Patterns of recombination in snakes reveal a tug-of-war between PRDM9 and promoter-like features.

In some mammals, notably humans, recombination occurs almost exclusively where the protein PRDM9 binds, whereas in vertebrates lacking an intact PRDM9, such as birds and canids, recombination rates are elevated near promoter-like features. To determine whether PRDM9 directs recombination in nonmammalian vertebrates, we focused on an exemplar species with a single, intact PRDM9 ortholog, the corn snake (Pantherophis guttatus). Analyzing historical recombination rates along the genome and crossovers in pedigrees, we found evidence that PRDM9 specifies the location of recombination events, but we also detected a separable effect of promoter-like features. These findings reveal that the uses of PRDM9 and promoter-like features need not be mutually exclusive and instead reflect a tug-of-war that is more even in some species than others.

Proliferative strongyloidiasis in a colony of colubrid snakes.

Strongyloides are small rhabditid nematodes primarily associated with enteric disease in a variety of animal species, including reptiles. Strongyloides spp life stages were associated with a disease outbreak in a large breeding colony of snakes. Multiple Pantherophis and Lampropeltis colubrids exhibited respiratory distress, anorexia, stomatitis, facial deformation, and waning body condition that resulted in death or necessitated euthanasia. Postmortem examinations of 13 snakes revealed epithelial hyperplasia and inflammation of the alimentary and respiratory tracts associated with varying numbers of adult and larval nematodes and embryonated or larvated ova. In a subset of snakes, aberrant nematode migration was also observed in the eye, genitourinary system, coelom, and vasculature. Histomorphology and gross examination of parasitic adult female nematodes from host tissues were consistent with a Strongyloides spp. Sedimented fecal material from 101/160 (63%) snakes housed in the affected facility was positive for nematodes and/or larvated ova. Polymerase chain reaction amplification and sequencing of portions of the 18S and 28S ribosomal ribonucleic acid (RNA) genes and the internal transcribed spacer region of adult female parasites and positive fecal samples supported the diagnosis of strongyloidiasis. Strongyloides spp possess a unique life cycle capable of alternating between parasitic (homogonic) and free-living (heterogonic) stages, resulting in the production of directly infective larvae. Commonly utilized husbandry practices in reptile collections can amplify the numbers of infective larvae generated in the captive environment, increasing the risk for rhabditid hyperinfections. This report documents morbidity, mortality, and non-enteric disease manifestations due to Strongyloides hyperinfections in a captive colubrid snake colony.

Evolution of Three-Finger Toxin Genes in Neotropical Colubrine Snakes (Colubridae).

Snake venom research has historically focused on front-fanged species (Viperidae and Elapidae), limiting our knowledge of venom evolution in rear-fanged snakes across their ecologically diverse phylogeny. Three-finger toxins (3FTxs) are a known neurotoxic component in the venoms of some rear-fanged snakes (Colubridae: Colubrinae), but it is unclear how prevalent 3FTxs are both in expression within venom glands and more broadly among colubrine species. Here, we used a transcriptomic approach to characterize the venom expression profiles of four species of colubrine snakes from the Neotropics that were dominated by 3FTx expression (in the genera Chironius, Oxybelis, Rhinobothryum, and Spilotes). By reconstructing the gene trees of 3FTxs, we found evidence of putative novel heterodimers in the sequences of Chironius multiventris and Oxybelis aeneus, revealing an instance of parallel evolution of this structural change in 3FTxs among rear-fanged colubrine snakes. We also found positive selection at sites within structural loops or "fingers" of 3FTxs, indicating these areas may be key binding sites that interact with prey target molecules. Overall, our results highlight the importance of exploring the venoms of understudied species in reconstructing the full evolutionary history of toxins across the tree of life.

A reference genome assembly for the continentally distributed ring-necked snake, Diadophis punctatus.

