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.

Disentangling the Pelodiscus axenaria complex, with the description of a new Chinese species and neotype designation for P. axenaria (Zhou, Zhang amp; Fang, 1991).

We describe a new species from the Pelodiscus axenaria complex from Hunan and Jiangxi Provinces, China. Also, the application of the name P. axenaria (Zhou, Zhang Fang, 1991) is clarified by designating a neotype for this species. Besides its genetic divergence, the new species differs from all other Pelodiscus species, including the two other taxa constituting the complex (P. axenaria, P. huangshanensis), in the following combination of morphological traits: (1) small adult size, <15 cm carapace length; (2) carapace distinctly keeled, more or less strongly tuberculated, usually olive clay-coloured and adorned with greenish black marbling; (3) plastron yellowish white, typically immaculate except for a blurred-edged blotch behind each axilla that does not extend to the entoplastron and some slight black suffusion along its anterior border; (4) underside of the leathery margin of the carapace with varying amounts of dark pigmentation; (5) head olive clay-coloured with numerous black splotches; (6) chin grey brown with pale stipples, throat dark grey, finely spotted with black; (7) neck with a wide yellow lateral band stretching from the tympanum posteriorly, which tends to fade with age; (8) entoplastron boomerang-shaped, the amount of bending of the transverse bar between the two posteriolaterally directed rami >90.

Effect of incubation temperature and substrate moisture on embryonic development, hatchling phenotypes and post-hatching growth in the Reeves' Turtle, Mauremys reevesii.

BACKGROUND: Reeves' Turtles (Mauremys reevesii) are economically important in aquaculture in China. Understanding the effects of incubation temperature and substrate moisture on embryos and hatchlings is of great significance for improving the artificial culture of M. reevesii. However, available studies have not yet determined the thermal and hydric optima for M. reevesii eggs, and the potential interaction between the two factors. METHODS: In this study, eggs of M. reevesii were incubated at five temperature levels (23, 26, 29, 32 and 35 °C, fluctuation range ± 0.5 °C). In each temperature level, there were three substrate moisture levels (1:0.5, 1:0.9 and 1:1.2, weight ratio of vermiculite to water). Thus, a total of 15 combinations of temperature and moisture were used to examine the effects of incubation temperature and substrate moisture on incubation duration, hatching success, hatchling phenotypes, post-hatching growth and hatchling survival. RESULTS: Substrate moisture did not significantly affect most development parameters (except incubation duration and carapace width of hatchlings). Eggs incubated at low moisture level (1:0.5) had a longer incubation duration and produced hatchlings with smaller carapace widths than those incubated at medium (1:0.9) or high (1:1.2) moisture levels. Incubation temperature had a significant effect on incubation duration, hatching success, hatchling phenotypes and hatchling survival. Incubation duration decreased as incubation temperature increased. Eggs incubated at 23, 26 and 29 °C showed higher hatching success than those incubated at 32 and 35 °C. Hatchlings incubated at 32 °C were smaller in body size and mass than those incubated at 23, 26 and 29 °C. At 12 months of age, incubation temperature had no long-lasting effect on body mass, but hatchlings incubated at 23 and 35 °C had lower survival rates than those incubated at 26, 29 and 32 °C. For the development of embryos and hatchlings, the interaction between incubation temperature and substrate moisture was not significant. CONCLUSIONS: Our results indicate that incubation temperature has a significant influence on the development of embryos and hatchlings of M. reevesii, while substrate moisture only significantly affects the incubation duration and carapace width of hatchlings. The combination of an incubation temperature of 29 ± 0.5 °C and a substrate moisture level of 1:1.2 represented optimal incubation conditions in this experiment. Such incubation conditions are helpful in obtaining higher hatching success, shorter incubation duration and higher survival rates for this aquaculture species.

Phylogenetic analyses of distantly related clades of bent-toed geckos (genus Cyrtodactylus) reveal an unprecedented amount of cryptic diversity in northern and western Thailand.

Cyrtodactylus species are the most diverse of the geckos and are widely distributed in Southeast Asia, including Thailand. However, their patterns of distribution, especially in northern and western parts of Thailand, remain unknown because few Cyrtodactylus species in these regions have been described. Thus, a data set of mitochondrial NADH dehydrogenase 2 (ND2) gene and flanking tRNAs from Cyrtodactylus found in northern and western Thailand, including contiguous areas, was assembled to elucidate phylogenetic relationships and identify the distribution patterns of these geckos. The results showed four well-supported clades, a northwestern clade (A), a northern clade (B), a western clade (C), and a special clade characterized by specific morphological features (D). Clades A-C were grouped with strong support by the geography of their localities from northern Thailand (Mae Hong Son and Chiang Mai Provinces) along the Tenasserim mountain ranges to Phang-Nga Province, Thailand. Clade D is a distinct clade of Cyrtodactylus species characterized by a tuberculate and prehensile tail and distributed widely in mainland Southeast Asia. Overall, the results suggest a pattern of geographic separation and distribution of Cyrtodactylus in northern and western Thailand. Additionally, this study provides evidence of a hidden biodiversity of Cyrtodactylus in these regions.

