Unveiling underestimated species diversity within the Central American Coralsnake, a medically important complex of venomous taxa.

Coralsnakes of the genus Micrurus are a diverse group of venomous snakes ranging from the southern United States to southern South America. Much uncertainty remains over the genus diversity, and understanding Micrurus systematics is of medical importance. In particular, the widespread Micrurus nigrocinctus spans from Mexico throughout Central America and into Colombia, with a number of described subspecies. This study provides new insights into the phylogenetic relationships within M. nigrocinctus by examining sequence data from a broad sampling of specimens from Mexico, Guatemala, Honduras, Nicaragua, Costa Rica, and Panama. The recovered phylogenetic relationships suggest that M. nigrocinctus is a species complex originating in the Pliocene and composed of at least three distinct species-level lineages. In addition, recovery of highly divergent clades supports the elevation of some currently recognized subspecies to the full species rank while others may require synonymization.

A new species of Dipsas (Serpentes, Dipsadidae) from central Panama.

A new species of Dipsas Laurenti, 1768, from Central Panama is described based on molecular analyses, hemipenial morphology, and external characters. This is the sixth species of Dipsas to be described for the country; the snake has been suspected to exist since 1977 and has not been thoroughly studied until now. Additionally, morphological comparations including scale counts are done with other species within the genus, and the current geographic distribution of Dipsastemporalis (Werner, 1909), the sister species, is updated. Finally, a key to the species of Dipsas currently known from Middle America is presented.

ResumenDescribimos una nueva especies de Dipsas Laurenti, 1768 de la región central de Panamá en base a análisis moleculares, morfología hemipenial y caracteres de morfología externa. Esta es la sexta especie del género Dipsas descrita para el país. Se sospechaba su existencia desde 1977 pero no había sido estudiada exhaustivamente hasta ahora. Adicionalmente, presentamos comparaciones morfológicas (incluyendo lepidosis) con otras especies del género y actualizamos la distribución geográfica de su especie hermana Dipsastemporalis (Werner, 1909). Finalmente, presentamos una clave para las especies de Dipsas distribuidas en Centroamérica.

A new species of Sceloporus of the torquatus group (Reptilia: Phrynosomatidae) from West Mexico.

We describe Sceloporus huichol sp. nov., a new species of spiny lizard of the genus Sceloporus, that is found in the mountainous regions of Jalisco and Nayarit in western Mexico. The new species belongs to the torquatus species group and has previously been confused with Sceloporus melanogaster, from which it differs by its smaller size, head color and nuchal collar. Additionally, Sceloporus huichol sp. nov. is different from the rest of its congeners by relevant genetic and morphologicall data. Despite having a complete nuchal collar, characteristic of Sceloporus torquatus, S. huichol sp. nov. is more closely related phylogenetically to S. melanogaster. Recognition of this new species brings the number of species in the torquatus group to nine.

A new species of Oreosaurus (Squamata: Gymnophthalmidae) from the Turimiquire Massif, northeastern Venezuela.

We describe a new species of gymnophthalmid lizard, Oreosaurus bisbali sp. nov., from the Turimiquire Massif in northeastern Venezuela. It is one of three reptile species known to be endemic to the region and the fifth species of Oreosaurus from Venezuela. It can be distinguished from all congeners by a combination of rectangular dorsal scales at midbody, having two scales separating femoral pores medially, and lacking a nasoloreal suture. The new species is likely to be restricted in distribution to the highlands of the Turimiquire Massif, which have been significantly degraded by agriculture.

Genome-wide SNPs clarify lineage diversity confused by coloration in coralsnakes of the Micrurus diastema species complex (Serpentes: Elapidae).

New world coralsnakes of the genus Micrurus are a diverse radiation of highly venomous and brightly colored snakes that range from North Carolina to Argentina. Species in this group have played central roles in developing and testing hypotheses about the evolution of mimicry and aposematism. Despite their diversity and prominence as model systems, surprisingly little is known about species boundaries and phylogenetic relationships within Micrurus, which has substantially hindered meaningful analyses of their evolutionary history. Here we use mitochondrial genes together with thousands of nuclear genomic loci obtained via ddRADseq to study the phylogenetic relationships and population genomics of a subclade of the genus Micrurus: The M. diastema species complex. Our results indicate that prior species and species-group inferences based on morphology and color pattern have grossly misguided taxonomy, and that the M. diastema complex is not monophyletic. Based on our analyses of molecular data, we infer the phylogenetic relationships among species and populations, and provide a revised taxonomy for the group. Two non-sister species-complexes with similar color patterns are recognized, the M. distans and the M. diastema complexes, the first being basal to the monadal Micrurus and the second encompassing most North American monadal taxa. We examined all 13 species, and their respective subspecies, for a total of 24 recognized taxa in the M. diastema species complex. Our analyses suggest a reduction to 10 species, with no subspecific designations warranted, to be a more likely estimate of species diversity, namely, M. apiatus, M. browni, M. diastema, M. distans, M. ephippifer, M. fulvius, M. michoacanensis, M. oliveri, M. tener, and one undescribed species.

