nuclear-encoded mitochondrial DNA)， rendering these data non-informative in this context (Spinks & Shaffer， 2007；Spinksetal.， 2009). Even a very recent study (Spinksetal.， 2012) that used nuclear markers could not resolve these issues， since the only potentialC.cyclornatain their study was the holotype ofC.cyclornataas designated by Blancketal. (2006)， for which Spinksetal. (2012) were unable to obtain genetic data. Specimens hypothesized by Spinks and Shaffer (2007)， Spinksetal. (2009， 2012) to be of wild Hong Kong origin actually originated from urban areas (Crow， pers. obs.) and were thus very likely from captive origin. In addition， Spinksetal. (2012) hypothesized thatC.mccordimight resemble a hybrid between (1)C.trifasciataand (2)C.aurocapitataorC.galbinifrons.

Yet another taxonomic uncertainty concerned the (morphologically) closely related speciesC.aurocapitata(Luo & Zong， 1988) andC.pani(Song， 1984)， which are hypothesized to be morphologically weakly distinguishable (Artner， 2004； Blanck & Tang， 2005)， extensively share mitochondrial haplotypes (Parhametal.， 2004； Spinksetal.， 2004)， and might potentially interbreed in an intergradation zone in western Anhui or Hubei province (Blanck & Tang， 2005).

These many uncertainties were addressed with high resolution microsatellite markers by Tiedmannetal.(2014). That study showed thatC.cyclornatais genetically distinct fromC.trifasciataand thatC.mccordiandC.zhouiare not of hybrid origin as speculated by Spinksetal. (2012) but are also genetically distinct. The genetic results of Tiedemannetal. (2014) and Lietal. (2015a， 2015b) agree perfectly with the available morphometric and colorimetric traits. In addition， that microsatellite study not only revalidatedC.cyclornatabut also showed that a potential third distinct group might exist. It clarified that specimens ofC.trifasciataassigned to Clade B by Blancketal. (2006) indeed represent a distinct genetic lineage clearly originating from Hainan Island. Given these date， it remains to be evaluated whether available mtDNA data are actually “numts” as hypothesized by Spinks and Shaffer (2007)， Spinksetal. (2009， 2012) or whether they constitute authentic mitochondrial sequences. If the latter were true， this would further corroborate the distinctiveness of Clade B. Finally， the microsatellite study also revealed two genetically distinct populations ofC.aurocapitatathat are colorimetrically and geographically distinct (Blanck & Tang， 2005).

Blancketal. (2006) also noticed additional variation withinC.cyclornata， and considered what they called possible intergrades betweenC.c.cyclornataandC.c.meieri. During the past nine years since the description ofC.cyclornata， the authors of this paper have studied hundreds of specimens representing all of the different forms ofC.cyclornataandC.trifasciata. Despite the rarity of these turtles in nature， wild-caught specimens with data collected by locals together with the genetic studies of Tiedemannetal.(2014) indicate that this third variety ofC.cyclornatais genetically more closely related toC.c.cyclornatathan toC.c.meieri. It seems not to be a hybrid/intergrade， but rather its own ESU (evolutionary significant unit).

Blancketal. (2006) reported that the Hainan population ofC.trifasciatadiffered in certain morphological characters from the mainland populations， but they lacked mtDNA data from known locality Hainan specimens. Whereas Spinks and Shaffer (2007)， Spinksetal. (2009， 2012) hypothesized that this clade was based on numts， the data provided by Tiedemannetal. (2014) clearly indicated that Hainan specimens are represented in Clade B and that they are divergent from Clade A on the mainland.

Using molecular techniques in the study of evolutionary histories has resulted in a gradual abandonment of morphological characters as the primary source of phylogenetic inference. However， morphological characters are valuable for phylogenetic reconstruction， especially for tracking adaptive changes across phylogeographic groups defined by genetic markers. With molecular techniques still evolving， critically endangered， morphologically identifiable ESU’s could disappear before our techniques finally confirm them to be unique (as in the case withC.cyclornata). While current genetics indicate only a minor divergence betweenC.c.cyclornataandC. cf.cyclornata(Tiedemannetal.， 2014)， morphometric， colorimetric and geographic differentiation is evident. Thus， it is important， especially for conservation efforts， to verify if this variety ofC.cyclornatawarrants subspecific designation.

Mayr and Ashlock (1991) stated that a subspecies is an aggregate of phenotypically similar populations of a species inhabiting a geographic subdivision of the range of that species and differing taxonomically from other populations of that species. In the sense of Mayr (1963) reproductive isolation means isolation by intrinsic mechanisms and not just by geographical barriers. Cracraft (1989) proposed that even a single fixed character difference may define a geographic form as a separate species since he sees them as separately evolving metapopulation lineages according to the general lineage concept or minimum diagnosable units.