Snakes in the family Colubridae include more than 2,000 currently recognized species, and comprise roughly 75% of the global snake species diversity on Earth. For such a spectacular radiation, colubrid snakes remain poorly understood ecologically and genetically. Two subfamilies, Colubrinae (788 species) and Dipsadinae (833 species), comprise the bulk of colubrid species richness. Dipsadines are a speciose and diverse group of snakes that largely inhabit Central and South America, with a handful of small-body-size genera that have invaded North America. Among them, the ring-necked snake, Diadophis punctatus, has an incredibly broad distribution with 14 subspecies. Given its continental distribution and high degree of variation in coloration, diet, feeding ecology, and behavior, the ring-necked snake is an excellent species for the study of genetic diversity and trait evolution. Within California, six subspecies form a continuously distributed "ring species" around the Central Valley, while a seventh, the regal ring-necked snake, Diadophis punctatus regalis is a disjunct outlier and Species of Special Concern in the state. Here, we report a new reference genome assembly for the San Diego ring-necked snake, D. p. similis, as part of the California Conservation Genomics Project. This assembly comprises a total of 444 scaffolds spanning 1,783 Mb and has a contig N50 of 8.0 Mb, scaffold N50 of 83 Mb, and BUSCO completeness score of 94.5%. This reference genome will be a valuable resource for studies of the taxonomy, conservation, and evolution of the ring-necked snake across its broad, continental distribution.

A taxonomic revision of Boiga multomaculata (Boie, 1827) and B. ochracea (Theobald, 1868), with the description of a new subspecies (Squamata, Serpentes, Colubridae).

The analyses of molecular genetic data (mtDNA markers 16S, ND4, CYTB, and the nuclear marker c-mos) provided evidence that the Asian cat snake taxa Boiga multomaculata and B. ochracea actually represent a single species. They form mixed clades of low intraclade genetic differentiation. This evidence for conspecificy is supported by the lack of differentiation in all examined pholidotic and morphometric characters. Therefore, we formally place Dipsas ochracea Theobald, 1868 in the synonymy of Dipsas multomaculata Boie, 1827. We provide a summary of the tangled taxonomic history of the taxa involved in this study. Also, we resurrect Dipsadomorphus stoliczkae Wall, 1909 from synonymy of B. ochracea, for specimens exhibiting 21 midbody dorsal scale rows. Boiga stoliczkae is found in the Himalayas north and west of the Brahmaputra valley. Finally, based on the detection of historical genetic lineages within the newly defined species Boiga multomaculata we recognize three subspecies: Boiga multomaculata multomaculata (Boie, 1827), Boiga multomaculata ochracea (Theobald, 1868), and Boiga multomaculata septentrionalis n. ssp. which is distributed in northern Myanmar and Assam and Nagaland, India. We designate BMNH 1946.1.2.60 (1) as neotype of Dipsas ochracea Theobald, (2) as lectotype of D. ochraceus Günther, and (3) as lectotype of Boiga ochracea walli Smith, thereby making these names objective synonyms. Finally, we designate BMNH 94.12.31.55 as lectotype of Dipsadomorphus stoliczkae Wall.

The Venom Composition of the Snake Tribe Philodryadini: 'Omic' Techniques Reveal Intergeneric Variability among South American Racers.