The roles of vicariance and dispersal in the differentiation of two species of the Rhinella marina species complex.

The high levels of Neotropical biodiversity are commonly associated with the intense Neogene-Quaternary geological events and climate dynamics. Here, we investigate the evolutionary history of two species of Neotropical closely related amphibians (R. horribilis and R. marina). We combine published data with new mitochondrial DNA sequences and multiple nuclear markers, including 12 microsatellites. The phylogenetic analyses showed support for grouping the samples in two main clades; R. horribilis (Central America and Mexico) and R. marina (South America east of the Andes). However, the phylogenetic inferences also show an evident mito-nuclear discordance. We use Approximate Bayesian Computation (ABC) to test the role of different events in the diversification between the two groups recovered. We found that both species were affected primarily by a recent Pleistocene divergence, which was similar to the divergence estimate revealed by the Isolation-with-Migration model, under persistent bidirectional gene flow through time. We provide the first evidence that R. horribilis is differentiated from the South American R. marina at the nuclear level supporting the taxonomic status of R. horribilis, which has been controversial for more than a century.

Systematics of the frogs allocated to Sarcohyla bistincta sensu lato (Cope, 1877), with description of a new species from Western Mexico.

A new species of hylid frog is described from the southwestern edge of the Mexican Plateau from the states of Morelos and Mexico through Michoacán and Jalisco, reaching the Sierra Madre Occidental in Sinaloa and western Durango. The new species is part of the widespread Mexican hylid Sarcohyla bistincta (sensu amplo) complex, comprised of S. bistincta, S. pentheter, S. calthula, and S. ephemera. One subspecies of S. bistincta (labeculata) was proposed for an isolated population in Oaxaca. We restrict the group's nominal species, S. bistincta (sensu stricto), to the Sierra Madre Oriental of Mexico and southward into the Sierra Madre del Sur of Guerrero and Oaxaca. Examination of type material places S. calthula and S. ephemera in the synonymy of S. labeculata (new combination). The species allied to S. bistincta, namely, S. bistincta, S. labeculata, S. pentheter, and the new species described herein, are diagnosed and described following recent suggested taxonomic changes and new available material.

Geographical features are the predominant driver of molecular diversification in widely distributed North American whipsnakes.

Allopatric divergence following the formation of geographical features has been implicated as a major driver of evolutionary diversification. Widespread species complexes provide opportunities to examine allopatric divergence across varying degrees of isolation in both time and space. In North America, several geographical features may play such a role in diversification, including the Mississippi River, Pecos River, Rocky Mountains, Cochise Filter Barrier, Gulf of California and Isthmus of Tehuantepec. We used thousands of nuclear single nucleotide polymorphisms (SNPs) and mitochondrial DNA from several species of whipsnakes (genera Masticophis and Coluber) distributed across North and Central America to investigate the role that these geographical features have played on lineage divergence. We hypothesize that these features restrict gene flow and separate whipsnakes into diagnosable genomic clusters. We performed genomic clustering and phylogenetic reconstructions at the species and population levels using Bayesian and likelihood analyses and quantified migration levels across geographical features to assess the degree of genetic isolation due to allopatry. Our analyses suggest that (i) major genetic divisions are often consistent with isolation by geographical features, (ii) migration rates between clusters are asymmetrical across major geographical features, and (iii) areas that receive proportionally more migrants possess higher levels of genetic diversity. Collectively, our findings suggest that multiple features of the North American landscape contributed to allopatric divergence in this widely distributed snake group.

Would behavioral thermoregulation enable pregnant viviparous tropical lizards to cope with a warmer world?

Sceloporus lizards depend on external heat to achieve their preferred temperature (Tsel ) for performing physiological processes. Evidence both in the field and laboratory indicates that pregnant females of this Genus select body temperatures (Tb ) lower than 34 °C as higher temperatures may be lethal to embryos. Therefore, thermoregulation is crucial for successful embryo development. Given the increase in global air temperature, it is expected that the first compensatory response of species that inhabit tropical climates will be behavioral thermoregulation. We tested whether viviparous Sceloporus formosus group lizards in the wild exhibited differences in thermoregulatory behavior to achieve the known Tsel for developing embryos regardless of local thermal conditions. We quantified field active body temperature, thermoregulatory behavior mechanisms (time of sighting, microhabitat used and basking time) and available microhabitat thermal conditions (i.e. operative temperature) for 10 lizard species during gestation, distributed along an altitudinal gradient. We applied both conventional and phylogenic analyses to explore whether Tb or behavioral thermoregulation could be regulated in response to different thermal conditions. These species showed no significant differences in field Tb during gestation regardless of local thermal conditions. In contrast, they exhibited significant differences in their behavioral thermoregulation associated with local environmental conditions. Based on these observations, the differences in thermoregulatory behavior identified are interpreted as compensatory adjustments to local thermal conditions. We conclude that these species may deal with higher temperatures predicted for the tropics by modulating their thermoregulatory behavior.