Vieitesetal. (2009) came up with the concept of distinguishing between Unconfirmed Candidate Species (UCS)， Confirmed Candidate Species (CCS) and Deep Conspecific Lineages (DCL). UCS lineages can be distinguished by molecular characters but cannot be confirmed by any other means. CCS lineages show detectable genetic differentiation plus distinctiveness characters based on Mayr (1963). DCL lineages show none or only slight differences in characters that mediate a reproductive barrier or species discrimination and/or indications of hybridisation with other species. Mirallesetal. (2011) suggest using three different lines of evidence when following this concept. Following this approach， subspecies status applies if a candidate species qualifies for only one line of evidence and species status if a candidate species qualifies for two or all lines (i.e. 75%-100%) of evidence. Hawlitscheketal. (2012) discussed all these different views in detail and came to the conclusion that taxonomic inflation in species can be problematic. According to him， describing species purely based on a personal and subjective interpretation of “existence as a separately evolving metapopulation lineage”， “minimum diagnosable unit” or lineage with its “own evolutionary tendencies and historical fate” can lead to very divergent species counts， the same problem for which the subspecies concept is now widely rejected. While species are given most attention in evolutionary studies， species lists and for conservation purposes (Phillimore & Owens， 2006)， describing every minimum diagnosable unit as a species， or elevating such subspecies to species level， bears the risk that diagnosable units no longer representing separately evolving entities. Species， in our opinion， have to represent real evolutionary entities， though. In our view， subspecies are evolutionary significant units. Even though they may eventually interbreed with other subspecies of the same species， such gene flow is sufficiently small to let them follow different evolutionary trajectories， often associated with local adaptation. Therefore， subspecies (if identified) warrant conservation.

When following the concept of Mirallesetal. (2011) and considering four important characters for turtles， i.e.， geographic isolation， genetic divergence， morphometric and colorimetric differences for which a minimum of 75% have to be fulfilled for species level， we come to the following conclusion：C.trifasciatacf.trifasciata(Clade B) shows significant genetic divergence (Tiedemannetal.， 2014)， is geograpically well isolated， and morphologically distinct to a certain extend， which is however not sufficient to warrant species status in our opinion. Thus， in our view this Clade qualifies for subspecific status. ForC.c. cf.cyclornatagenetic divergence is sufficient for subspecific ranking but not near species level， colorimetric differences are significant but morphometric characters and geographic isolation are not as significant. Thus， this taxon also qualifies as a subspecies.C.aurocapitataClade B yet shows sufficient genetic divergence for subspecific differentiation， and morphometric as well as colorimetric characters are significantly different (although its geographic status needs further evaluation) and it also qualifies as a subspecies.

Since the subspecies concept is still commonly and widely accepted in turtles (TTWG， 2014)， we suggest that the three discussed taxa are best classified as subspecies.

While the concept of subspecies is controversial as can be seen above， and has been neglected in many species concepts other than the biological species concept， there is certainly utility in its application for conservation as reviewed by Haigetal. (2006). The only quantitative metric they found for defining a subspecies was the 75% rule (Amadon， 1949； Patten & Unitt， 2002)； i.e.， 75% of the individuals of one subspecies must be distinguishable from those from the other.

Our data suggest that the three taxa discussed above comply with this rule.This leads us to the conclusion that three new subspecies of threeCuoraspecies are in need of description.

1 Materials & Methods

1.1Materials

Altogether 365 carapaces/323 plastra of adult primarily wild-caught specimens of sevenCuoraspecies， namely the aquatic to semiaquatic species of this genus， were used in this study (15/31C.aurocapitataClade A， 11/23C.aurocapitataClade B， 8/8C.aurocapitataintermediates， 40/43C.c.cyclornata， 18/21C.c.meieri， 23/24C.c. cf.cyclornata， 45/51C.mccordi， 26/25C.pani， 77/31C.trifasciataA， 55/22C.trifasciataB， 23/22C.yunnanensis， and 24/22C.zhoui). Data from a carapace or plastron with abnormalities were discarded as were those where the plastron was in a closed condition. Nearly a quarter of the specimens were also genetically analyzed by the Agriculture， Fisheries and Conservation Department of Hong Kong (AFCD) Project at Hong Kong University and Tiedemannetal. (2014).

1.2Geometricmorphometricmethods

Mid-line straight carapace length (SCL in mm) was measured in all possible specimens with calipers to the nearest mm. Digital images of the carapace and the plastrons were obtained for each specimen. Following the classification of Bookstein (1997)， 17 type 1 anatomical landmarks were established on the plastron and 25 type 1 and one of type 3 landmarks were established on the carapace (Fig. 3). These were recorded using TPSdig (Rohlf， 2008). Each set was then symmetrized across the axis of symmetry and one half was removed using BigFix6 (Sheets， 2003). Statistical examination was then performed on the remaining half of landmark sets. To remove the effects of position， orientation， and scale， using sets of X， Y coordinates of landmarks from each specimen， we employed the Procrustes superimposition method (Zelditchetal.， 2004) performed in CoordGen6 (Sheets， 2003). To remove the effects of size (only in separate analysis of plastron and carapace shape inC.trifasciataClades A and B，C.c.cyclornata，C.c.meieriandC.c. cf.cylornata) and sex (in all analysis) we used standardization on mean carapace length or sex (for each species and sex， separately) applied in the program Standard6 (Sheets， 2003). Partial warp scores for further statistical analysis were generated using PCAGen6 (Sheets， 2003). Differences in shell shape among all species were tested and visualized in the program Statistica 6 (Weiß， 2007).