Snakes of the Philodryadini tribe are included in the Dipsadidae family, which is a diverse group of rear-fanged snakes widespread in different ecological conditions, including habitats and diet. However, little is known about the composition and effects of their venoms despite their relevance for understanding the evolution of these snakes or even their impact on the occasional cases of human envenoming. In this study, we integrated venom gland transcriptomics, venom proteomics and functional assays to characterize the venoms from eight species of the Philodryadini tribe, which includes the genus Philodryas, Chlorosoma and Xenoxybelis. The most abundant components identified in the venoms were snake venom metalloproteinases (SVMPs), cysteine-rich secretory proteins (CRISPs), C-type lectins (CTLs), snake endogenous matrix metalloproteinases type 9 (seMMP-9) and snake venom serinoproteinases (SVSPs). These protein families showed a variable expression profile in each genus. SVMPs were the most abundant components in Philodryas, while seMMP-9 and CRISPs were the most expressed in Chlorosoma and Xenoxybelis, respectively. Lineage-specific differences in venom composition were also observed among Philodryas species, whereas P. olfersii presented the highest amount of SVSPs and P. agassizii was the only species to express significant amounts of 3FTx. The variability observed in venom composition was confirmed by the venom functional assays. Philodryas species presented the highest SVMP activity, whereas Chlorosoma species showed higher levels of gelatin activity, which may correlate to the seMMP-9 enzymes. The variability observed in the composition of these venoms may be related to the tribe phylogeny and influenced by their diets. In the presented study, we expanded the set of venomics studies of the Philodryadini tribe, which paves new roads for further studies on the evolution and ecology of Dipsadidae snakes.

Independent Recruitment of Different Types of Phospholipases A2 to the Venoms of Caenophidian Snakes: The Rise of PLA2-IIE within Pseudoboini (Dipsadidae).

Snake venoms harbor a wide and diverse array of enzymatic and nonenzymatic toxic components, allowing them to exert myriad effects on their prey. However, they appear to trend toward a few optimal compositional scaffolds, dominated by four major toxin classes: SVMPs, SVSPs, 3FTxs, and PLA2s. Nevertheless, the latter appears to be restricted to vipers and elapids, as it has never been reported as a major venom component in rear-fanged species. Here, by investigating the original transcriptomes from 19 species distributed in eight genera from the Pseudoboini tribe (Dipsadidae: Xenodontinae) and screening among seven additional tribes of Dipsadidae and three additional families of advanced snakes, we discovered that a novel type of venom PLA2, resembling a PLA2-IIE, has been recruited to the venom of some species of the Pseudoboini tribe, where it is a major component. Proteomic and functional analyses of these venoms further indicate that these PLA2s play a relevant role in the venoms from this tribe. Moreover, we reconstructed the phylogeny of PLA2s across different snake groups and show that different types of these toxins have been recruited in at least five independent events in caenophidian snakes. Additionally, we present the first compositional profiling of Pseudoboini venoms. Our results demonstrate how relevant phenotypic traits are convergently recruited by different means and from homologous and nonhomologous genes in phylogenetically and ecologically divergent snake groups, possibly optimizing venom composition to overcome diverse adaptative landscapes.

Venom phenotype conservation suggests integrated specialization in a lizard-eating snake.

Biological specialization reduces the size of niche space while increasing efficiency in the use of available resources. Specialization often leads to phenotypic changes via natural selection aligning with niche space constraints. Commonly observed changes are in size, shape, behavior, and traits associated with feeding. One often selected trait for dietary specialization is venom, which, in snakes, often shows variation dependent on diet across and within species. The Neotropical Blunt-headed Treesnake (Imantodes cenchoa) is a highly specialized, rear-fanged, arboreal, lizard hunter that displays a long thin body, enlarged eyes, and a large Duvernoy's gland. However, toxin characterization of I. cenchoa has never been completed. Here, we use RNA-seq and mass spectrometry to assemble, annotate, and analyze the venom gland transcriptomes of four I. cenchoa from across their range. We find a lack of significant venom variation at the sequence and expression levels, suggesting venom conservation across the species. We propose this conservation provides evidence of a specialized venom repertoire, adapted to maximize efficiency of capturing and processing lizards. Importantly, this study provides the most complete venom gland transcriptomes of I. cenchoa and evidence of venom specialization in a rear-fanged snake, giving insight into selective pressures of venom across all snake species.

Hepatozoon Miller, 1908 parasites in the Colubridae snakes Clelia clelia (Daudin, 1803) and Drymarchon corais (Boie, 1827) from the Eastern Amazonia.

