The leaf turtle population of Phnom Kulen National Park (northwestern Cambodia) has genetic and morphological signatures of hybridization

Introduction

Until recently, the Southeast Asian leaf turtle genus Cyclemys (Testudines: Geoemydidae) was thought to contain only one or two species, Cyclemys dentata (Gray, 1831) and C. tcheponensis (Bourret, 1939). However, starting in the mid-1990s, several studies resurrected and described additional leaf turtle species (Fritz et al. 1996, 1997, 2008; Iverson and McCord 1997; Stuart and Fritz 2008), so that currently seven distinct species are recognized (van Dijk et al. 2014). Cambodia is thought to harbour three species: C. atripons Iverson and McCord, 1997; C. oldhamii Gray, 1863 and C. pulchristriata Fritz et al., 1997; although the exact distribution ranges are not well understood (van Dijk et al. 2014). Yellow-bellied leaf turtles from southwestern Cambodia represent C. atripons, while yellow-bellied leaf turtles from the southeast of the country belong to C. pulchristriata, a species found mainly in Vietnam. Dark-bellied leaf turtles from the inland regions of Cambodia are assigned to C. oldhamii (Iverson and McCord 1997; Fritz et al. 2008; Stuart and Fritz 2008; van Dijk et al. 2014).

Leaf turtles are among the more frequently encountered turtle species in Cambodia, as indicated both by wildlife surveys and confiscations from the illegal wildlife trade (Som et al. 2006; Som and Kheng 2007; TRAFFIC 2014). They can be found in various freshwater habitats, including rivers and streams, swamps, grassland ponds and flooded rice paddies. Adult leaf turtles also utilize terrestrial habitats nearby freshwater (Durkin 2012). Leaf turtles are heavily traded and common in seizures of smuggled wildlife en route from Cambodia to food and traditional medicine markets in Vietnam, indicating that wild populations continue to be depleted. In 2013, all Cyclemys species were placed onto CITES Appendix II (CITES 2013). In Cambodia, confiscated turtles are often released (pers. observ.). To avoid introduction of non-native turtles with the risks of competition and hybridization, the exact knowledge of the local distribution of the different leaf turtle species is of great importance.

Durkin et al. (2010) reported dark-bellied leaf turtles from Phnom Kulen National Park in northwestern Cambodia which they tentatively identified as C. aff. atripons. However, the turtles' morphology conflicted with this determination, and based on coloration and pattern, Kim (2011) and Durkin (2012) assigned the Phnom Kulen population to C. oldhamii. Phnom Kulen National Park is located in between the known distribution ranges of C. atripons and C. oldhamii and is geographically isolated in terms of Cyclemys records. Thus, the taxonomic identity of the Phnom Kulen population remains unclear. In this study, we compare Cyclemys samples from Phnom Kulen National Park with previously published mitochondrial DNA (mtDNA) sequence data of all currently recognized Cyclemys species. In addition, we compare the Phnom Kulen turtles with representatives of C. atripons by genotyping them at 17 unlinked microsatellite loci and compare their coloration and pattern with C. atripons and C. oldhamii.

Materials and Methods

Results

Mitochondrial relationships

All individuals of the Phnom Kulen population yielded the same mitochondrial haplotype (k1; Fig. 1; Appendix) which differed in parsimony network analysis by seven mutational steps from the haplotypes of Cyclemys atripons. However, among the haplotypes of C. atripons, up to 12 mutations were observed (Fig. 1). Accordingly, in ML analyses, the haplotype of the Phnom Kulen population clustered with maximum support with the sequences of C. atripons and within this clade was sister to the remaining haplotypes (Fig. S1).

Coloration and pattern

The leaf turtles from Phnom Kulen National Park had straight carapace lengths from 5.8 to 21.0 cm. All corresponded in coloration and pattern perfectly to the eastern coloration morph of Cyclemys oldhamii as described in Fritz et al. (2008). Phnom Kulen leaf turtles had a speckled pattern on the crown of the head and salmon striping on the neck on a dark background. The throat typically showed a mottled dark pattern on salmon background. The carapace of Phnom Kulen turtles was generally dark brownish. The plastron showed in the smallest individuals a spotted pattern on yellow background. With increasing size and age, this pattern was replaced by densely radiating dark streaks on each plastral scute. Aged individuals showed a gradient of patterns from dense dark brown radiations on a yellow background to a uniformly dark coloured plastron, as typical for C. oldhamii (Fig. 2).

Discussion

The taxonomy and species number of Southeast Asian leaf turtles was recently much in flux, with several species described as new to science or resurrected from synonymy (Fritz et al. 1996, 1997, 2008; Iverson and McCord 1997). The leaf turtles of Phnom Kulen National Park morphologically closely resemble Cyclemys oldhamii, a dark-bellied leaf turtle species which has been recorded at a distance of approximately 190 km from the national park in the Prey Long forest of central Cambodia (Som and Kheng 2007). Yet, in phylogenetic analyses the mitochondrial haplotypes of the Phnom Kulen turtles do not cluster with C. oldhamii (Fig. S1), but with C. atripons, another leaf turtle species that is morphologically clearly distinct and known to occur in the Cardamom Mountains of southwest Cambodia (Stuart and Platt 2004; Som et al. 2006). The nearest unambiguous record of C. atripons to Phnom Kulen National Park are two specimens collected approximately 175 km away in Phnom Samkos Wildlife Sanctuary in the Cardamom Mountains and deposited at the Centre for Biodiversity Conservation in Phnom Penh under collection numbers CBC 00576 and CBC 01515. However, a questionable extraterritorial record for C. atripons has recently been published from Krong Samraong District in northwestern Cambodia (Brakels et al. 2016), approximately 90 km from the park.