Fig. 3 Dorsal (A) and ventral (B) view of a shell showing the landmarks placement on the carapace and plastron in this study

1.3Principalcomponentanalysis(PCA)ofmicrosatellitedata

The microsatellite data of Tiedemannetal. (2014) were reanalyzed using the software PCAGEN 1.2， which performs principal component analysis (PCA) on gene frequency data.

1.4Nomenclaturalacts

The article conforms to the requirements of the amended International Code of Zoological Nomenclature， and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank， the online registration system for the ICZN. The LSID (Life Science Identifier) for this publication is： urn：lsid：zoobank.org：pub：EB370CB0-B26A-4579-89B8-72F1A56762E2.The electronic edition of this work was published in a journal with an ISSN， and has been archived.

2 Results

Discriminant analyses of the shell shape differences in theC.cyclornataandC.trifasciatacomplex (with both sex and size removed) were significant both for carapace (Wilks’ Lambda=0.13，F(100， 720)=4.76，P<0.000 1) and plastron (Wilks’ Lambda=0.07，F(84， 421)=4.82，P<0.000 1). We found significant differences in the shape of the carapace and plastron among all species and potential subspecies (Table 1). Discrimination was successful for 80.90% in carapace and 74.17% in plastron (Discrimination rate for each species in Table 2). Differences among species are demonstrated in the CVA for carapace (Fig. 4) and plastron (Fig. 5).

Discriminant function analysis of the shell shape differences among allCuoraspecies (size was not removed) was significant both for carapace (Wilks’ Lambda=0.006，F(150， 1887)=17.88，P<0.000 1)and plastron (Wilks’ Lambda=0.004，F(225， 2637)=10.93，P<0.000 1). We found significant differences in the shape of the carapace and plastron among all taxa (P<0.000 1； Table 1). Discrimination was successful for 85.25% of specimens based on carapace and for 86.83% based on plastron (discrimination rate for each species in Table 3). Differences among species are demonstrated in the Canonical plots for carapace (Fig. 6) and plastron (Fig. 7).

Table 1 P-levels from the analysis of the carapace and plastron shape in Cuora cyclornataand C. trifasciata and their subspecies

Table 2 Classifications from the analysis of the carapace and plastron shape in Cuora cyclornata and C. trifasciata and their subspecies

Note： Observed classifications are in rows， predicted classifications are in columns.

Fig. 4 Canonical plot for carapace shape in Cuora cyclornata，C. trifasciata and their subspecies

Fig. 5 Canonical plot for plastron shape in Cuora cyclornata,C. trifasciata, their subspecies

Discriminant analysis of the shell shape differences inC.aurocapitataandC.paniwas significant for both carapace (Wilks’ Lambda=0. 043，F(50， 50)=3.80，P<0.000 1) and plastron (Wilks’ Lambda=0.21，F(42， 112)=3.15，P<0.000 1). We found significant differences in both the shape of the carapace and plastron (Table 3) among all species and subspecies. Disc-rimination was successful on 96.15% in carapace and 82.28% in plastron. Differences among species are demonstrated in the Canonical plot for carapace (Fig. 8) and plastron (Fig. 9).

Fig. 6 Canonical plot for carapace shape in all seven Cuora species

Fig. 7 Canonical plots for plastron shape in all seven Cuora species

A principal component analysis (PCA) of the entire microsatellite data of Tiedemannetal. (2014) shows distinction in almost complete agreement with morphological assignments， pointing towards a strong genetic signal confirming the species delimitation (Fig. 10). If the putatively polytypic species are analyzed in separate PCAs， there is confirmation for two morphologically defined types inC.trifasciata(Fig. 11； with a single mismatching specimen)， for three types inC.cyclornata(Fig. 12)， and for two types inC.aurocapitata(Fig. 13). In essence， with the exception of a single specimen， the species-specific PCAs on microsatellite data provide a perfect match with the morphological assignments.

Fig. 8 Canonical plot for carapace shape in Cuoraaurocapitata and C. pani

Fig. 9 Canonical plot for plastron shape in Cuoraaurocapitata and C. pani

The molecular data by Tiedemannetal. (2014， Fig. 1)， the multivariate analysis of the morphometric data in this study， the PCA data， plus our colorimetric analyses (see below) clearly provide evidence thatC.cyclornatais a distinct species and comprised of3 subspecies， whileC.trifasciataandC.aurocapitataare each comprised of two subspecies， and thatC.aurocapitataandC.paniare not as closely related as previously believed (Artner， 2004； Parhametal.， 2004； Spinksetal.， 2004； Blanck & Tang， 2005).

Table 3 Classifications for the analysis of the carapace and plastron shape in seven Cuora species

Note： Observed classifications are in rows， predicted classifications are in columns.