Based on the genetic, morphological, and morphometric data of blood gamonts, we identified Hepatozoon parasites in colubrid snakes sampled in the Eastern Amazon region. Hepatozoon trigeminum was detected in the mussurana snake Clelia clelia and exhibited wide and elongated gamonts (mean dimensions: 14.25±0.65 × 4.31±0.43 µm) with an evident parasitophorous vacuole. Hepatozoon odwyerae sp. nov. was described in the indigo snake Drymarchon corais, whose gamonts have elongated and thin bodies (mean dimensions: 13.41±0.79 × 3.72±0.35 µm) with one end more tapered than the other. Phylogenetic analyses, based on the amplification of a 441 bp fragment of the 18S rRNA gene, revealed that the novel sequences of Hepatozoon spp. from our study were closely related to hemogregarine lineages found in lizards and snakes from Brazil, forming a well-supported monophyletic clade with them. The present study provides the first species description of Hepatoozon in D. corais and a new record of a host species for C. clelia using the integrated taxonomic data. We also highlight the importance of further investigations into the diversity of Hepatozoon in snakes, a rich but underestimated group of parasites, especially in the Amazonian biome.

Considering admixture when producing draft genomes: an example in North American ratsnakes (Pantherophis alleghaniensis/Pantherophis obsoletus).

The number of reference genomes of snakes lags behind several other vertebrate groups (e.g. birds and mammals). However, in the last two years, a concerted effort by researchers from around the world has produced new genomes of snakes representing members from several new families. Here, we present a high-quality, annotated genome of the central ratsnake (Pantherophis alleghaniensis), a member of the most diverse snake lineage, Colubroidea. Pantherophis alleghaniensis is found in the central part of the Nearctic, east of the Mississippi River. This genome was sequenced using 10X Chromium synthetic long reads and polished using Illumina short reads. The final genome assembly had an N50 of 21.82 Mb and an L50 of 22 scaffolds with a maximum scaffold length of 82.078 Mb. The genome is composed of 49.24% repeat elements dominated by long interspersed elements. We annotated this genome using transcriptome assemblies from 14 tissue types and recovered 28,368 predicted proteins. Finally, we estimated admixture proportions between two species of ratsnakes and discovered that this specimen is an admixed individual containing genomes from the western (Pantherophis obsoletus) and central ratsnakes (P. alleghaniensis). We discuss the importance of considering interspecific admixture in downstream approaches for inferring demography and phylogeny.

Conspicuous coloration of toxin-resistant predators implicates additional trophic interactions in a predator-prey arms race.

Antagonistic coevolution between natural enemies can produce highly exaggerated traits, such as prey toxins and predator resistance. This reciprocal process of adaptation and counter-adaptation may also open doors to other evolutionary novelties not directly involved in the phenotypic interface of coevolution. We tested the hypothesis that predator-prey coevolution coincided with the evolution of conspicuous coloration on resistant predators that retain prey toxins. In western North America, common garter snakes (Thamnophis sirtalis) have evolved extreme resistance to tetrodotoxin (TTX) in the coevolutionary arms race with their deadly prey, Pacific newts (Taricha spp.). TTX-resistant snakes can retain large amounts of ingested TTX, which could serve as a deterrent against the snakes' own predators if TTX toxicity and resistance are coupled with a conspicuous warning signal. We evaluated whether arms race escalation covaries with bright red coloration in snake populations across the geographic mosaic of coevolution. Snake colour variation departs from the neutral expectations of population genetic structure and covaries with escalating clines of newt TTX and snake resistance at two coevolutionary hotspots. In the Pacific Northwest, bright red coloration fits an expected pattern of an aposematic warning to avian predators: TTX-resistant snakes that consume highly toxic newts also have relatively large, reddish-orange dorsal blotches. Snake coloration also seems to have evolved with the arms race in California, but overall patterns are less intuitively consistent with aposematism. These results suggest that interactions with additional trophic levels can generate novel traits as a cascading consequence of arms race coevolution across the geographic mosaic.

