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.

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.

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.

Molecular phylogeny of Neotropical Parrot Snakes (Serpentes: Colubrinae: Leptophis) supports underestimated species richness.

Tetrapod taxa with broad geographic distributions across the Neotropics are often composed of multiple evolutionary lineages. In this paper, we present the most complete phylogeny of Leptophis to date and assess morphology-based species limits within the broadly distributed green parrot snake Leptophis ahaetulla sensu lato, which occurs from Mexico to Argentina. Although L. ahaetulla sensu stricto, L. nigromarginatus and L. occidentalis were recovered as paraphyletic, tree topology tests failed to reject their monophyly. Monophyly of L. bocourti, L. coeruleodorsus, L. cupreus, L. depressirostris, L. marginatus, L. riveti and L. sp. nov. was strongly supported. Our phylogenetic trees support recognition of multiple species within Leptophis ahaetulla sensu lato and suggest that color evolution and the uplift of the Andes played an important role in the diversification of parrot snakes.

Philodryas (Serpentes: Dipsadidae) Envenomation, a Neglected Issue in Chile.

Snakebite envenomation is considered a neglected tropical disease, although it also occurs outside the tropics. In this work, we analyzed the literature on Philodryas species in Chile (Philodryaschamissonis, P.simonsii, and P.tachymenoides) from 1834 to 2019, searching for epidemiological, clinical, and molecular aspects of envenomation. Ninety-one percent of the studies found regarded taxonomy, ecology, and natural history, suggesting that snakebites and venom toxins are a neglected issue in Chile. All snakebite cases reported and toxicological studies concerned the species Philodryaschamissonis. Using 185 distributional records from the literature and museum collections for this species, we show for the first time that the reported snakebite cases correlate with human population density, occurring in the Valparaiso and Metropolitan regions in Central Chile. The reduced number of snakebite cases, which were previously considered as having a low incidence in Chile, may be a consequence of under-reported cases, probably due to the inadequate publication or scarce research on this issue. Absence of information about official pharmacological treatment, post-envenoming sequels, clinical management of particular patient groups (e.g., with non-communicable diseases, pregnant women, and the elderly) was also detected. In conclusion, despite having over 185 years of literature on Chilean snakes, knowledge on the envenomation of Philodryas genus remains scarce, seriously affecting adequate medical handling during an ophidic accident. This review highlights the need to develop deep research in this area and urgent improvements to the management of this disease in Chile.

Phylogeny, diversity and biogeography of Neotropical sipo snakes (Serpentes: Colubrinae: Chironius).

Neotropical sipo snakes (Chironius) are large diurnal snakes with a long tail and big eyes that differ from other Neotropical snakes in having 10 or 12 dorsal scale rows at midbody. The 22 currently recognized species occur from Central America south to Uruguay and northeastern Argentina. Based on the largest geographical sampling to date including ∼90% of all species, we analyzed one nuclear and three mitochondrial genes using phylogenetic methods to (1) test the monophyly of Chironius and some of its widely distributed species; (2) identify lineages that could represent undescribed species; and (3) reconstruct ancestral distributions. Our best hypothesis placed C. grandisquamis (Chocoan Rainforest) + C. challenger (Pantepui) as sister to all other species. Based on phylogeny and geographic distribution, we identified 14 subclades as putative species within Chironius fuscus, C. multiventris (including C. foveatus and C. laurenti), C. monticola, and C. exoletus. Under current taxonomy, these species show nearly twice as much genetic diversity as other species of Chironius for ND4. Biogeographical analyses using BioGeoBEARS suggest that current distribution patterns of Chironius species across South America resulted from multiple range expansions. The MRCA of the clade C. challenger + C. grandisquamis was most likely distributed over the Pantepui region, the Andes, and the Chocoan Rainforest, whereas the remaining lineages probably evolved from an Amazonian ancestor.

The Harderian gland transcriptomes of Caraiba andreae, Cubophis cantherigerus and Tretanorhinus variabilis, three colubroid snakes from Cuba.

The Harderian gland is a cephalic structure, widely distributed among vertebrates. In snakes, the Harderian gland is anatomically connected to the vomeronasal organ via the nasolacrimal duct, and in some species can be larger than the eyes. The function of the Harderian gland remains elusive, but it has been proposed to play a role in the production of saliva, pheromones, thermoregulatory lipids and growth factors, among others. Here, we have profiled the transcriptomes of the Harderian glands of three non-front-fanged colubroid snakes from Cuba: Caraiba andreae (Cuban Lesser Racer); Cubophis cantherigerus (Cuban Racer); and Tretanorhinus variabilis (Caribbean Water Snake), using Illumina HiSeq2000 100 bp paired-end. In addition to ribosomal and non-characterized proteins, the most abundant transcripts encode putative transport/binding, lipocalin/lipocalin-like, and bactericidal/permeability-increasing-like proteins. Transcripts coding for putative canonical toxins described in venomous snakes were also identified. This transcriptional profile suggests a more complex function than previously recognized for this enigmatic organ.

