Platysternon Research Articles

Threatened Species of Amphibians and Reptiles from Vu Quang National Park and Their Conservation Values

Based on five field surveys in 2023 and 2024, we herein provide a list of 24 threatened species of amphibians and reptiles from Vu Quang National Park, Ha Tinh Province. Among them, four species are listed in the Governmental Decree No. 64/2019/ND-CP, 13 species are listed in the Governmental Decree No. 84/2021/ND-CP, 13 species are listed in the CITES Appendices, 17 species are listed in the Vietnam Red Data Book (2007), and 13 species are listed in the IUCN Red List (2024). Seven species with the highest level of conservation priority due to their small populations are Python molurus, Ophiophagus hannah, Platysternon megacephalum, Cuora cyclornata, Cuora galbinifrons, Sacalia quadriocellata, and Indotestudo elongata. The major threat to the herpetofauna of the national park is illegal hunting.

Fine-scale geographic difference of the endangered Big-headed Turtle (Platysternon megacephalum) fecal microbiota, and comparison with the syntopic Beale’s Eyed Turtle (Sacalia bealei)

BackgroundStudies have elucidated the importance of gut microbiota for an organism, but we are still learning about the important influencing factors. Several factors have been identified in helping shape the microbiome of a host, and in this study we focus on two factors—geography and host. We characterize the fecal microbiota of the Big-headed Turtle (Platysternon megacephalum) and compare across a relatively fine geographic scale (three populations within an 8-km radius) and between two syntopic hosts (P. megacephalum and Sacalia bealei). Both species are endangered, which limits the number of samples we include in the study. Despite this limitation, these data serve as baseline data for healthy, wild fecal microbiotas of two endangered turtle species to aid in conservation management.ResultsFor geography, the beta diversity of fecal microbiota differed between the most distant sites. The genus Citrobacter significantly differs between sites, which may indicate a difference in food availability, environmental microbiota, or both. Also, we identify the common core microbiome for Platysternon across Hong Kong as the shared taxa across the three sites. Additionally, beta diversity differs between host species. Since the two species are from the same site and encounter the same environmental microbiota, we infer that there is a host effect on the fecal microbiota, such as diet or the recruitment of host-adapted bacteria. Lastly, functional analyses found metabolism pathways (KEGG level 1) to be the most common, and pathways (KEGG level 3) to be statistically significant between sites, but statistically indistinguishable between species at the same site.ConclusionsWe find that fecal microbiota can significantly differ at a fine geographic scale and between syntopic hosts. Also, the function of fecal microbiota seems to be strongly affected by geographic site, rather than species. This study characterizes the identity and function of the fecal microbiota of two endangered turtle species, from what is likely their last remaining wild populations. These data of healthy, wild fecal microbiota will serve as a baseline for comparison and contribute to the conservation of these two endangered species.

Microhabitat characteristics of the critically endangered big-headed turtle Platysternon megacephalum.

Understanding the microhabitat requirements of an animal is vital for ensuring the success of targeted conservation and microhabitat restoration measures. The big-headed turtle (Platysternon megacephalum) is a freshwater species that is distributed across Southeast Asia. Owing to the human threats posed by illegal pet trade and overharvesting for food and medicinal purposes, the species has undergone rapid decline. However, in Hainan, their microhabitat characteristics are still unknown, which is neither conducive to the conservation of the species nor to the establishment of the Hainan Tropical Rainforest National Park. This study examined the microhabitat characteristics of P. megacephalum using sample plot methods in the Diaoluo Mountain area of Hainan Tropical Rainforest National Park. Our results indicated that P. megacephalum prefers stream microhabitats with rocky substrates, several caves, and a high diversity of food sources. Microhabitat characteristics did not differ significantly between adults and juveniles. Our results suggest that protecting microhabitats and main food sources is important for the conservation of P. megacephalum. Our findings provide a reference for the protection of this species in Jianfeng Ridge, Yingge Ridge, and other areas in Hainan Tropical Rainforest National Park.