Rediscovery and systematics of the rarely encountered Blue-bellied kukri snake (Oligodon melaneus Wall, 1909) from Assam, India.

We report the rediscovery of Oligodon melaneus 112 years after its original description and document the third, and only non-type, specimen for the species. The new specimen was found 267 km east of the type locality (Tindharia, West Bengal state) from Assam state, India. We designate a lectotype for the species, and provide an extended description of a freshly collected male specimen. Phylogenetic analyses of 16s and cytb mitochondrial genes provide support for O. melaneus being closely related to the widespread South Asian endemic O. arnensis.

Reference genome of the California glossy snake, Arizona elegans occidentalis: A declining California Species of Special Concern.

The glossy snake (Arizona elegans) is a polytypic species broadly distributed across southwestern North America. The species occupies habitats ranging from California's coastal chaparral to the shortgrass prairies of Texas and southeastern Nebraska, to the extensive arid scrublands of central México. Three subspecies are currently recognized in California, one of which is afforded state-level protection based on the extensive loss and modification of its preferred alluvial coastal scrub and inland desert habitat. We report the first genome assembly of A. elegans occidentalis as part of the California Conservation Genomics Project (CCGP). Consistent with the reference genome strategy of the CCGP, we used Pacific Biosciences HiFi long reads and Hi-C chromatin-proximity sequencing technologies to produce a de novo assembled genome. The assembly comprises a total of 140 scaffolds spanning 1,842,602,218 base pairs, has a contig NG50 of 61 Mb, a scaffold NG50 of 136 Mb, and a BUSCO complete score of 95.9%, and is one of the most complete snake genome assemblies. The A. e. occidentalis genome will be a key tool for understanding the genomic diversity and the basis of adaptations within this species and close relatives within the hyperdiverse snake family Colubridae.

Divergent Specialization of Simple Venom Gene Profiles among Rear-Fanged Snake Genera (Helicops and Leptodeira, Dipsadinae, Colubridae).

Many venomous animals express toxins that show extraordinary levels of variation both within and among species. In snakes, most studies of venom variation focus on front-fanged species in the families Viperidae and Elapidae, even though rear-fanged snakes in other families vary along the same ecological axes important to venom evolution. Here we characterized venom gland transcriptomes from 19 snakes across two dipsadine rear-fanged genera (Leptodeira and Helicops, Colubridae) and two front-fanged genera (Bothrops, Viperidae; Micrurus, Elapidae). We compared patterns of composition, variation, and diversity in venom transcripts within and among all four genera. Venom gland transcriptomes of rear-fanged Helicops and Leptodeira and front-fanged Micrurus are each dominated by expression of single toxin families (C-type lectins, snake venom metalloproteinase, and phospholipase A2, respectively), unlike highly diverse front-fanged Bothrops venoms. In addition, expression patterns of congeners are much more similar to each other than they are to species from other genera. These results illustrate the repeatability of simple venom profiles in rear-fanged snakes and the potential for relatively constrained venom composition within genera.

Uncovering Cryptic Coevolution.

AbstractStudies of coevolution in the wild have largely focused on reciprocally specialized species pairs with striking and exaggerated phenotypes. Textbook examples include interactions between toxic newts and their garter snake predators, long-tongued flies and the flowers they pollinate, and weevils with elongated rostra used to bore through the defensive pericarp of their host plants. Although these studies have laid a foundation for understanding coevolution in the wild, they have also contributed to the widespread impression that coevolution is a rare and quirky sideshow to the day-to-day grind of ecology and evolution. In this perspective, we argue that the focus of coevolution has been biased toward the obvious and ignored the cryptic. We have focused on the obvious-studies of reciprocally specialized species pairs with exaggerated phenotypes-mainly because we have lacked the statistical tools required to study coevolution in more generalized and phenotypically mundane systems. Building from well-established coevolutionary theory, we illustrate how model-based approaches can be used to remove this barrier and begin estimating the strength of coevolutionary selection indirectly using routinely collected data, thus uncovering cryptic coevolution in more typical communities. By allowing the distribution of coevolutionary selection to be estimated across genomes, phylogenies, and communities and over deep timescales, these novel approaches have the potential to revolutionize the way we study coevolution. As we develop a road map to these next-generation approaches, we highlight recent studies making notable progress in this direction.