Comparative cytogenetic analysis in Erythrolamprus snakes (Serpentes: Dipsadidae) from Argentina.

We described the karyotypes of five snake taxa from Argentina: Erythrolamprus almadensis, E. ceii, E. poecilogyrus caesius, E. p. schotti and E. p. sublineatus, and also intergrading individuals between the last two subspecies by conventional staining, chromosome bandings and fluorescent in situ hybridization (FISH) with 28S ribosomal DNA probes. Erythrolamprus ceii and E. almadensis share a diploid chromosome number of 2n= 28, whereas in E. poecilogyrus intraspecific variations were observed: E. p. caesius has 2n= 28, E. p. schotti and E. p. sublineatus as well as in the intergrading individuals have 2n= 32. In E. almadensis and E. p. caesius, the 2nd and 6th chromosome pairs respectively are heteromorphic by size, morphology and C-banding pattern. These results allow us to suggest that these chromosome pairs might be considered as the ZW sex chromosomes in these species. The present comparative cytogenetic analyzes contributes to the already remarkable karyotypic variability in Erythrolamprus genus and propose a hypothesis about potential mechanisms involved in the chromosome evolution among taxa analyzed. Furthermore, the karyotypic differences observed between E. p. caesius (2n= 28) and E. p. schotti and E. p. sublineatus (2n= 32) might play a causal role in speciation.

The Biogeography of Deep Time Phylogenetic Reticulation.

Most phylogenies are typically represented as purely bifurcating. However, as genomic data have become more common in phylogenetic studies, it is not unusual to find reticulation among terminal lineages or among internal nodes (deep time reticulation; DTR). In these situations, gene flow must have happened in the same or adjacent geographic areas for these DTRs to have occurred and therefore biogeographic reconstruction should provide similar area estimates for parental nodes, provided extinction or dispersal has not eroded these patterns. We examine the phylogeny of the widely distributed New World kingsnakes (Lampropeltis), determine if DTR is present in this group, and estimate the ancestral area for reticulation. Importantly, we develop a new method that uses coalescent simulations in a machine learning framework to show conclusively that this phylogeny is best represented as reticulating at deeper time. Using joint probabilities of ancestral area reconstructions on the bifurcating parental lineages from the reticulating node, we show that this reticulation likely occurred in northwestern Mexico/southwestern US, and subsequently, led to the diversification of the Mexican kingsnakes. This region has been previously identified as an area important for understanding speciation and secondary contact with gene flow in snakes and other squamates. This research shows that phylogenetic reticulation is common, even in well-studied groups, and that the geographic scope of ancient hybridization is recoverable.

Daily activity patterns influence retinal morphology, signatures of selection, and spectral tuning of opsin genes in colubrid snakes.

BACKGROUND: Morphological divergences of snake retinal structure point to complex evolutionary processes and adaptations. The Colubridae family has a remarkable variety of retinal structure that can range from all-cone and all-rod to duplex (cone/rod) retinas. To explore whether nocturnal versus diurnal activity is responsible for constraints on molecular evolution and plays a role in visual opsin spectral tuning of colubrids, we carried out molecular evolution analyses of the visual opsin genes LWS, RH1, and SWS1 from 17 species and performed morphological analyses. RESULTS: Phylogenetic reconstructions of the RH1 and LWS recovered major clades characterized by primarily diurnal or primarily nocturnal activity patterns, in contrast with the topology for SWS1, which is very similar to the species tree. We found stronger signals of purifying selection along diurnal and nocturnal lineages for RH1 and SWS1, respectively. A blue-shift of the RH1 spectral peak is associated with diurnal habits. Spectral tuning of cone opsins did not differ among diurnal and nocturnal species. Retinas of nocturnal colubrids had many rows of photoreceptor nuclei, with large numbers of rods, labeled by wheat germ agglutinin (WGA), and two types of cones: large cones sensitive to long/medium wavelengths (L/M) and small cones sensitive to ultra-violet/violet wavelengths (UV/VS). In contrast, retinas of diurnal species had only one row of photoreceptor nuclei, with four types of cones: large and double L/M cones, small UV/VS cones, and a second group of small cones, labeled by WGA. CONCLUSIONS: For LWS gene, selection tests did not confirm different constraints related to activity pattern. For SWS1, stronger purifying selection in nocturnal lineages indicates divergent evolutionary pressures related to the activity pattern, and the importance of the short wavelength sensitivity at low light condition. Activity pattern has a clear influence on the signatures of selection and spectral tuning of RH1, with stronger purifying selection in diurnal lineages, which indicates selective pressure to preserve rhodopsin structure and function in pure-cone retinas. We suggest that the presence of four cone types in primarily diurnal colubrids might be related to the gain of color discrimination capacity.