Genetic Diversity of the Critically Endangered Big-Headed Turtle (Platysternon megacephalum) Based on Wild and Traded Samples: Implications for Conservation

Overexploitation has driven catastrophic declines in most turtle species in Asia. Many turtles are seized annually by law enforcement officers; unfortunately, seized turtles often lack associated information on their provenance. There is also taxonomic confusion regarding the clear delineation of turtle species and subspecies in Asia. This lack of provenance data and taxonomic uncertainty is problematic as it may preclude release, make appropriate release sites hard to select, or result in genetic pollution when animals are released. In this study, we amassed and analyzed the largest number of genetic samples of the critically endangered big-headed turtle (Platysternon megacephalum) to date. Our phylogenetic and network results based on nearly 500 sequences of a mitochondrial ND4 fragment corroborate the earlier hypothesis that only two of the three currently recognized subspecies have strong molecular support and that there is greater genetic structuring within one subspecies than has been previously reported. The vast majority of P. megacephalum confiscated from the trade in Vietnam belong to Subclade 3 of P. m. peguense, and this subclade is the most broadly distributed taxon of the big-headed turtle in Vietnam based on samples with known localities. Nonetheless, for the other two subclades, all their samples originate from the trade and could not be assigned to any geographic provenance because of the lack of wild-caught samples. In addition, our results reveal two trade centers of the species, Quang Ninh Province on the border between China and Vietnam and Kon Tum Province in the Central Highlands. However, smaller animal seizures occur across the range of the species in Vietnam. While the data presented here have facilitated the selection of appropriate release sites for confiscated P. megacephalum, the significant costs of genetic screening and the shortage of samples from wild animals with good localities hinder the rewilding efforts, especially for conservation practitioners working in countries where conservation activities are not well funded and research on poorly studied species is still limited. Nevertheless, we urge that wildlife translocations include robust pre-translocation genetic screening to ensure that all individual animals are reintroduced at appropriate sites.

Genomic analyses reveal three phylogenetic species and their evolutionary histories in the big-headed turtle

The critically endangered big-headed turtle (Platysternon megacephalum) is currently classified into three subspecies. However, the classification is still controversial and their evolutionary histories are still unclear. Here, multiple genetic analyses consistently revealed three phylogenetic groups with substantial genetic divergences and distinct demographic histories, suggesting three phylogenetic species (P.megacephalum, P.peguense, and Baise clade). Phylogeographical analyses revealed that the Red River plains and Guangxi basins are largely coincident with the boundaries between the three phylogenetic species, highlighting the key role of lowland areas in driving speciation in the big-headed turtle. The Baise clade is characterized by high-linkage disequilibrium but the lowest effective population size, indicating that the cryptic phylogenetic species is more vulnerable to human activities and environmental disturbance, and urgently needs more protection. Our findings provide fundamental insights into the taxonomy and scientific conservation of the family Platysternidae.

Genomic insights into the genetic basis of eagle-beak jaw, large head, and long tail in the big-headed turtle.

The big-headed turtle (Platysternon megacephalum) is an endemic chelonian species in Asia. Unlike most other turtles in the world, P. megacephalum is characterized with eagle-beak jaw, large head, and long tail. Although these unique characteristics are well recognized, the underlying genetic basis remains largely elusive. Here, we performed comparative genomic analysis between P. megacephalum and other representative species, aiming to reveal the genetic basis of the unique morphological features. Our results revealed that the eagle-beak jaw is most likely enabled by combined effects of expansion of SFRP5, extraction of FGF11, and mutation of both ZFYVE16 and PAX6. Large head is supported by mutations of SETD2 and FGRF2 and copy number variations of six head circumference modulation-related genes (TGFBR2, Twist2, Rdh10, Gas1, Chst11, and SNAP25). The long tail is probably involved in a genetic network comprising Gdf11, Lin 28, and HoxC12, two of which showed a consistent expression pattern with a model organism (mice). These findings suggest that expansion, extraction, and mutation of those genes may have profound effects on unique phenotypes of P. megacephalum.

An assessment of turtle communities in Bach Ma National Park, Vietnam

Most of the extraordinary biodiversity richness of South-East Asian countries is concentrated in the national parks and other Protected Areas, with species-rich turtle communities surviving mostly in these reserves where their natural habitats are better preserved. However, very few studies have documented the turtle species richness of the various Protected Areas in Vietnam, which is not only one of the hotspots of chelonian diversity in the world but also one of the countries where their exploitation is the highest. Here, the diversity of turtles is studied in the Bach Ma National Park in central Vietnam, mainly characterised by forested hills and mountains, with ponds and streams of various shapes and structures. The study was carried out by conducting (i) semi-structured interviews of hunters recruited through a snow-ball procedure in local villages, (ii) inspection of turtle individuals in their hands, (iii) field surveys along random transects inspecting the various microhabitats used by these reptiles. We observed a total of 15 species, out of which 14 (93.3%) are threatened, based on IUCN Red List. A natural hybrid of Cuora bourreti × C. mouhotii obsti (Cuora «serrata») was observed. Three species (Cuora bourreti, Manouria impressa, Platysternon megacephalum) were the most frequently encountered species according to our interviewees. We conclude that, in order to enhance the conservation status of the turtle communities at the local level, it would be important (i) to increase the number of rangers patrolling the study area in the rainy season (from April to September) and (ii) to list the hunter's names in the villages surrounding the Bach Ma National Park and to convince them to sign an agreement with the local authorities to avoid hunting turtles within the Protected Area.

A population genetic analysis of the Critically Endangered Madagascar big-headed turtle, Erymnochelys madagascariensis across captive and wild populations

Erymnochelys madagascariensis is a Critically Endangered turtle endemic to Madagascar. Anthropogenic activity has depleted the wild population by 70% in the last century, and effective conservation management is essential to ensuring its persistence. Captive breeding was implemented to augment depleted populations in the southern part of Ankarafantsika National Park (ANP), when no genetic data were available for E. madagascariensis. It is unknown how much of the natural population’s diversity is encapsulated in captivity. We used eight microsatellite loci and fragments of two mitochondrial genes to identify the genetic structure of E. madagascariensis in the wild. Captive bred turtles were compared with wild populations in order to assess the representativeness of this ex situ conservation strategy for ANP. Six microsatellite clusters, ten cytochrome b, and nine COI haplotypes were identified across wild populations, with high genetic divergence found between populations in two groups of watersheds. Captive bred individuals represent three out of six sampled microsatellite clusters found in the wild and just one mitochondrial haplotype, possibly due to genetic drift. To improve genetic representation, the strategy of frequent interchange between captive and wild breeders within ANP should be revitalised and, as originally planned, hatchlings or juveniles should not be released beyond ANP.

Temporal and Spatial Stability on the Population Structure of Consumed and Illegally Traded Big-Headed Amazon River Turtle in the Negro River Basin, Central Amazon, Brazil

Freshwater turtles are a valuable food resource for riverine human communities and have been historically overharvested throughout all major tropical large river basins, with consequent gradual population decreases. Even species considered to be abundant are declining, and in many cases were brought to a condition of near extinction. The collection of adult females during breeding season on nesting beaches is considered a major factor in population decline and subsequent loss of food sources for humans. There is growing consensus that adult females constitute the category which turtle populations can least afford to lose. In the Negro River Basin, the podocnemidid big-headed Amazon River turtle,Peltocephalus dumerilianus, is heavily exploited for consumption and poached for illegal trade among riverine communities and cities. Between 1997 and 2002 and in 2019, we measured live turtles and carapaces of big-headed turtles in the city of Barcelos and its surroundings, and among the riverine families living in the Jaú National Park. We compared body sizes and sex ratios between areas, periods, and between consumed and traded individuals. We found no differences between areas, even those close to Barcelos and the ones belonging to remote areas where pressure levels are lower. The individuals consumed in Jaú National Park are larger than those poached for illegal trade in both areas. There was an increase in average size between 1997 and 2002. Sex ratio was slightly skewed toward males, which were larger, and did not differ between areas and periods. Results indicate stability on size of harvested populations, which may be supporting current extraction levels. Data suggest this could be related to the absence of adult female capture during nesting for this species. We recommend protection strategies for other Amazon Podocnemidid species that focus on the protection of nesting beaches and surrounding areas where adults occupy, specifically in areas under communal protection.

Using eDNA techniques to find the endangered big-headed turtle (Platysternon megacephalum).