Integrating and updating wildlife conservation in China.

China has about 11% of the world's total wildlife species, so strengthening China's wildlife conservation is of great significance to global biodiversity. Despite some successful cases and conservation efforts, 21.4% of China's vertebrate species are threatened by human activities. The booming wildlife trade in China has posed serious threat to wildlife in China and throughout the world, while leading to a high risk of transmission of infectious zoonotic diseases. China's wildlife conservation has faced a series of challenges, two of which are an impractical, separated management of wildlife and outdated protected species lists. Although the Wildlife Protection Law of China was revised in 2016, the issues of separated management remain, and the protected species lists are still not adequately revised. These issues have led to inefficient and overlapping management, waste of administrative resources, and serious obstacles to wildlife protection. In this article, we analyze the negative effects of current separated management of wildlife species and outdated protected species lists, and provide some suggestions for amendment of the laws and reform of wildlife management system.

Turtles and Tortoises Are in Trouble.

Turtles and tortoises (chelonians) have been integral components of global ecosystems for about 220 million years and have played important roles in human culture for at least 400,000 years. The chelonian shell is a remarkable evolutionary adaptation, facilitating success in terrestrial, freshwater and marine ecosystems. Today, more than half of the 360 living species and 482 total taxa (species and subspecies combined) are threatened with extinction. This places chelonians among the groups with the highest extinction risk of any sizeable vertebrate group. Turtle populations are declining rapidly due to habitat loss, consumption by humans for food and traditional medicines and collection for the international pet trade. Many taxa could become extinct in this century. Here, we examine survival threats to turtles and tortoises and discuss the interventions that will be needed to prevent widespread extinction in this group in coming decades.

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.

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.

Millennium-old farm breeding of Chinese softshell turtles (Pelodiscus spp.) results in massive erosion of biodiversity.

Chinese softshell turtles (Pelodiscus spp.) are widely distributed, ranging from the Amur and Ussuri Rivers in the Russian Far East through the Korean Peninsula, Japan, and eastern, central, and southern China to southern Vietnam. In East and Southeast Asia, Chinese softshell turtles are traditionally exploited for food and have been farm-bred in China since the Spring and Autumn Period, more than 2400 years ago. Currently, the annual production of Pelodiscus amounts to 340,000 t in China alone. Using mitochondrial DNA (2428 bp) and five nuclear loci (3704 bp), we examined broad sampling of wild and farm-bred Pelodiscus to infer genetic and taxonomic differentiation. We discovered four previously unknown mitochondrial lineages, all from China. One lineage from Jiangxi is deeply divergent and sister to the mitochondrial lineage of Pelodiscus axenaria. The nuclear loci supported species status for P. axenaria and the new lineage from Jiangxi. Pelodiscus maackii and P. parviformis, both harboring distinct mitochondrial lineages, were not differentiated from P. sinensis in the studied nuclear markers. The same is true for two new mitochondrial lineages from Zhejiang, China, represented by only one individual each, and another new lineage from Anhui, Guangdong, Jiangxi and Zhejiang, China. However, Vietnamese turtles yielding a mitochondrial lineage clustering within P. sinensis were distinct in nuclear markers, suggesting that these populations could represent another unknown species with introgressed mitochondria. Its species status is also supported by the syntopic occurrence with P. sinensis in northern Vietnam and by morphology. In addition, we confirmed sympatry of P. axenaria and P. parviformis in Guangxi, China, and found evidence for sympatry of P. sinensis and the new putative species from Jiangxi, China. We also discovered evidence for hybridization in turtle farms and for the occurrence of alien lineages in the wild (Zhejiang, China), highlighting the risk of genetic pollution of native stock. In the face of the large-scale breeding of Pelodiscus, we claim that the long-term survival of distinct genetic lineages and species can only be assured when an upscale market segment for pure-bred softshell turtles is established, making the breeding of pure lineages lucrative for turtle farms. Our findings underline that the diversity of Pelodiscus is currently underestimated and threatened by anthropogenic admixture. We recommend mass screening of genetic and morphological variation of Chinese softshell turtles as a first step to understand and preserve their diversity.

Melanin deposition ruled out as cause of color changes in the red-eared sliders (Trachemys scripta elegans).