Evolutionary analysis of Chironius snakes unveils cryptic diversity and provides clues to diversification in the Neotropics.

Recent hypotheses to explain tropical diversity involves the Neogene and Quaternary geoclimatic dynamics, but the absence of unambiguous data permitting the choice between alternative hypotheses makes a general theory for the origin of tropical biodiversity far to be achieved. The occurrence of Chironius snakes in well-defined biogeographical regions led us to adopt Chironius as a model to unveil patterns of vertebrate diversification in the Neotropics. Here, we used molecular markers and records on geographic distribution to investigate Chironius evolution and, subsequently, providing hints on diversification in the Neotropics. To avoid analyzing nominal species that do not constitute exclusive evolutionary lineages, we firstly conducted a species delimitation study prior to carrying out the species distribution modeling analysis. We generated 161 sequences of 12S, 16S, c-mos and rag2 for 15 species and 50 specimens, and included additional data from GenBank yielding a matrix of 137 terminals, and performed the following evolutionary analyses: inference of a concatenated gene tree, estimation of gene divergence times, inference of the coalescent-based phylogeny of Chironius, estimation of effective population sizes and modeling potential distribution of species across the last millennia. We tested for species boundaries within Chironius by implementing a coalescent-based Bayesian species delimitation approach. Our analyses supported the monophyly of Chironius, although our findings underscored cryptic candidate species in C. flavolineatus and C. exoletus. The inferred timetree suggested that Chironius snakes have evolved in the early Miocene (ca. 20.2Mya) and began to diversify from the late Miocene to the early Pliocene, values that are much older than previously reported. Following genetic divergence of virtually all extant Chironius species investigated, the effective sizes of the populations have expanded when compared to their MRCAs. The evolutionary and SDM data from C. brazili and C. diamantina provided additional evidence of the origin of species in the Neotropics. We argue that temperature, instead of altitude, has been the major driving factor in the evolution of both species, and thus we present a case for the consequences of global warming.

Trends in the Evolution of Snake Toxins Underscored by an Integrative Omics Approach to Profile the Venom of the Colubrid Phalotris mertensi.

Only few studies on snake venoms were dedicated to deeply characterize the toxin secretion of animals from the Colubridae family, despite the fact that they represent the majority of snake diversity. As a consequence, some evolutionary trends observed in venom proteins that underpinned the evolutionary histories of snake toxins were based on data from a minor parcel of the clade. Here, we investigated the proteins of the totally unknown venom from Phalotris mertensi (Dipsadinae subfamily), in order to obtain a detailed profile of its toxins and to appreciate evolutionary tendencies occurring in colubrid venoms. By means of integrated omics and functional approaches, including RNAseq, Sanger sequencing, high-resolution proteomics, recombinant protein production, and enzymatic tests, we verified an active toxic secretion containing up to 21 types of proteins. A high content of Kunitz-type proteins and C-type lectins were observed, although several enzymatic components such as metalloproteinases and an L-amino acid oxidase were also present in the venom. Interestingly, an arguable venom component of other species was demonstrated as a true venom protein and named svLIPA (snake venom acid lipase). This finding indicates the importance of checking the actual protein occurrence across species before rejecting genes suggested to code for toxins, which are relevant for the discussion about the early evolution of reptile venoms. Moreover, trends in the evolution of some toxin classes, such as simplification of metalloproteinases and rearrangements of Kunitz and Wap domains, parallel similar phenomena observed in other venomous snake families and provide a broader picture of toxin evolution.

Colubrid Venom Composition: An -Omics Perspective.