The road not taken: Evolution of tetrodotoxin resistance in the Sierra garter snake (Thamnophis couchii) by a path less travelled.

The repeated evolution of tetrodotoxin (TTX) resistance provides a model for testing hypotheses about the mechanisms of convergent evolution. This poison is broadly employed as a potent antipredator defence, blocking voltage-gated sodium channels (Nav ) in muscles and nerves, paralysing and sometimes killing predators. Resistance in taxa bearing this neurotoxin and a few predators appears to come from convergent replacements in specific Nav residues that interact with TTX. This stereotyped genetic response suggests molecular and phenotypic evolution may be constrained and predictable. Here, we investigate the extent of mechanistic convergence in garter snakes (Thamnophis) that prey on TTX-bearing newts (Taricha) by examining the physiological and genetic basis of TTX resistance in the Sierra garter snake (Th. couchii). We characterize variation in this predatory adaptation across populations at several biological scales: whole-animal TTX resistance; skeletal muscle resistance; functional genetic variation in three Nav encoding loci; and levels of gene expression for one of these loci. We found Th. couchii possess extensive geographical variation in resistance at the whole-animal and skeletal muscle levels. As in other Thamnophis, resistance at both levels is highly correlated, suggesting convergence across the biological levels linking organism to organ. However, Th. couchii shows no functional variation in Nav loci among populations or difference in candidate gene expression. Local variation in TTX resistance in Th. couchii cannot be explained by the same relationship between genotype and phenotype seen in other taxa. Thus, historical contingencies may lead different species of Thamnophis down alternative routes to local adaptation.

Toxic Relationships and Arms-Race Coevolution Revisited.

Toxin evolution in animals is one of the most fascinating and complex subjects of scientific inquiry today. Gaining an understanding of toxins poses a multifaceted challenge given the diverse modes of acquisition, evolutionary adaptations, and abiotic components that affect toxin phenotypes. Here, we highlight some of the main genetic and ecological factors that influence toxin evolution and discuss the role of antagonistic interactions and coevolutionary dynamics in shaping the direction and extent of toxicity and resistance in animals. We focus on toxic Pacific newts (family Salamandridae, genus Taricha) as a system to investigate and better evaluate the widely distributed toxin they possess, tetrodotoxin (TTX), and the hypothesized model of arms-race coevolution with snake predators that is used to explain phenotypic patterns of newt toxicity. Finally, we propose an alternative coevolutionary model that incorporates TTX-producing bacteria and draws from an elicitor-receptor concept to explain TTX evolution and ecology.

Every breath you take: assessing metabolic costs of toxin resistance in garter snakes (Thamnophis).