Fig. 10 PCA of the specimens studied in Tiedemann et al.， 2014

As it can be observed in Fig.1， for some specimens morphological and genetic data do not match. This could be caused by multiple factors. First， Fig. 1 is based on a neighbor-joining (NJ) algorithm， rather than a model-based program such as multiple likelihood or Bayesian analysis. Second， intergradation zones frequently occur between turtle subspecies as goes introgression between species (Fritzetal.， 2002). Based upon our morphological data the herein described subspecies show a convincing level of distinction， even though full reciprocal monophyly of the proposed subspecies is not evident in the NJ analysis.

While the subspecies ofC.cyclornataandC.trifasciataare distinct clusters in the NJ， this is not the case forC.aurocapitata， in which Clade A is not confined to a single cluster， while Clade B is. Further phylogenetic analysis of more samples could resolve this issue more completely. A subspecies in our opinion (see also Phillimore & Owens， 2006) cannot necessarily be expected to comprise a genetically monophyletic clade， as different subspecies may overlap in their distribution， allowing for reciprocal introgression.

Fig. 11 PCA of the two Cuora trifasciata clades from Tiedemann et al.， 2014

Fig.12 PCA of the three Cuora cyclornata clades from Tiedemann et al.， 2014

Fig. 13 PCA of the two Cuora aurocapitata clades from Tiedemann et al.， 2014

Monophyly with regard to microsatellite gene genealogies is generally difficult (or even impossible) to establish， for a combination of reasons inherent to this type of data：

•Under the infinite-allele model， monophyly at a microsatellite locus could only be assumed if (1) all individuals of any taxon share the same allele (i.e.， they are fixed for this allele) and (2) this allele is only found in this taxon. This situation is rarely encountered within sexually reproducing species.

•Under the stepwise-mutation model， monophyly at a microsatellite locus could only be assumed if (1) all individuals of any taxon share the same allele range (i.e.， all alleles are within this length range) and (2) this allele range is non-overlapping with the allele ranges of any other taxon.

•Both models assume that alleles of the same size are identical by descent， in other words， there is no homoplasy. This assumption is rarely met for real microsatellite loci (and not either under the stepwise-mutation model).

This is the reason why applications of microsatellites are (1) restricted to closely related species/subspecies and (2) microsatellite trees are typically based on distance measures (like NJ trees). Optimality-criterion based approaches (Maximum Likelihood， Maximum Parsimony) are-to our best knowledge-not directly applied to microsatellite data in the established and widespread phylogenetic software. In principle， one could specify a mutation model for the microsatellites (stepwise mutation model) and incorporate this into distance estimations (e.g.， RST). In phylogenetic contexts， these measures have been found to perform worse than those based on the infinite allele model (see Richard & Thorpe， 2001). This is likely due to the invalid assumption of homoplasy-free data. In addition， this approach is also distance based.

In essence， our NJ tree on microsatellites appears to be the established approach to infer a signal of divergence from microsatellite data. This can be interpreted in the context of taxonomic hypotheses， but does not provide phylogenetical information about plesio-or apomorphy， due to the inherent limitation of distance-based trees and the mutation mode of microsatellites. The NJ tree is pretty well resolved and congruent with morphology， although， as correctly noted， it does not verify monophyly.

Besides the evidence presented in our NJ tree， we consider the congruence between genetic， morphological， and geographic data in the structure assignment included in Tiedemannetal. 2014 are particularly relevant. Indeed， this software uses a Maximum Likelihood approach to identify clusters， within which there is Hardy Weinberg equilibrium (HWE). HWE is indicative of random mating， while departure from HWE is indicative-among other reasons-of lack of interbreeding.

Note that our structure clusters are solely based on the genetic data， not a priori assignment to taxa or locations. Nonetheless， they perfectly match both phenotypes and locations. Note also that we had hardly any evidence for interbreeding among these clusters.

In fact， these structure clusters comprise genetically， phenotypically， and geographically distinct units， within which random mating is inferred， but among which genetic exchange is extremely limited or absent.

If one adopts the idea of defining genetically， phenotypically， and geographically distinct units as subspecies， we present evidence to do so in some species ofCuora.

In any case， the morphological characters are clearly sufficient to diagnose the subspecies at the 75% threshold.Given this 75% rule as stated above， it is not surprising that each subspecies is not perfectly reciprocally monophyletic in this tree. When combining the morphologic， structure， PCA and genetic data we provide above， subspecies are well supported. We herein describe a new subspecies in each ofC.cyclornata，C.trifasciataandC.aurocapitata.

2.1Cuoracyclornataannamiticassp.nov.

ZooBank LSID： urn：lsid：zoobank.org：act： E454BADB-4CA9-46F1-A0BF-6A58CD568BCA

Holotype： Vinh University Museum (VUM) R.Em.04 a sub-adult male (Fig. 14) from Vietnam， Nghe An province， Tan Ky district， near Ky Son village， 146 mm SCL， collected in July 1986 by Prof. Hoang Xuan Quang & Ngyen Tat So’n near a stream.