Genetic surfing, not allopatric divergence, explains spatial sorting of mitochondrial haplotypes in venomous coralsnakes.

Strong spatial sorting of genetic variation in contiguous populations is often explained by local adaptation or secondary contact following allopatric divergence. A third explanation, spatial sorting by stochastic effects of range expansion, has been considered less often though theoretical models suggest it should be widespread, if ephemeral. In a study designed to delimit species within a clade of venomous coralsnakes, we identified an unusual pattern within the Texas coral snake (Micrurus tener): strong spatial sorting of divergent mitochondrial (mtDNA) lineages over a portion of its range, but weak sorting of these lineages elsewhere. We tested three alternative hypotheses to explain this pattern-local adaptation, secondary contact following allopatric divergence, and range expansion. Collectively, near panmixia of nuclear DNA, the signal of range expansion associated sampling drift, expansion origins in the Gulf Coast of Mexico, and species distribution modeling suggest that the spatial sorting of divergent mtDNA lineages within M. tener has resulted from genetic surfing of standing mtDNA variation-not local adaptation or allopatric divergence. Our findings highlight the potential for the stochastic effects of recent range expansion to mislead estimations of population divergence made from mtDNA, which may be exacerbated in systems with low vagility, ancestral mtDNA polymorphism, and male-biased dispersal.

Estimating the temporal and spatial extent of gene flow among sympatric lizard populations (genus Sceloporus) in the southern Mexican highlands.

Interspecific gene flow is pervasive throughout the tree of life. Although detecting gene flow between populations has been facilitated by new analytical approaches, determining the timing and geography of hybridization has remained difficult, particularly for historical gene flow. A geographically explicit phylogenetic approach is needed to determine the overlap of ancestral populations. In this study, we performed population genetic analyses, species delimitation, simulations and a recently developed approach of species tree diffusion to infer the phylogeographic history, timing and geographic extent of gene flow in lizards of the Sceloporus spinosus group. The two species in this group, S. spinosus and S. horridus, are distributed in eastern and western portions of Mexico, respectively, but populations of these species are sympatric in the southern Mexican highlands. We generated data consisting of three mitochondrial genes and eight nuclear loci for 148 and 68 individuals, respectively. We delimited six lineages in this group, but found strong evidence of mito-nuclear discordance in sympatric populations of S. spinosus and S. horridus owing to mitochondrial introgression. We used coalescent simulations to differentiate ancestral gene flow from secondary contact, but found mixed support for these two models. Bayesian phylogeography indicated more than 60% range overlap between ancestral S. spinosus and S. horridus populations since the time of their divergence. Isolation-migration analyses, however, revealed near-zero levels of gene flow between these ancestral populations. Interpreting results from both simulations and empirical data indicate that despite a long history of sympatry among these two species, gene flow in this group has only recently occurred.

Phylogenetic relationships of the enigmatic longtailed rattlesnakes (Crotalus ericsmithi, C. lannomi, and C. stejnegeri).

The longtailed rattlesnakes of western Mexico represent an enigmatic group of poorly known venomous snake species: Crotalus ericsmithi, C. lannomi, and C. stejnegeri. In the 120 years since their discovery, fewer than twenty individuals have been deposited in natural history collections worldwide. These three species share similar morphological traits, including a particularly long tail that has been interpreted as either an ancestral condition among rattlesnakes or as derived within the longtailed group. An understanding of the phylogenetic distinctiveness and relationships among the longtailed rattlesnakes, and their relationships to other rattlesnake groups, has previously been hampered by a dearth of comparative material and tissues for collection of DNA sequence data. Facilitated by the recent availability of tissue samples from multiple individuals of each species, we estimate the phylogenetic relationships among the longtailed rattlesnakes and their placement among other rattlesnake groups, using DNA sequence data from three mitochondrial and three nuclear gene fragments. We explore phylogenetic signal in our data using Bayesian and maximum likelihood methods, species tree analyses and hypothesis testing. Our results strongly support the monophyly of longtailed rattlesnakes and suggest the three species diverged from each other during the mid to late Pliocene or early Pleistocene (~1.5-5.6 mya). Contrary to prevailing hypotheses, we find no evidence for an early or basal divergence of the longtailed clade within the rattlesnake tree, and instead estimate that it diverged relatively recently (~6.8 mya) from its sister lineage, composed of the diamondback rattlesnakes (C. atrox group) and the prairie rattlesnakes (C. viridis group). With our added sampling of lineages and identification of previously used problematic sequences, we provide a revised hypothesis for relationships among Crotalus species, yet underscore the need for future studies and new data to deliver a well-supported robust estimate of rattlesnake relationships.

Isolation, characterization, cloning and expression of an alpha-neurotoxin from the venom of the Mexican coral snake Micrurus laticollaris (Squamata: Elapidae).

A new member of short chain α-neurotoxic protein family from venom of the Mexican coral snake, