Large-scale snake genome analyses provide insights into vertebrate development.

Snakes are a remarkable squamate lineage with unique morphological adaptations, especially those related to the evolution of vertebrate skeletons, organs, and sensory systems. To clarify the genetic underpinnings of snake phenotypes, we assembled and analyzed 14 de novo genomes from 12 snake families. We also investigated the genetic basis of the morphological characteristics of snakes using functional experiments. We identified genes, regulatory elements, and structural variations that have potentially contributed to the evolution of limb loss, an elongated body plan, asymmetrical lungs, sensory systems, and digestive adaptations in snakes. We identified some of the genes and regulatory elements that might have shaped the evolution of vision, the skeletal system and diet in blind snakes, and thermoreception in infrared-sensitive snakes. Our study provides insights into the evolution and development of snakes and vertebrates.

DNA barcoding of Chinese snakes reveals hidden diversity and conservation needs.

DNA barcoding has greatly facilitated studies of taxonomy, biodiversity, biological conservation, and ecology. Here, we establish a reliable DNA barcoding library for Chinese snakes, unveiling hidden diversity with implications for taxonomy, and provide a standardized tool for conservation management. Our comprehensive study includes 1638 cytochrome c oxidase subunit I (COI) sequences from Chinese snakes that correspond to 17 families, 65 genera, 228 named species (80.6% of named species) and 36 candidate species. A barcode gap analysis reveals gaps, where all nearest neighbour distances exceed maximum intraspecific distances, in 217 named species and all candidate species. Three species-delimitation methods (ABGD, sGMYC, and sPTP) recover 320 operational taxonomic units (OTUs), of which 192 OTUs correspond to named and candidate species. Twenty-eight other named species share OTUs, such as Azemiops feae and A. kharini, Gloydius halys, G. shedaoensis, and G. intermedius, and Bungarus multicinctus and B. candidus, representing inconsistencies most probably caused by imperfect taxonomy, recent and rapid speciation, weak taxonomic signal, introgressive hybridization, and/or inadequate phylogenetic signal. In contrast, 43 species and candidate species assign to two or more OTUs due to having large intraspecific distances. If most OTUs detected in this study reflect valid species, including the 36 candidate species, then 30% more species would exist than are currently recognized. Several OTU divergences associate with known biogeographic barriers, such as the Taiwan Strait. In addition to facilitating future studies, this reliable and relatively comprehensive reference database will play an important role in the future monitoring, conservation, and management of Chinese snakes.

Genomic evidence reveals intraspecific divergence of the hot-spring snake (Thermophis baileyi), an endangered reptile endemic to the Qinghai-Tibet plateau.

Understanding how and why species evolve requires knowledge on intraspecific divergence. In this study, we examined intraspecific divergence in the endangered hot-spring snake (Thermophis baileyi), an endemic species on the Qinghai-Tibet Plateau (QTP). Whole-genome resequencing of 58 sampled individuals from 15 populations was performed to identify the drivers of intraspecific divergence and explore the potential roles of genes under selection. Our analyses resolved three groups, with major intergroup admixture occurring in regions of group contact. Divergence probably occurred during the Pleistocene as a result of glacial climatic oscillations, Yadong-Gulu rift, and geothermal fields differentiation, while complex gene flow between group pairs reflected a unique intraspecific divergence pattern on the QTP. Intergroup fixed loci involved selected genes functionally related to divergence and local adaptation, especially adaptation to hot spring microenvironments in different geothermal fields. Analysis of structural variants, genetic diversity, inbreeding, and genetic load indicated that the hot-spring snake population has declined to a low level with decreased diversity, which is important for the conservation management of this endangered species. Our study demonstrated that the integration of demographic history, gene flow, genomic divergence genes, and other information is necessary to distinguish the evolutionary processes involved in speciation.

East palearctic treefrog past and present habitat suitability using ecological niche models.

Ecological niche modeling is a tool used to determine current potential species' distribution or habitat suitability models which can then be used to project suitable areas in time. Projections of suitability into past climates can identify locations of climate refugia, or areas with high climatic stability likely to contain the highest levels of genetic diversity and stable populations when climatic conditions are less suitable in other parts of the range. Modeling habitat suitability for closely related species in recent past can also reveal potential periods and regions of contact and possible admixture. In the east palearctic, there are five Dryophytes (Hylid treefrog) clades belonging to two groups: Dryophytes japonicus group: Clades A and B; and Dryophytes immaculatus group: Dryophytes immaculatus, Dryophytes flaviventris, and Dryophytes suweonensis. We used maximum entropy modeling to determine the suitable ranges of these five clades during the present and projected to the Last Glacial Maximum (LGM) and Last Interglacial (LIG) periods. We also calculated climatic stability for each clade to identify possible areas of climate refugia. Our models indicated suitable range expansion during the LGM for four clades with the exclusion of D. immaculatus. High climatic stability in our models corresponded to areas with the highest numbers of recorded occurrences in the present. The models produced here can additionally serve as baselines for models of suitability under climate change scenarios and indicate species ecological requirements.