Fig. 14 VUM R.Em.04， the holotype of Cuora cyclornata annamiticain lateral (A)， dorsal (B) and ventral (C) view(Photos by Hoang Ngoc Thao)

Diagnosis： A large subspecies ofC.cyclornatawith a maximum SCL of 350 mm， distinguished from the other subspecies by the combination of the following characters： 1) chin amber orange， lateral head yellowish-orange (both orange in the nominate subspecies， and white inC.c.meieri， and yellow to cream inC.trifasciata)； 2) predominantly to fully black plastral fore lobe， closely resembling the fully black one inC.c.meieri(never so inC.c.cyclornatawhere less than 40% of the humeral scute width is covered by black)； 3) black pattern on gular scutes interconnected sometimes with a yellow spot along the intergular seam (black pattern always well separated inC.c.cyclornata； always fully connected without any yellow inC.c.meieri)； 4) strongly reduced black pattern on the head (similar toC.c.cyclornata)； 5) The black vertebral stripe often exhibiting arrow-like thickened areas (which are usually present inC.c.cyclornatatoo， but usually to an even stronger extent， always absent inC.c.meierithat also shows much finer stripes)； and 6) a rather flat-domed， dorsally oval to rounded carapace (rather similar to that ofC.c.cyclornata， less rounded inC.c.meieri).

Large males reach 18-26 cm SCL， females 22-35 cm SCL.C.c.cyclornatareaches similar sizes， while inC.c.meierimales are 17-24 cm and females 20-30 cm SCL.

Description of holotype：Semi-adult male specimen with a straight carapace length of 146.02 mm and a maximum carapace width of 104.38 mm at M6/7. The carapace color is light brown and seems slightly faded； the three black carapacial stripes have faded to dark brown but are well developed and the vertebral stripes show an arrow shape on the hind part of vertebrals three to five. No sign of dark spots or diagonal stripes are present on the lateral scutes. Plastron pattern is faded to dark brown. The fore lobe of the plastron is mainly black， the humerals are covered by about 95% black with a small yellow stripe along the front， and a small yellow blotch is present on the intergular seam. The head nearly completely lacks black pigmentation and the light brownish posterior head-stripes are connected to the dorsal olive-yellow head； chin color appears pinkish.

Derivatio nominis： The subspecies nameannamitica(from Annam) refers to the distribution of this subspecies within the Annamite mountain range of central Vietnam and adjacent Laos.

Distribution：Cuorac.annamiticassp. nov. is currently only known by specimens from Vietnam but almost certainly occurs in adjacent Laos (Bolikhamsai， Khammuan provinces) in the Annamite mountain range. In Vietnam it is known to occur in Thanh Hoa (photographic evidence)， Nghe An (voucher specimens)， Ha Tinh (photographic evidence， Fig. 15) and northern Quang Binh (photographic evidence) provinces. Current data indicate that it occurs northwards to the delta of the Red River. The greater Ca River basin combined with the high mountain ridge in Nghe An province seem to form the subspecies boundary betweenC.c.annamiticaandC.c.cyclornataand likely prevents the species from extending into much of adjacent Laos. Further fieldwork is required to define the range boundaries ofC.c.meieri，C.c.annamiticaandC.c.cyclornatamore precisely.

Remarks： Captive breeding has shown that the color characteristics are generally inherited (Blanck pers. obs.， Meier pers. comm.) and stable in the offspring in all subspecies. InC.c.annamiticathe amount of (black) pattern on the plastron fore lobe can be triggered with incubation temperature， similar to the head pattern onGraptemyssp. (Meier pers. comm.， Vogt, 1978， 1993). Specimens incubated at temperatures above 29 ℃ showed a plastral pattern resembling that ofC.c.cyclornatawhile specimens incubated at lower temperatures show the typical plastral pattern ofC.c.annamiticaas described above. The plastral forelobe in specimens with theC.c.cyclornatapattern darken with age and become predominantly black at an age of about five years. This variation caused by incubation temperature has not been observed in eitherC.c.cyclornataorC.c.meieri， and thus seems to be unique forC.c.annamitica. The chin coloration is not significantly affected by dietary components such as carotenoids and even remains unchanged in specimens that are in captivity since decades (Meier pers. comm.， Blanck pers. obs.)， the same is true for captive bred specimens， even if they are raised on a diet low in carotenoids. When hybridizingC.c.cyclornataandC.c.annamiticawith each other， 50% of the offspring resembleC.c.cyclornataand 50%C.c.annamitica. The plastral pattern in these specimens stays constant and does not darken in the specimens resemblingC.c.cyclornata.

Fig. 15 Adult female Cuora cyclornata annamitica from Huong Khe district， Ha Tinh province (Photo by Mai Van Que)

2.2Cuoratrifasciataluteocephalassp.nov.

ZooBank LSID： urn：lsid：zoobank.org：act： 6252307C-37E7-4959-87C0-D69A81202612

Holotype： Chengdu Institute of Biology (CIB) 64III5279 a nearly adult female (Fig. 16) from China， Hainan province， Dan county， 300 m elevation，152 mm SCL， collected 18 May 1964 by Prof. Ermi Zhao in a stream.