Snake venoms have been subjected to increasingly sensitive analyses for well over 100 years, but most research has been restricted to front-fanged snakes, which actually represent a relatively small proportion of extant species of advanced snakes. Because rear-fanged snakes are a diverse and distinct radiation of the advanced snakes, understanding venom composition among "colubrids" is critical to understanding the evolution of venom among snakes. Here we review the state of knowledge concerning rear-fanged snake venom composition, emphasizing those toxins for which protein or transcript sequences are available. We have also added new transcriptome-based data on venoms of three species of rear-fanged snakes. Based on this compilation, it is apparent that several components, including cysteine-rich secretory proteins (CRiSPs), C-type lectins (CTLs), CTLs-like proteins and snake venom metalloproteinases (SVMPs), are broadly distributed among "colubrid" venoms, while others, notably three-finger toxins (3FTxs), appear nearly restricted to the Colubridae (sensu stricto). Some putative new toxins, such as snake venom matrix metalloproteinases, are in fact present in several colubrid venoms, while others are only transcribed, at lower levels. This work provides insights into the evolution of these toxin classes, but because only a small number of species have been explored, generalizations are still rather limited. It is likely that new venom protein families await discovery, particularly among those species with highly specialized diets.

Molecular phylogeny of Caribbean dipsadid (Xenodontinae: Alsophiini) snakes, including identification of the first record from the Cay Sal Bank, The Bahamas.

We document the first specimen of a dipsadid snake from the Anguilla Cays, Cay Sal Bank, The Bahamas. We analyze 3,426 base pairs (bp) of sequence data derived from five mitochondrial loci and one nuclear locus using Maximum Likelihood (ML) and Bayesian Inference (BI) methods. Our molecular data agree with some aspects of morphology (e.g., scale counts, dentition, and color pattern) supporting identification of this specimen as the Cuban Racer, Cubophis cantherigerus cantherigerus (Bibron 1840), a species previously regarded as endemic to Cuba. This discovery provides another example of the strong Cuban affinities of the terrestrial vertebrate fauna of Bahamian islands.

Fuzzy boundaries: color and gene flow patterns among parapatric lineages of the western shovel-nosed snake and taxonomic implication.

Accurate delineation of lineage diversity is increasingly important, as species distributions are becoming more reduced and threatened. During the last century, the subspecies category was often used to denote phenotypic variation within a species range and to provide a framework for understanding lineage differentiation, often considered incipient speciation. While this category has largely fallen into disuse, previously recognized subspecies often serve as important units for conservation policy and management when other information is lacking. In this study, we evaluated phenotypic subspecies hypotheses within shovel-nosed snakes on the basis of genetic data and considered how evolutionary processes such as gene flow influenced possible incongruence between phenotypic and genetic patterns. We used both traditional phylogenetic and Bayesian clustering analyses to infer range-wide genetic structure and spatially explicit analyses to detect possible boundary locations of lineage contact. Multilocus analyses supported three historically isolated groups with low to moderate levels of contemporary gene exchange. Genetic data did not support phenotypic subspecies as exclusive groups, and we detected patterns of discordance in areas where three subspecies are presumed to be in contact. Based on genetic and phenotypic evidence, we suggested that species-level diversity is underestimated in this group and we proposed that two species be recognized, Chionactis occipitalis and C. annulata. In addition, we recommend retention of two subspecific designations within C. annulata (C. a. annulata and C. a. klauberi) that reflect regional shifts in both genetic and phenotypic variation within the species. Our results highlight the difficultly in validating taxonomic boundaries within lineages that are evolving under a time-dependent, continuous process.

Consuming viscous prey: a novel protein-secreting delivery system in neotropical snail-eating snakes.

BACKGROUND: Efficient venom delivery systems are known to occur only in varanoid lizards and advanced colubroidean snakes among squamate reptiles. Although components of these venomous systems might have been present in a common ancestor, the two lineages independently evolved strikingly different venom gland systems. In snakes, venom is produced exclusively by serous glands in the upper jaw. Within the colubroidean radiation, lower jaw seromucous infralabial glands are known only in two distinct lineages-the basal pareatids and the more advanced Neotropical dipsadines known as "goo-eating snakes". Goo-eaters are a highly diversified, ecologically specialized clade that feeds exclusively on invertebrates (e.g., gastropod molluscs and annelids). Their evolutionary success has been attributed to their peculiar feeding strategies, which remain surprisingly poorly understood. More specifically, it has long been thought that the more derived Dipsadini genera Dipsas and Sibynomorphus use glandular toxins secreted by their infralabial glands to extract snails from their shells. RESULTS: Here, we report the presence in the tribe Dipsadini of a novel lower jaw protein-secreting delivery system effected by a gland that is not functionally related to adjacent teeth, but rather opens loosely on the oral epithelium near the tip of the mandible, suggesting that its secretion is not injected into the prey as a form of envenomation but rather helps control the mucus and assists in the ingestion of their highly viscous preys. A similar protein-secreting system is also present in the goo-eating genus Geophis and may share the same adaptive purpose as that hypothesized for Dipsadini. Our phylogenetic hypothesis suggests that the acquisition of a seromucous infralabial gland represents a uniquely derived trait of the goo-eating clade that evolved independently twice within the group as a functionally complex protein-secreting delivery system. CONCLUSIONS: The acquisition by snail-eating snakes of such a complex protein-secreting system suggests that the secretion from the hypertrophied infralabial glands of goo-eating snakes may have a fundamental role in mucus control and prey transport rather than envenomation of prey. Evolution of a functional secretory system that combines a solution for mucus control and transport of viscous preys is here thought to underlie the successful radiation of goo-eating snakes.