Genomic adaptations for arboreal locomotion in Asian flying treefrogs.

SignificanceTo adapt to arboreal lifestyles, treefrogs have evolved a suite of complex traits that support vertical movement and gliding, thus presenting a unique case for studying the genetic basis for traits causally linked to vertical niche expansion. Here, based on two de novo-assembled Asian treefrog genomes, we determined that genes involved in limb development and keratin cytoskeleton likely played a role in the evolution of their climbing systems. Behavioral and morphological evaluation and time-ordered gene coexpression network analysis revealed the developmental patterns and regulatory pathways of the webbed feet used for gliding in Rhacophorus kio.

Transcriptome Analysis Reveals Olfactory System Expression Characteristics of Aquatic Snakes.

Animal olfactory systems evolved with changes in habitat to detect odor cues from the environment. The aquatic environment, as a unique habitat, poses a formidable challenge for olfactory perception in animals, since the higher density and viscosity of water. The olfactory system in snakes is highly specialized, thus providing the opportunity to explore the adaptive evolution of such systems to unique habitats. To date, however, few studies have explored the changes in gene expression features in the olfactory systems of aquatic snakes. In this study, we carried out RNA sequencing of 26 olfactory tissue samples (vomeronasal organ and olfactory bulb) from two aquatic and two non-aquatic snake species to explore gene expression changes under the aquatic environment. Weighted gene co-expression network analysis showed significant differences in gene expression profiles between aquatic and non-aquatic habitats. The main olfactory systems of the aquatic and non-aquatic snakes were regulated by different genes. Among these genes, RELN may contribute to exploring gene expression changes under the aquatic environment by regulating the formation of inhibitory neurons in the granular cell layer and increasing the separation of neuronal patterns to correctly identify complex chemical information. The high expression of TRPC2 and V2R family genes in the accessory olfactory systems of aquatic snakes should enhance their ability to bind water-soluble odor molecules, and thus obtain more information in hydrophytic habitats. This work provides an important foundation for exploring the olfactory adaptation of snakes in special habitats.

A new species of the Achalinus rufescens complex (Xenodermidae: Achalinus) from Fujian Province, China.

A new species of the xenodermid snake genus Achalinus Peters, 1869 is described from Fujian Province, China, based on six specimens. Bayesian inference and maximum likelihood analyses based on a mitochondrial DNA fragment (CO1) indicated the new taxon is different from its congeners (pdistance ≥ 18.5%). Morphologically, the new species can be diagnosed from the other species by a combination of following characters: (1) dorsal scales 23 rows throughout, strongly keeled, the most outer rows on both sides also keeled and slightly enlarged; (2) tail relatively longer, TaL/TL ratio 0.260.29 in males, 0.210.22 in females; (3) maxillary teeth 3033; (4) length of suture between internasals significantly longer than that between prefrontals; (5) nasal divided into two sections by nasal cleft; (6) a single loreal; (7) SPO 1, seldom 2; (8) SPL 6, the fourth and fifth contacting eye; (9) IFL5, rarely 6, the first three touching the first pair of chin shields; (10) TMP 79, arranged in three rows; (11) VS 142149 in males, VS 152154 in females; (12) SC 7481 in males, SC 6365 in females, arranged in a single row; (13) cloacal entire; (14) greyish brown above, pale yellow beneath; (15) dorsum with an indistinct longitudinal vertebral stripe. The description of the new species brings the total species of Achalinus to 19.

Taxonomic revision of Amolops chunganensis (Pope, 1929) (Amphibia: Anura) and description of a new species from southwestern China, with discussion on Amolops monticola group and assignment of species groups of the genus Amolops.

Amolops chunganensis is a species complex and reported widely from eastern, southern, and southwestern China. Based on molecular data of 19 populations of A. chunganensis sensu lato from China, including the population from Mt. Wuyi (type locality), we recognize A. chunganensis sensu stricto and provide an expanded description based on the topotypic specimens. Combining morphological and molecular data, we describe a new species, Amolops chaochin sp. nov., from southwestern China, which was previously identified as A. chunganensis. The new species is distinguished from all other species in the A. monticola group by: (1) moderate body size, SVL 35.3-39.2 mm in males (n=7), and 50.5-54.4 mm in females (n=7); (2) distinct tympanum, larger than half of eye diameter; (3) small tooth-like projection on anteromedial edge of mandible; (4) circummarginal groove on all fingers; (5) white tubercles on dorsal side of posterior body in both sexes; (6) distinct tubercles on dorsal thigh and white spinose tubercles on dorsal tibia in both sexes; (7) white tubercles on posterior region of tympanum in males; (8) toe webbing reaching disk by dermal fringe on inner side of toe II; (9) vomerine teeth present; (10) transverse bands on dorsal limbs; (11) external vocal sacs present in males. We further reviewed the assignment of Amolops groups, with an overall revision of membership and diagnosis of all species groups.