Paratypes： American Museum of Natural History (AMNH) 30126-30153， all from Nodoa (Danzhou)，Hainan collected by Pope 1922/1923； FMNH6614-6621 same as AMNH origin； Museum of Comparative Zoology Harvard (MCZ) 20689 same as AMNH； Museum of Vertebrate Zoology， Berkeley (MVZ) 23931 from Kachek (Qionghai)， Hainan， collected by Gressit on 06 August 1935； MVZ23932 from Dwa Bi (Tai Pin)， Hainan collected by Gressit on 24 July 1935； CIB64III6083 & CIB64III6845 from Lingshui Li aut. County， Hainan collected by Zhao in 1964； and CIB64III6532 & CIB64III6533 from the same locality， data and collector as the holotype.

Fig. 16 CIB64III5279， the holotype of Cuora trifasciata luteocephalain (A) dorsolateral， (B) dorsal and (C) ventral view(Photo A & B by Zhou Ting, photo C by Hou Mian)

Diagnosis： A large subspecies ofC.trifasciatawith a maximum SCL of 26 cm， distinguished from its congeners by the combination of the following characters： 1) a fine dark brown to grayish spotted pattern (usually forming fine darkish diagonal strips on the costal and vertebral scutes) is present on the otherwise brown carapace (also appearing in juvenile nominate specimens which apparently lose this pattern with age； never occurring inC.cyclornata)； 2) usually fine blackish， often slightly radiating black diagonal stripes extending from the dorso-posterior part of the costal scute， always connected to the dorsolateral longitudinal black stripe on each side， to the bottom front third of the costal scute (occasionally also occurs in mainland specimens， but these usually exhibit a much wider non-radiating stripe；C.cyclornatadoes not exhibit this pattern)； 3) an intense yellow dorsal head pattern and a yellow chin [nominate specimens usually have a duller straw yellow pattern sometimes with an olive sheen and always with a darker (olive brownish) pattern at the tip of the nose； the chin in nominate specimens is cream-white to yellow， milder than inC.t.luteocephala； the dorsal head pattern ofC.cyclornatais citrine-brownish and the chin is white (C.c.meieri) or orange]； 4) the amount of black pattern on the head is well developed， with most specimens exhibiting a pronounced V shaped dorsal yellow head pattern bordered by black (usually less developed in the nominate subspecies)； 5) the limbs and soft parts are orange to pinkish with varying intensity in both subspecies， but inC.t.luteocephalathey can occasionally also be yellow， a pattern uniquely occurring in this subspecies； 6) the humerals are mainly black with a yellow anterolateral border along the periphery， involving approximately 15% of the scute’s lateral border forming a dent-like shape (nominate specimens can exhibit a rather similar pattern， where a fine yellow stripe is formed along the anterolateral area of the humeral scutes， but the dent-like shape is not formed， rather a longitudinal stripe； nominate specimens often also show an entirely black humeral scute)； 7) males usually exhibit a darker brown ground color of the carapace than females (not yet observed in the nominate subspecies)； 8) Maximum carapace width occurs at marginal 7/8-8 in females， at M8-M8/9 in males， and at M8 in juveniles (M7-7/8 in females and M7/8-8 in males in the nominate subspecies)； 9) adult males tend to have a flared posterior carapace (not present in mainland specimens)； 10) carapace dorsally more elongated (pear-like shape in the nominate subspecies and also more highly domed with a steeper decreasing angle laterally).

Large males are 18-23 cm SCL， females 20-26 cm SCL； in the nominate subspecies males are 15-21 cm SCL and females 16-23 cm SCL.

Description of holotype： CIB64III5279 is a moderately large female， 152 mm in straight carapace length. The maximum carapace width is 109 mm at marginal scute 7. Maximum carapace height 56 mm. Plastron length 153 mm. The carapace is light brown with the typical dark brownish fine spotted pattern on each carapace scute. The three black longitudinal stripes are fully developed. The two dorsolateral black stripes extend from the first to the fourth costal scutes. A finely radiated diagonal black stripe is present on costal one and two. The black pattern on the dorsolateral head is well developed and forms a V shape. The plastron shows some yellow radiating patches along the central seams. The humeral scutes are 98% black with a fine anterior yellow patch present.

Derivatio nominis： Derived from the latin word “luteus” meaning golden-yellow and the greek “κεφαλ(cephale)” meaning head； i.e.， golden-yellow head， referring to the intensely golden-yellow head pattern.