Cyclemys atripons belongs to a phylogenetically well-supported clade comprised of three yellow-bellied species, C. atripons, C. dentata and C. pulchristriata. Cyclemys atripons is the sister taxon to C. dentata, a species distributed across the Great Sunda Islands and the Malay Peninsula (Fritz et al. 2008). The leaf turtles from Phnom Kulen National Park harbour mitochondrial haplotypes of C. atripons, but our analyses of microsatellite DNA provide evidence that they are genetically distinct from the latter species. Thus, we can dismiss the hypothesis that the Phnom Kulen turtles belong to the species C. atripons and that this species is polymorphic. Considering that the Phnom Kulen turtles are morphologically indistinguishable from C. oldhamii, it seems reasonable to hypothesize that the Phnom Kulen population represents a hybrid swarm of C. atripons and C. oldhamii. This hypothesis is also supported by the pronounced linkage disequilibrium found in the Phnom Kulen turtles and by the lack of Hardy–Weinberg equilibrium at several loci (Tables 1 and 2), as typical for hybridized populations. That also some loci of C. atripons are not in Hardy–Weinberg equilibrium is not astonishing because our sample of this species is very small and does not represent a natural population (unlike the Phnom Kulen turtles), but an assemblage of individuals from several distinct populations.

The illegal wildlife trade in Southeast Asia is extensive, and confiscations over the last two decades have included many hundreds of kilograms of live turtles and tortoises being smuggled from Cambodia to Vietnam and China for the food and traditional medicine markets (Compton 2000; own unpubl. observ.). Seizures of live animals are often released back into the wild without knowledge of their origin. For example, in 2008, 419 kg of live pythons and turtles of uncertain origin confiscated in Battambang, Cambodia, were released to the wild, including the Tonle Sap lake (TRAFFIC 2014). This practice has obvious implications for the genetic integrity of species and can threaten the persistence of morphologically or genetically distinct populations through gene pool mixing (Laikre et al. 2010). In certain conservation contexts, a low level of assisted immigration can produce fitness benefits for populations with outbreeding depression (‘genetic rescue’; Tallmon et al. 2004), but for species that do not require immediate conservation intervention, gene pool mixing can erode evolutionarily significant units (ESUs) and complicate baseline taxonomic work. While to our knowledge no bulk releases of leaf turtles have occurred in Phnom Kulen National Park, the study site Kbal Spean is a culturally significant site, with a history of ceremonial human presence dating back over 1000 years (Freeman et al. 1999). In Cambodia, the tradition of releasing captured or purchased turtles for religious reasons persists today (Ihlow et al. 2016). Whether releases of turtles occurred at Kbal Spean over the centuries due to human intervention is unknown. However, it is unlikely that the genetic situation found in the Phnom Kulen population is caused by the translocation of a few alien leaf turtles because the entire population seems affected. All studied leaf turtles were genetically similar in that they harboured the same mitochondrial haplotype and were assigned to the same cluster in structure analyses. In addition, both the Phnom Kulen population and C. atripons possess many private alleles (Table 2). This would not be expected if the Phnom Kulen population is derived from recent hybridization because in the latter case, a high number of shared alleles with C. atripons would be expected.

Unfortunately, no samples of Cambodian C. oldhamii were available for genetic examination, and our conclusion that the Phnom Kulen turtles are a hybrid swarm remains somewhat speculative. In particular, the inferred genetic impact of C. oldhamii is backed only by morphology. However, morphology alone is very convincing in this case and introgressed mitochondria have been previously described for another pair of yellow-bellied and dark-bellied Cyclemys species, C. dentata and C. enigmatica. The yellow-bellied C. dentata occurs throughout its distribution range sympatrically with a dark-bellied leaf turtle of which only few individuals have been characterized genetically (Fritz et al. 2008). All of these turtles yielded mitochondrial haplotypes of C. dentata, while nuclear genomic markers were distinct, which is why Fritz et al. (2008) described these dark-bellied turtles as the distinct species C. enigmatica. Thus, we also cannot exclude the possibility that the Phnom Kulen turtles represent an undescribed species with introgressed mitochondria from C. atripons. However, for the time being we refrain from taxonomic consequences. Further research, including extensive sampling of other leaf turtle populations from Cambodia, especially of C. oldhamii, is needed to clarify this situation.

Our study highlights that the taxonomy of leaf turtles is still incompletely known and that additional investigations will contribute to a significantly better understanding of their taxonomic and genetic diversity. Even though our results do not support the hypothesis that the observed genetic constitution of the leaf turtle population in Phnom Kulen National Park has been noticeably impacted by the introduction of leaf turtles from elsewhere, we provide evidence for a complicated genetic variation in wild populations that may become increasingly threatened by translocations of confiscated individuals.