Animal coloration primarily depends on the presence of pigments and the mixing ratio of eumelanin and pheomelanin. The color of red-eared slider's carapace varies with age, from an olive green to a yellow green, and then to a yellow brown in juveniles, generally. The purpose of the present study was to investigate whether this color change is related to the difference in melanin expression. Melanin deposition levels were examined in the carapace, skin, eye and muscle of the three color-types using hematoxylin and eosin staining. Moreover, the full-length coding sequence (CDS) of red-eared slider turtle melanin biosynthesis regulatory genes TYR, TYRP1, MITF and SLC24A5 were cloned, sequenced and quantitatively analyzed. Both histological view of melanin deposition and quantitative real-time PCR test of melanin-regulated gene expressions showed that there are significant differences among different tissues of red-eared slider, but no significant difference among different color-types, indicating that melanin deposition is not associated with ontogenetic color change in the carapace of red-eared slider. This study initially explore the melanin deposition and the mRNA expression of melanin biosynthesis regulatory genes in red-eared slider, which serve as a foundation for further insight into the pigmentation patterns and the mechanism of body color change in turtles.

A new species of the genus Takydromus (Squamata: Lacertidae) from Tianjingshan Forestry Station, northern Guangdong, China.

Many early descriptions of species of the genus Takydromus were based on limited diagnostic characteristics. This has caused considerable challenges in accurate species identification, meaning that a number of cryptic species have been erroneously identified as known species, resulting in substantially underestimated species diversity. We have integrated evidence from morphology and DNA sequence data to describe a new species of the Asian Grass Lizard, Takydromus albomaculosus sp. nov., based on two specimens from Tianjingshan Forestry Station, Ruyuan County, Guangdong Province, China. The new species can be distinguished from other known Takydromus species by distinctive morphological differences and significant genetic divergence in the mitochondrial COI gene. Morphologically, the new species differs from the known 22 congeners in the following combination of characters: relatively large body size, SVL 70.9 mm in female, 65.5 mm in male; presence of three or four pairs of femoral pores and four pairs of chin-shields; supraoculars three, rarely four, the posterior loreal either in contact with the anteriormost supraocular or not; the presence of supraciliary granules; the presence of six rows of ventral scales, strongly keeled in the male, and smooth but with the outermost row keeled in the female; the presence of enlarged and keeled lateral scales in a row above the ventrals, but shorter than the ventrals; subdigital lamellae 23-24 under the fourth finger and 29-30 under the fourth toe; dorsal surface brown, ventral surface green, flank dark brown or brown black with several prominent white round spots; the presence of a pair of white longitudinal dorsolateral stripes and a pair of white longitudinal ventrolateral stripes. Takydromus albomaculosus sp. nov. represents the twenty-third species of this genus, of which six species occur in northern Guangdong, where the biodiversity level of Takydromus is the second highest in the world, just below that of Taiwan.

Disappearance of endangered turtles within China's nature reserves.

China ranks first among Northern hemisphere countries for species richness, but approximately 43% of its species are threatened [1], with harvesting being the major threat to vertebrates [2]. To protect its biodiversity, China has established about 2,700 nature reserves covering 1.46 million km2 ( about 15% of China's territory, a percentage higher than the world average [3]). With increasing habitat destruction and harvesting, nature reserves are the final refugia for threatened species. However, many Chinese nature reserves are poorly managed, leaving them vulnerable to poaching and other human encroachment [4]. In this study, we conducted a 12-year (2002-2013) case study on turtles to illustrate the damaging impacts China's nature reserves have on wildlife conservation. We discovered that poaching occurred in all of the 56 reserves surveyed, resulting in dramatically reduced turtle populations. In a majority of the reserves, the reserve staff themselves were involved in poaching. Although nature reserves were created to protect plants and animals, they have become part of the problem due to weak enforcement of rules.

Captive breeding of pangolins: current status, problems and future prospects.

Pangolins are unique placental mammals with eight species existing in the world, which have adapted to a highly specialized diet of ants and termites, and are of significance in the control of forest termite disaster. Besides their ecological value, pangolins are extremely important economic animals with the value as medicine and food. At present, illegal hunting and habitat destruction have drastically decreased the wild population of pangolins, pushing them to the edge of extinction. Captive breeding is an important way to protect these species, but because of pangolin's specialized behaviors and high dependence on natural ecosystem, there still exist many technical barriers to successful captive breeding programs. In this paper, based on the literatures and our practical experience, we reviewed the status and existing problems in captive breeding of pangolins, including four aspects, the naturalistic habitat, dietary husbandry, reproduction and disease control. Some recommendations are presented for effective captive breeding and protection of pangolins.

Spirometra (Pseudophyllidea, Diphyllobothriidae) severely infecting wild-caught snakes from food markets in Guangzhou and Shenzhen, Guangdong, China: implications for public health.