Distribution：Cuorat.luteocephalais currently only known from the mountainous areas on Hainan Island， China. It might also (have) occur(ed) in southeastern Guangxi Zhuang Autonomous Region， China， but no specimen ofC.trifasciatahas been seen in the area since the early 1980’s. Previously Mell (1938) mentioned that specimens from south of the West (Xi) River of the Pearl River system were different from specimens to the north. TwoC.trifasciata(MNHN9102 & 9103) in the MNHN collected from Macao， China by Fontanier in 1850， presumably from a market and at the same time， represent both subspecies as was shown by Tiedemannetal. (2014). Since at that time the species was only traded from the mainland (Zhao， pers. comm.) it strongly indicates the occurrence ofC.t.luteocephalasouth of the Xi River. The Hainan Turtle fauna is often regarded as comprising intermediate varieties of species/subspecies from Vietnam and southern China， as has been suggested forC.galbinifrons(Bourret， 1939) by Lehretal. (1998)， based on market specimens. According to Blanck (2013)，C.galbinifronson Hainan is very similar to mainland specimens occurring in North Vietnam， northern Laos and southwest Guangxi， China， and is by no means intermediate toC.bourreti(Obst & Reimann， 1994) as hypothesized by Lehretal. (1998). This was also shown by genetic studies (Stuart & Parham， 2004). Shietal. (2008) found a similar pattern with the genusSacalia； specimens from Hainan were genetically similar to specimens from eastern Guangxi Zhuang Autonomous Region and southwestern Guangdong province. This is likely also true for HainanC.trifasciata. In general the distributional pattern of the genusSacaliaand its genetic variation reflect the distribution pattern ofC.trifasciataandC.cyclornatawhich are sympatric withSacaliasp. across their ranges. The effects of the last ice age in Asia need to be evaluated further in order to understand the complex distributional patterns of East Asian turtle species. Hainan currently seems to harbor a general gene pool of several turtle species/subspecies that also occurred in the now heavily depleted areas of southern Guangdong province and Guangxi Zhuang Autonomous Region， China. Hainan was connected to the Chinese mainland during the last ice age until about 17 000 years ago (Voris， 2000). The connection to Vietnam was disconnected many times during the last ice age， generally ended earlier， and furthermore， it was intersected by the Red River paleo drainage basin， a potential barrier (Voris， 2000). Thus， it is far more plausible that most of the Hainan fauna dispersed to the island from the adjacent Chinese mainland.

Remarks：C.t.trifasciataandC.t.luteocephalaare not as easy to differentiate on morphometric and colorimetric grounds as are the different subspecies ofC.cyclornata， despite them being genetically further separated (making this new subspecies slightly cryptic). This has also been described in other species/subspecies. For example，Emystrinacris(Fritzetal.， 2005， 2006) andE.orbiculariswhich are morphologically nearly indistinguishable， but are genetically distinct at the species level. While the pattern is very constant withinC.t.luteocephala， the nominate subspecies can exhibitluteocephala-like traits in some specimens； however， the majority are easy to differentiate based upon the given diagnosis， especially when combining several of the diagnostic characters. It seems possible that the nominate subspecies still harbors a wide morphologic variability lacking in the Hainan population. This seems especially true in Hong Kong (compare Blancketal.， 2006) where specimens are more often quite similar to Hainan specimens in color (but not morphometrics and size) than on the remaining mainland. Still， Hong Kong animals are genetically clearly assignable to the nominate subspecies. In order to distinguish the subspecies at least three of the above mentioned diagnostic characteristics should be present to assign specimens morphologically.

2.3Cuoraaurocapitatadabieshanissp.nov.

ZooBank LSID： urn：lsid：zoobank.org：act： E4A9421F-475D-4C31-B9EA-B841897E987E

Holotype： Natural History Museum of Vienna (NMW) 32987： 2-a large male (Fig. 17) from Anhui province， China. 110.4 mm SCL， donated by Heinz Weissinger in 1993.

Diagnosis： A large subspecies ofC.aurocapitatawith a maximum SCL of 195 mm， distinguished from the nominate subspecies by the combination of the following characters： 1) the carapace color pattern differs from the nominate subspecies in that vertebral and costal scutes 2 to 4 are dark brown to greyish black (olive-brown to orange-reddish， usually with a black line along the seams， in the nominate subspecies)； carapace ground color generally darker than in the nominate subspecies； 2) the plastral pattern forms a interconnected black pattern closely resembling that ofC.panibut differentiated from it by the general lack of the black intergular stripe always present inC.pani(Blanck & Tang， 2005)， while the nominate subspecies exhibits a black pattern consisting of irregularly shaped and not fully interconnected black stripes and blotches commonly referred to by Chinese as a “bamboo pattern” (i.e.， resembling bamboo leaves)； 3) the carapace ofC.a.dabieshaniis slightly more domed than in the nominate subspecies； 4) the head pattern usually shows some

Fig. 17 NMW32987： 2， the holotype of Cuora aurocapitata dabieshaniin (A) lateral， (B) dorsal and (C) ventral view

brown areas and more greyish-black stripes than in the nominate subspecies， which can often exhibit a nearly completely golden yellow head； and 5) each ventral marginal scute with a triangular black spot often interconnecting to a black band on M2-M6/7 but with single triangles on all remaining scutes (in the nominate subspecies no triangles are present， some show a faint black stripe on some posterior marginals， but a black band extending from M2 to M6/7 is always present in this subspecies).