Trait specialization often comes at the expense of original trait function, potentially causing evolutionary tradeoffs that may render specialist populations vulnerable to extinction. However, many specialized adaptations evolve repeatedly, suggesting selection favors specialization in specific environments. Some garter snake (Thamnophis) populations possess specialized mutations in voltage-gated sodium channels that allow them to consume Pacific newts (Taricha) defended by a highly potent neurotoxin (tetrodotoxin). These mutations, however, also decrease protein and muscle function, suggesting garter snakes may suffer evolutionary tradeoffs. We measured a key physiological process, standard metabolic rate (SMR), to investigate whether specialized adaptations in toxin-resistant garter snakes affect baseline energy expenditure. In snakes, skeletal muscles influence metabolism and power ventilation, so inefficiencies of sodium channels in these muscles might impact whole-animal energy expenditure. Further, because sodium channels are membrane-bound proteins, inefficiencies of channel kinetics and performance might be exacerbated at suboptimal temperatures. We measured SMR in 2 species, Thamnophis atratus and Thamnophis sirtalis, that independently evolved tetrodotoxin resistance through unique mutations, providing replicate experiments with distinct underlying genetics and potential physiological costs. Despite our expectations, neither resistance phenotype nor sodium channel genotype affected metabolism and resistant snakes did not perform worse under suboptimal body temperature. Instead, T. atratus and T. sirtalis show nearly identical rates of mass-adjusted energy expenditure at both temperatures, despite differing eco-morphologies, life histories, and distant phylogenetic positions. These findings suggest SMR may be a conserved feature of Thamnophis, and that any organismal tradeoffs may be compensated to retain whole-animal function.

Phylogenomic analyses resolve relationships among garter snakes (Thamnophis: Natricinae: Colubridae) and elucidate biogeographic history and morphological evolution.

Garter snakes (Thamnophis) are a successful group of natricines endemic to North America. They have become important natural models for ecological and evolutionary research, yet prior efforts to resolve phylogenetic relationships have resulted in conflicting topologies and weak support for certain relationships. Here, we use genomic data generated with a reduced representation double-digest RADseq approach to reassess evolutionary relationships across Thamnophis. We then use the resulting phylogeny to better understand how biogeography and feeding ecology have influenced lineage diversification and morphological evolution. We recovered highly congruent and strongly supported topologies from maximum likelihood and Bayesian analyses, but some discordance with a multispecies coalescent approach. All phylogenomic estimates split Thamnophis into two clades largely defined by northern and southern North American species. Divergence time estimates and biogeographic analyses indicate a mid-Miocene origin of Thamnophis in Mexico. In addition, historic vicariant events thought to explain biogeographic patterns in other lineages (e.g., Isthmus of Tehuantepec, Rocky Mountain Range, and Trans-Mexican Volcanic Belt) appear to have influenced patterns of diversification in Thamnophis as well. Analyses of morphological traits associated with feeding ecology showed moderate to strong phylogenetic signal. Nevertheless, phylogenetic ANOVA suggested significant differences in certain cranial morphologies between aquatic specialists and garter snakes that are terrestrial-aquatic generalists, independent of evolutionary history. Our new estimate of Thamnophis phylogeny yields an improved understanding of the biogeographic history and morphological evolution of garter snakes, and provides a robust framework for future research on these snakes.

Cryptic diversity and phylogeography of the Rhabdophis nuchalis group (Squamata: Colubridae).

Previous studies, have found that the rapid uplift of the Tibetan plateau accelerated the diversification of species. However, there are few relevant biogeographic data for the Colubridae in this region. We conducted a comprehensive study of the Rhabdophis nuchalis Group, which presently contains four nominal species, R. nuchalis, R. pentasupralabialis, R. leonardi, and R. chiwen. Building upon previous studies with specimens we have recently examined, greater interspecific and intraspecific diversity has been revealed. Here we address three questions: (1) Do the intraspecific differences represent only geographic variation within lineages, or are there cryptic species? (2) What are the interspecific relationships among members of the R. nuchalis Group? (3) What has been the biogeographic history of this species group? To resolve these questions we used four mitochondrial gene sequences and one nuclear sequence to investigate the molecular phylogenetic and geographic relationships among populations. Our molecular analysis reveals cryptic species diversity within the R. nuchalis Group, and seven clades were identified in the analysis. Ancestral area estimation suggests that the R. nuchalis Group originated in the Hengduan Mountains approximately 6.24 Mya and expanded its range northward to the Qinling-Daba Mountains. The Sichuan Basin appears to have been a barrier to migration. Species divergence seems to have been related to the rapid uplift of the Qinghai-Tibet Plateau.