We evaluate the efficacy of environmental DNA (eDNA) techniques to locate wild populations and estimate the population size of the endangered big-headed turtle (Platysternon megacephalum) in Hong Kong. The results from this study are important for identifying priority sites for protection and further research. Additionally, we assess the impact of two environmental variables (temperature and pH) on eDNA quantity. We surveyed 34 streams for three years, sampling four times each year. Four new populations were first identified with eDNA analysis, and then verified by field surveys. Our multi-year survey highlights that eDNA detection can be inconsistent over time, even in streams with known populations. There was no significant relationship between eDNA quantity and the environmental variables tested. Lastly, our results suggest that eDNA methods remain promising to estimate population size, since number of positive detections were positively correlated with population size in streams with known populations. We conclude that eDNA methods are powerful, but care must be taken when interpreting field results as they are affected by species ecology and environmental conditions.

A Survey of Freshwater Turtles in Diaoluoshan Nature Reserve with Conservation Implications for the Endangered Big-Headed Turtle

We conducted population surveys of freshwater turtles in Diaoluoshan National Nature Reserve, Hainan Province, China, from 2012 to 2019, capturing 20 individuals belonging to 4 species, including 2 native species (big-headed turtle [Platysternon megacephalum] and Hainan four-eyed turtle [Sacalia insulensis]) and 2 alien species (red-eared slider [Trachemys scriptia elegans] and common snapping turtle [Chelydra serpentia]). We captured 12 big-headed turtles and estimated their population density (1.6514 individuals/km) and abundance (14 individuals), both of which indicate that Diaoluoshan National Nature Reserve is an important area for turtle conservation, especially for big-headed turtles.

Home-site fidelity and homing behavior of the big-headed turtle Platysternon megacephalum.

Site fidelity refers to the restriction of dispersal distance of an animal and its tendency to return to a stationary site. To our knowledge, the homing ability of freshwater turtles and their fidelity is reportedly very low in Asia. We examined mark–recapture data spanning a 4‐year period in Diaoluoshan National Nature Reserve, Hainan Province, China, to investigate the site fidelity and homing behavior of big‐headed turtles Platysternon megacephalum. A total of 11 big‐headed turtles were captured, and all individuals were used in this mark–recapture study. The site fidelity results showed that the adult big‐headed turtles (n = 4) had a 71.43% recapture rate in the original site after their release at the same site, whereas the juveniles (n = 1) showed lower recapture rates (0%). Moreover, the homing behavior results showed that the adults (n = 5) had an 83.33% homing rate after displacement. Adult big‐headed turtles were able to return to their initial capture sites (home) from 150 to 2,400 m away and precisely to their home sites from either upstream or downstream of their capture sites or even from other streams. However, none of the juveniles (n = 4) returned home, despite only being displaced 25–150 m away. These results indicated that the adult big‐headed turtles showed high fidelity to their home site and strong homing ability. In contrast, the juvenile turtles may show an opposite trend but further research is needed.

An alternative interpretation of the Paleogene turtle Cardichelyon rogerwoodi as a hinged kinosternoid

AbstractCardichelyon rogerwoodi is an enigmatic fossil turtle from the late Paleocene to early Eocene of North America. Previous analyses suggested affiliation with Testudinoidea, in particular the big-headed turtle Platysternon megacephalum, based on the presence of multiple musk-duct foramina and a large head. We here highlight previously undocumented characteristics for this turtle, notably the presence of short costiform processes, a rib-like axillary process, and a posterior plastral hinge. Phylogenetic analysis places Cardichelyon rogerwoodi within Testudinoidea, but the exclusion of testudinoids suggest an affiliation with Dermatemydidae. Using consilience with external data we favor placement within Kinosternoidea. Cardichelyon rogerwoodi is therefore an aberrant, hinged kinosternoid that developed in situ in North America during the Paleocene long before the arrival of testudinoids on this continent in the early Eocene.

Unique multiple paternity in the endangered big-headed turtle (Platysternon megacephalum) in an ex situ population in South China.