Large males are 11-13.5 cm SCL， females 14-19.5 cm SCL； males of the nominate subspecies are10-12.5 cm and females 13-16 cm SCL.

Description of holotype： NMW 32987：2； A large male with a SCL of 110.4 mm， a maximum carapace width of 74.5 mm and a plastron length of 102.1 mm. The carapace is dark greyish-black with dark reddish-brown spots on vertebrals 2 and 3 as well as costals 2. The horn-colored plastron shows a black bar along each scute seam except the intergular seam. The black pattern along the interhumeral seam is very thin compared to all other black bars. A black triangular spot is present on each marginal scute. Head yellowish with a darker blotch behind the eyes surrounded by fine black stripes.

Derivatio nominis： The subspecies namedabieshani(from Dabie Shan) refers to the distribution of this subspecies within the Dabie Shan Mountain range in Anhui province， China.

3 Distribution

This subspecies seems to be restricted to the Dabie Shan Mountain range extending north of the Yangtze River in Anhui province and into adjacent Henan and Hubei provinces. The adjacent Tongbai Shan in the north is inhabited byC.pani(Huang & Huang， 2011；Blanck pers. obs.). Detailed distribution records are absent so far and require further surveys. The nominate subspecies is confined to the Jiuhua Shan and Huang Shan Mountain ranges south of the Yangtze River in China’s Anhui province.

Remarks：C.aurocapitatais functionally extinct in the wild despite estimates by Zhang & Wu (2005， 2006) that about 400 specimens could remain in the wild. Blanck and Zhang surveyed the last known habitats of this species in the Jiuhua Shan range in 2013 and were unable to find any natural habitat remaining. The rivers and streams have been heavily impacted by sand mining and dam building， etc. A few scattered individuals seem to remain but a viable population is no longer existent. The status in the Dabie Shan range has not really been assessed yet. As was reported recently， the morphometrically and colorimetrically closely related but genetically well-separatedC.paniis not only restricted to the Qin Ling Mountain range as it was long believed (see Blanck & Tang， 2005)， but also occurs in the Tongbai Shan range which borders directly to the Dabie Shan in the north (Huang & Huang， 2011； Blanck pers. obs.). HenceC.panimay possibly occur in northern Dabie Shan， and an intergradation zone withC.aurocapitatamight occur. The plastral pattern differences between the two subspecies ofC.aurocapitataare not temperature nor sex induced based on captive breeding efforts (Meier， pers. comm.)， but the “bamboo” pattern of the nominate subspecies is colorimetrically dominant when the two subspecies are hybridized in captivity.

4 Final Remarks

The description of these three new subspecies has a critical impact on the conservation of these three already critically endangered species and should act as a basis for the management of studbooks for these taxa and potential future release projects. To our knowledge a large majority of specimens maintained in studbooks are already being maintained and bred following the patterns of color differentiation already recognized many years ago (Meier， 1997； Blanck & Tang， 2005； Blancketal.， 2006) and finally described herein. However many facilities and private keepers still mix specimens of the different subspecies producing offspring that are unsuitable for potential future release programs and also form a major danger to studbooks if such specimens are not correctly identified and thus included into such breeding programs. First steps are already in preparation forin-siturelease projects and it is of highest importance that only 100% pure specimens are released into their native habitats where still a few lonely survivors might have escaped human collection. This subspecies description further clarifies the variability within the genusCuoraand provides a key for identifying specimens of unknown provenance， as it is very common with East Asian turtle species that， due to their rarity in the wild， originate from the pet or food trade without any further data.

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NewSubspeciesofCuoracyclornata(Blanck，Mccord&Le，2006)，Cuoratrifasciata(Bell，1825)andCuoraaurocapitata(Luo&Zong，1988)

Torsten Blanck1*， ZHOU Ting2， LI Yi3， Tomáš Protiva4， Paul Crow5， Ralph Tiedemann6

Recent genetic studies have revalidatedCuoracyclornataand shown that a third， yet undescribed genetic subgroup which can be differentiated colorimetrically and morphometrically from the previously recognized 2 subspecies. Similarly forC.trifasciataa divergent genetic group was identified that can be clearly assigned to the specimens from Hainan Island， China. The same is true forC.aurocapitatawhere a colorimetrical well-distinguished variety has long been known， and this is supported by microsatellite markers (Tiedemannetal.， 2014) and by morphometrics. Here we use geometric morphological methods for the discriminant function analysis. Both the genetic results and the morphometric analysis of the shell clearly support the distinctiveness betweenC.trifasciataandC.cyclornataand the undescribed subspecies. These3 groups are herein described as the new subspeciesC.cyclornataannamitica，C.trifasciataluteocephalaandC.aurocapitatadabieshani， respectively.

Testudines； Geoemydidae；Cuorassp. nov.； taxonomy； Vietnam； China； microsatellite； morphometrics

2016-09-16接受日期：2017-04-26

*通信作者Corresponding author， E-mail：cuora_yunnanensis@yahoo.com

10.11984/j.issn.1000-7083.20160249

Q959.6

： A

： 1000-7083(2017)04-0368-18