Sparganosis is a zoonotic disease caused by the spargana of Spirometra, and snake is one of the important intermediate hosts of spargana. In some areas of China, snake is regarded as popular delicious food, and such a food habit potentially increases the prevalence of human sparganosis. To understand the prevalence of Spirometra in snakes in food markets, we conducted a study in two representative cities (Guangzhou and Shenzhen), during January-August 2013. A total of 456 snakes of 13 species were examined and 251 individuals of 10 species were infected by Spirometra, accounting for 55.0% of the total samples. The worm burden per infected snake ranged from 1 to 213, and the prevalence in the 13 species was 0∼96.2%. More than half (58.1%) of the spargana were located in muscular tissue, 25.6% in subcutaneous tissue, and 16.3% in coelomic cavity. The results indicated that Spirometra severely infected snakes in food markets in Guangzhou and Shenzhen, implying that eating snakes has great health risk and improper cooking methods may increase the risk of Spirometra infection in humans in China. Additional steps should be considered by the governments and public health agencies to prevent the risk of snake-associated Spirometra infections in humans.

Isolation and characterization of 14 polymorphic microsatellite loci in the big-headed turtle (Platysternon megacephalum).

The big-headed turtle (Platysternon megacephalum) is critically endangered because of overharvesting, illegal trade, and habitat destruction. Assessment of genetic variability in existing populations becomes very important to the taxonomy and conservation of this species. Here we describe 14 microsatellite loci isolated from an enriched genomic library of the big-headed turtle, and the polymorphisms of these loci were assessed in 28 individuals from Huizhou, Heyuan, Zhaoqing, and Shaoguan of Guangdong, China. The range of polymorphism information content is 0.305-0.738, and no evidence of significant linkage disequilibrium was found among any pairs of loci. These 14 new polymorphic microsatellite loci can be used in population genetics, taxonomy, phylogeography, behavior ecology, and conservation efforts of Platysternon megacephalum.

Highly pathogenic Salmonella Pomona was first isolated from the exotic red-eared slider (Trachemys scripta elegans) in the wild in China: Implications for public health.

Salmonella Pomona, a highly pathogenic serotype, can cause severe human salmonellosis, especially in children. Turtles and other reptiles are reservoirs for S. Pomona, and these cold-blooded animals remain a source of human Salmonella infections. Since the 1980s, this serotype has become a significant public health concern because of the increasing number of cases of S. Pomona infection in humans. To date, outbreaks of Salmonella Pomona infection in humans have mainly occurred in the United States, with some in other countries (e.g. Belgium, Germany, Canada), and most of the infections in humans were associated with turtles and other reptiles. In China, S. Pomona was first isolated from the feces of an infant in Shanghai in 2000, and two further cases of S. Pomona infection in humans were later found in Guangzhou. No one knew the source of S. Pomona in China. In this study, for the first time we isolated S. Pomona from free-living exotic red-eared sliders in the wild in China. Salmonella serotype (S. Pomona) was isolated from 16 turtle samples. The total carrying rate of S. Pomona in the collected red-eared sliders was 39% (n = 41) overall: 40% (n = 25) in juveniles and 38% (n = 16) in adult turtles. This study suggests that the widespread exotic red-eared sliders may impact on public health and ecosystems of China by transmitting S. Pomona. Additional steps should be considered by the governments and public health agencies to prevent the risk of turtle-associated Salmonella infections in humans in China.

Severe infection of wild-caught snakes with Spirometra erinaceieuropaei from food markets in Guangzhou, China involves a risk for zoonotic sparganosis.

Wild-caught snakes are a popular and traditional food in China. However, little known to the public, snakes are also intermediate hosts of Spirometra erinaceieuropaei, a food- and water-borne pathogen of sparganosis. Therefore, we investigated the prevalence of S. erinaceieuropaei in 10 popular species of wild-caught snakes in Guangzhou City (Guangdong Province) between July 2009 and July 2010. One hundred and twenty-four specimens of 10 species (including Enhydris plumbea, Zoacys dhumnades, Elaphe radiate, Elaphe taeniura, Elaphe carinata, Ptyas mucosus, Ptyas korros, Naja naja atra, Bungarus fasciatus, and Bungarus multicinctus) were randomly selected from a total of 1,160 wild-caught snakes. They were obtained from food markets in 5 representative districts (Huadou, Panyu, Tianhe, Haizhu, and Conghua). The specimens were killed, necropsied, and examined for parasitic helminths. Of the snakes examined, 29.8% were infected by spargana and the worm burden per infected snake ranged from 1 to 221. Most species were infected except for En. plumbea, B. fasciatus, and B. multicinctus. Prevalence even reached 100% in Zoacys dhumnades. More than half (53.5%) of the spargana were located in muscular tissue, 36.4% in subcutaneous tissue, and 10.1% in the coelomic cavity. The study revealed the potential risk for the zoonotic sparganosis by eating wild-caught snakes and will be helpful in arousing public health concern about the consumption of snake meat.