Understanding the mating system and reproductive strategies of an endangered species is critical to the success of captive breeding. The big‐headed turtle (Platysternon megacephalum) is one of the most threatened turtle species in the world. Captive breeding and reintroduction are necessary to re‐establish wild populations of P. megacephalum in some of its historical ranges in China, where the original populations have been extirpated. However, the captive breeding of P. megacephalum is very difficult and this may be due to its mysterious reproductive strategies and special behavior (e.g., aggressive temperament and territoriality). In this study, we achieved successful captive breeding of P. megacephalum by creating a habitat that mimics natural conditions and then investigated its mating system using microsatellite makers. A total of 16 clutches containing 79 eggs of P. megacephalum were collected, and 52 were hatched successfully over two breeding seasons. Of the 15 effective clutches, 6 clutches (40%) exhibited multiple paternity. There was no significant correlation between clutch size and multiple paternity, and no significant difference in hatching success between multiple‐sired and single‐sired clutches. However, there was significant correlation between male body size and the number of offspring, with higher‐ranked males contributing to more clutches. Our results provide the first evidence of multiple paternity and male hierarchy in P. megacephalum. These findings suggest that multiple paternity and male hierarchy should be considered in captive breeding programs for P. megacephalum, and creating a habitat that mimics natural conditions is an effctive way to achieve successful captive breeding and investigate the mating systems of this species.

Observations on carapace color change in the juvenile big-headed turtle (Platysternon megacephalum).

The carapace color of newborn big-headed turtles (Platysternon megacephalum) is polymorphic and usually consists of two phenotypes: yellowish brown and olive green. As the turtles grew, over the first year of life, its carapace gradually turned from yellowish brown to chestnut brown, or from olive green to dark brown, depending on the phenotype. Meanwhile, the turtle’s plastron remained an orange and black pattern and did not change much. In this study, we primarily used HE staining to observe the carapace color change with age in big-headed turtle juveniles. We took the carapace marginal scute tissues twice from the same turtles before and after the carapace color change. Histological observations show that in the marginal scutes of the four tested turtles with different carapace color phenotypes, melanin granules are all concentrated in the dermal layer underneath the dorsal corneous layer, but rarely on the ventral side. Melanin deposits in the dorsal corneous layer were found to increase as the corneous layers thickened, while the melanin deposits in the ventral corneous layer did not change significantly. However, there was no significant difference in melanin deposition in the epidermis and dermis of the carapace among the yellowish brown, chestnut brown, olive green, and dark brown big-headed turtles. The results of our study indicate that the carapace color darkening in big-headed turtles may not be due to changes in melanin content of the carapace, but is the result of melanin accumulation and superposition in the dorsal corneous layer.

The Complete Mitochondrial Genome of Platysternon megacephalum peguense and Molecular Phylogenetic Analysis.

Platysternon megacephalum is the only living representative species of Platysternidae and only three subspecies remain: P. m. megalorcephalum, P. m. shiui, and P. m. peguense. However, previous reports implied that P. m. peguense has distinct morphological and molecular features. The characterization of the mitogenome has been accepted as an efficient means of phylogenetic and evolutionary analysis. Hence, this study first determined the complete mitogenome of P. m. peguense with the aim to identify the structure and variability of the P. m. peguense mitogenome through comparative analysis. Furthermore, the phylogenetic relationship of the three subspecies was tested. Based on different tRNA gene loss and degeneration of these three subspecies, their rearrangement pathways have been inferred. Phylogenetic analysis showed that P. m. peguense is a sister group to (P. m. megalorcephalum and P. m. shiui). Furthermore, the divergence time estimation of these three subspecies coincided with the uplift of the Tibetan Plateau. This study shows that the genetic distances between P. m. peguense and the other two subspecies are comparable to interspecific genetic distances, for example within Mauremys. In general, this study provides new and meaningful insights into the evolution of the three Platysternidae subspecies.

Developing quantitative PCR assays to detect threatened and invasive freshwater turtles in Hong Kong using environmental DNA

Environmental DNA (eDNA) techniques show promise in facilitating the study and conservation of threatened species. The advantages of high sensitivity and high efficiency have been demonstrated by studies across various taxa. Asian turtles, one of the most threatened vertebrate groups, are facing extinction due to overexploitation for use as traditional Chinese medicine, food, and pets. Most species are extirpated across their ranges, but Hong Kong is unique because wild turtle populations persist. Due to the rarity and secretiveness of turtles, data on the location and size of populations are incomplete, even for a small place like Hong Kong. The result is that research and conservation are hindered. In this study, quantitative PCR eDNA assays were developed for three turtle species: two native [big-headed turtle (Platysternon megacephalum) and Chinese softshell turtle (Pelodiscus sinensis)] and one invasive [red-eared slider (Trachemys scripta elegans)]. For each assay, we tested species specificity, optimized reactions, and benchmarked analytical sensitivity for eDNA surveys. The assays will help in the conservation of Hong Kong turtles by identify priority sites for protection and research, provide another tool for population monitoring, and clarify the impact of the invasive T. s. elegans on native species.