Review of the emGekko/em (emJaponigekko/em) emsubpalmatus/em complex (Squamata, Sauria, Gekkonidae), with description of a new species from China.

The gecko species Gekko (Japonigekko) subpalmatus was previously recorded with a relatively wide distribution from eastern, southern, and southeastern China. However, the populations in southern China are currently recognized as another valid species G. (J.) melli. In this study, we conduct a detailed morphological examination and phylogenetic analysis of the populations currently treated as G. (J.) subpalmatus or G. (J.) melli, which are collectively designated as the G. (J.) subpalmatus complex. Our results reveal that the G. (J.) subpalmatus complex comprises three evolutionarily independent taxa. The populations from Zhejiang, eastern China are G. (J.) subpalmatus, those from southern China are G. (J.) melli, while those from the Sichuan Basin, southwestern China represent a cryptic species, Gekko (Japonigekko) cib sp. nov.. Gekko (Japonigekko) cib sp. nov. can be distinguished from all congeners, by its divergence from other complex members in the CYTB and 16S genes, and a combination of morphological characteristics, especially in hemipenial morphology. Historic records of G. (J.) subpalmatus complex are also reviewed.

Yellow sea mediated segregation between North East Asian Dryophytes species.

While comparatively few amphibian species have been described on the North East Asian mainland in the last decades, several species have been the subject of taxonomical debates in relation to the Yellow sea. Here, we sampled Dryophytes sp. treefrogs from the Republic of Korea, the Democratic People's Republic of Korea and the People's Republic of China to clarify the status of this clade around the Yellow sea and determine the impact of sea level change on treefrogs' phylogenetic relationships. Based on genetics, call properties, adult morphology, tadpole morphology and niche modelling, we determined the segregated status species of D. suweonensis and D. immaculatus. We then proceeded to describe a new treefrog species, D. flaviventris sp. nov., from the central lowlands of the Republic of Korea. The new species is geographically segregated from D. suweonensis by the Chilgap mountain range and known to occur only in the area of Buyeo, Nonsan and Iksan in the Republic of Korea. While the Yellow sea is the principal element to the current isolation of the three clades, the paleorivers of the Yellow sea basin are likely to have been the major factor for the divergences within this clade. We recommend conducting rapid conservation assessments as these species are present on very narrow and declining ranges.

A new species of the genus Dendrelaphis (Squamata: Colubridae) from Yunnan Province, China, with discussion of the occurrence of D. cyanochloris (Wall, 1921) in China.

A new species of the genus Dendrelaphis is described from Xishuangbanna, southern Yunnan, China, based on molecular and morphological data. The new species can be differentiated from other congeners by the following combination of characters: 1) ground color of body bronze, a black postocular stripe extending onto the neck only; 2) pale and dark ventrolateral stripe absent; 3) relatively indistinct transverse bands on the anterior part of lateral body; 4) loreal single; 5) vertebral scales strongly enlarged; 6) dorsal scale rows 15-15-11, all smooth; 7) ventrals 193-197, subcaudals 130-135, paired; 8) SVL/TOL ratio 0.292-0.301; 9) supralabials 9, 4th through 6th touching the eye; 10) the outermost row of dorsal scales the same color as other dorsal scales; 11) retracted hemipenis extending to the 6-7th subcaudal scales. According to molecular and morphological data, D. ngansonensis likely belongs to the D. cyanochloris complex. We further discussed D. cyanochloris complex from Tibet, Yunnan and Hainan, China. A key to Chinese species of Dendrelaphis is provided.

The Genome of Shaw's Sea Snake (Hydrophis curtus) Reveals Secondary Adaptation to Its Marine Environment.