Refugial isolation and divergence in the Narrowheaded Gartersnake species complex (Thamnophis rufipunctatus) as revealed by multilocus DNA sequence data.

Glacial-interglacial cycles of the Pleistocene are hypothesized as one of the foremost contributors to biological diversification. This is especially true for cold-adapted montane species, where range shifts have had a pronounced effect on population-level divergence. Gartersnakes of the Thamnophis rufipunctatus species complex are restricted to cold headwater streams in the highlands of the Sierra Madre Occidental and southwestern USA. We used coalescent and multilocus phylogenetic approaches to test whether genetic diversification of this montane-restricted species complex is consistent with two prevailing models of range fluctuation for species affected by Pleistocene climate changes. Our concatenated nuDNA and multilocus species analyses recovered evidence for the persistence of multiple lineages that are restricted geographically, despite a mtDNA signature consistent with either more recent connectivity (and introgression) or recent expansion (and incomplete lineage sorting). Divergence times estimated using a relaxed molecular clock and fossil calibrations fall within the Late Pleistocene, and zero gene flow scenarios among current geographically isolated lineages could not be rejected. These results suggest that increased climate shifts in the Late Pleistocene have driven diversification and current range retraction patterns and that the differences between markers reflect the stochasticity of gene lineages (i.e. ancestral polymorphism) rather than gene flow and introgression. These results have important implications for the conservation of T. rufipunctatus (sensu novo), which is restricted to two drainage systems in the southwestern US and has undergone a recent and dramatic decline.

Biological and proteomic analysis of venom from the Puerto Rican Racer (Alsophis portoricensis: Dipsadidae).

The Puerto Rican Racer Alsophis portoricensis is known to use venom to subdue lizard prey, and extensive damage to specific lizard body tissues has been well documented. The toxicity and biochemistry of the venom, however, has not been explored extensively. We employed biological assays and proteomic techniques to characterize venom from A. portoricensis anegadae collected from Guana Island, British Virgin Islands. High metalloproteinase and gelatinase, as well as low acetylcholinesterase and phosphodiesterase activities were detected, and the venom hydrolyzed the alpha-subunit of human fibrinogen very rapidly. SDS-PAGE analysis of venoms revealed up to 22 protein bands, with masses of approximately 5-160 kDa; very little variation among individual snakes or within one snake between venom extractions was observed. Most bands were approximately 25-62 kD, but MALDI-TOF analysis of crude venom indicated considerable complexity in the 1.5-13 kD mass range, including low intensity peaks in the 6.2-8.8 kD mass range (potential three-finger toxins). MALDI-TOF/TOF MS analysis of tryptic peptides confirmed that a 25 kDa band was a venom cysteine-rich secretory protein (CRiSP) with sequence homology with tigrin, a CRiSP from the natricine colubrid Rhabdophis tigrinus. The venom was quite toxic to NSA mice (Mus musculus: LD(50)=2.1 microg/g), as well as to Anolis lizards (A. carolinensis: 3.8 microg/g). Histology of the venom gland showed distinctive differences from the supralabial salivary glands (serous vs. mucosecretory), and like the Brown Treesnake (Boiga irregularis), another rear-fanged snake, serous secretory cells are arranged in densely packed secretory tubules, with little venom present in tubule lumina. These results clearly demonstrate that venom from A. portoricensis shares components with venoms of front-fanged snakes as well as with other rear-fanged species. Venom from A. portoricensis, in particular the prominent metalloproteinase activity, likely serves an important trophic function by facilitating prey handling and predigestion of prey.

Preliminary biochemical characterization of the venomsof five Colubridae species from Brazil

The paraphyletic family Colubridae comprises several species of rear-fanged snakes with toxin-secreting Duvernoy’s gland, some of them able to cause human envenomation withsystemic and/or local damage. In this work we have explored some aspects of biochemical composition and activity of the venoms of five species from Colubridae family from Brazil.Taken together our results suggest distinct features in colubrid venoms, which could be related to the presence of still unknown toxins.