Draft genome of the big-headed turtle Platysternon megacephalum

The big-headed turtle, Platysternon megacephalum, as the sole member of the monotypic family Platysternidae, has a number of distinct characteristics including an extra-large head, long tail, flat carapace, and a preference for low water temperature environments. We performed whole genome sequencing, assembly, and gene annotation of an adult male big-headed turtle based on the Illumina HiSeq X genomic sequencing platform. We generated ~497.1 Gb of raw sequencing data (×208.9 depth) and produced a draft genome with a total length of 2.32 Gb and contig and scaffold N50 sizes of 41.8 kb and 7.22 Mb, respectively. We also identified 924 Mb (39.84%) of repetitive sequences, 25,995 protein-coding genes, and 19,177 non-coding RNAs. We generated the first de novo genome of the big-headed turtle; these data will be essential to the further understanding and exploration of the genomic innovations and molecular mechanisms contributing to its unique morphology and physiological features.

Detecting coevolution of positively selected in turtles sperm-egg fusion proteins

Physically interacting sperm-egg proteins have been identified using gene-modified animals in some mammal species. Three proteins are essential for sperm-egg binding: Izumo1 on the sperm surface, and JUNO and CD9 on the egg surface. Most proteins linked to reproductive function evolve rapidly among species by positive selection, and have correlated evolutionary rates to compensate for changes on both the sperm and egg. Up to now, interactions between sperm and egg proteins have not been identified in non-mammalian vertebrates, such as turtles that have interspecific hybrids that can produce surviving F1 generations. To explore the potential physical interactions of sperm-egg proteins in turtle species, the coding region of Izumo1, JUNO, and CD9 homologous genes (named Tu-Izumo1, Tu-JUNO, and Tu-CD9) in six turtle species (Mauremys reevesii, M. mutica, M. sinensis, Cistoclemmys flavomarginata, Platysternon megacephalum and Chrysemys picta bellii) were identified, amplified, and sequenced, and tissue-specific expression was analyzed in M. reevesii. We constructed phylogenetic trees and analyzed the signatures of coevolution between sperm-egg protein pairs using MirrorTree Server and linear regression methods. The results showed that Tu-Izumo1, Tu-JUNO, and Tu-CD9 proteins have correlated evolutionary rates, and that the area where Tu-Izumo1 interacts with Tu-JUNO has only one positive selection site in some turtle species. These results suggest there is a potential interaction between Tu-Izumo1 and Tu-JUNO among turtles that can interbreed, and that a significantly lower positive selection in the interaction region may be one of the reasons why turtle hybrids are so common. Further studies are required to uncover Tu-Izumo1, Tu-JUNO and Tu-CD9 protein biological functions during gamete fusion.

A Review of the Fossil Record of North American Turtles of the Clade Pan-Testudinoidea

Turtles of the clade Pan-Testudinoidea have a rich fossil record in North America, including the Caribbean, ranging from the late Paleocene to the Holocene. All earlier reports cannot be substantiated herein. The earliest members of this clade probably immigrated in multiple waves from Asia. Current phylogenies of crown Testudinoidea recognize four primary clades: Pan-Emydidae, Pan-Geoemydidae, Pan-Testudinidae, and the lineage leading to Platysternon megacephalum. An updated global phylogeny allows attribution of fossils to these lineages with confidence that allows the discernment of new diversity trends and biogeographic patterns. The diversity of North American Pan-Testudinidae increased consistently throughout the Cenozoic and reached its peak in the early Miocene. The extinction of many testudinids at the end of the Pleistocene, however, decreased tortoise diversity toward its extant levels. The diversity of North American Pan-Emydidae and Pan-Geoemydidae shows opposite patterns. Pan-Emydidae are remarkably diverse today, but their diversity was low in the Eocene and only increased dramatically from the Oligocene and onwards. Pan-Geoemydidae, on the other side, were diverse in the late Paleocene to Eocene, but their diversity decreases to their extremely low present levels starting with the Oligocene. A taxonomic review of 191 named North and Central American pan-testudinoid taxa finds 57 nomina valida, 69 nomina invalida, 64 nomina dubia, and 1 nomen nudum.