The transition of terrestrial snakes to marine life ∼10 Ma is ideal for exploring adaptive evolution. Sea snakes possess phenotype specializations including laterally compressed bodies, paddle-shaped tails, valvular nostrils, cutaneous respiration, elongated lungs, and salt glands, yet, knowledge on the genetic underpinnings of the transition remains limited. Herein, we report the first genome of Shaw's sea snake (Hydrophis curtus) and use it to investigate sea snake secondary marine adaptation. A hybrid assembly strategy obtains a high-quality genome. Gene family analyses date a pulsed coding-gene expansion to ∼20 Ma, and these genes associate strongly with adaptations to marine environments. Analyses of selection pressure and convergent evolution discover the rapid evolution of protein-coding genes, and some convergent features. Additionally, 108 conserved noncoding elements appear to have evolved quickly, and these may underpin the phenotypic changes. Transposon elements may contribute to adaptive specializations by inserting into genomic regions around functionally related coding genes. The integration of genomic and transcriptomic analyses indicates independent origins and different components in sea snake and terrestrial snake venom; the venom gland of the sea snake harbors the highest PLA2 (17.23%) expression in selected elapids and these genes may organize tandemly in the genome. These analyses provide insights into the genetic mechanisms that underlay the secondary adaptation to marine and venom production of this sea snake.

Taxonomic re-evaluation of the monotypic genus Pararhabdophis Bourret, 1934 (Squamata: Colubridae: Natricinae) with discovery of its type species, P. chapaensis, from China.

The Asian monotypic genus Pararhabdophis Bourret, 1934 has long been known from a single holotype of the type species Pararhabdophis chapaensis Bourret, 1934 only. The limited available information hampered the identification of the natricine species. On the basis of eight newly collected specimens of P. chapaensis from the type locality in Sa Pa, Lao Cai Province, Vietnam and from Pingbian, Yunnan Province in southwestern China, the taxonomic position of the genus Pararhabdophis was re-evaluated using both morphological and molecular datasets for the first time. Pararhabdophis chapaensis is nested within the genus Hebius Thompson, 1913 with strong support, and morphologically indistinguishable from the latter genus. As a consequence, we herein synonymize the genus Pararhabdophis with Hebius and discuss about the existing paraphyly of some Hebius species. In addition we report Hebius chapaensis for the first time from China and provide redescription and natural history data of this poorly known species. To facilitate future taxonomic work, an identification key to all known natricine genera from China and Vietnam is also provided.

Comparative genomic investigation of high-elevation adaptation in ectothermic snakes.

Several previous genomic studies have focused on adaptation to high elevations, but these investigations have been largely limited to endotherms. Snakes of the genus Thermophis are endemic to the Tibetan plateau and therefore present an opportunity to study high-elevation adaptations in ectotherms. Here, we report the de novo assembly of the genome of a Tibetan hot-spring snake (Thermophis baileyi) and then compare its genome to the genomes of the other two species of Thermophis, as well as to the genomes of two related species of snakes that occur at lower elevations. We identify 308 putative genes that appear to be under positive selection in Thermophis We also identified genes with shared amino acid replacements in the high-elevation hot-spring snakes compared with snakes and lizards that live at low elevations, including the genes for proteins involved in DNA damage repair (FEN1) and response to hypoxia (EPAS1). Functional assays of the FEN1 alleles reveal that the Thermophis allele is more stable under UV radiation than is the ancestral allele found in low-elevation lizards and snakes. Functional assays of EPAS1 alleles suggest that the Thermophis protein has lower transactivation activity than the low-elevation forms. Our analysis identifies some convergent genetic mechanisms in high-elevation adaptation between endotherms (based on studies of mammals) and ectotherms (based on our studies of Thermophis).

A new species of the Southeast Asian genus Opisthotropis (Serpentes: Colubridae: Natricinae) from western Hunan, China.

A new species of natricine snake of the Southeast Asian genus Opisthotropis Gü nther, 1872 is described from western Hunan Province of China based on both mitochondrial DNA and morphological data. The new species is morphologically most similar and genetically most closely related to O. cheni Zhao, 1999 and O. latouchii (Boulenger, 1899), but possesses considerable genetic divergence (p-distance 5.1%-16.7%) and can be differentiated from all other congeners by a combination of the following morphological characters: (1) body size large (total length 514-586 mm) and strongly built; (2) dorsal scale rows 17 throughout, feebly keeled anteriorly and moderately keeled posteriorly; (3) ventral scales 147-152, subcaudal scales 54-62; (4) preocular absent, loreal elongated and touching orbit; (5) supralabials 8-9, fifth and sixth entering obit; (6) anterior temporals short, length 1.74-2.04 times longer than width; (7) maxillary teeth subequal, 28-30; (8) dorsal surface of head with distinct irregular yellow stripes and markings edged with ochre; (9) body with clear black and yellow longitudinal streaks, partly fused to several lighter patches or thicker stripes anteriorly; and (10) venter pale yellow, with asymmetric blackish speckles along outer margin. We present an updated diagnostic key to all members of the genus Opisthotropis, and recommendations on the ecological study for the group are provided.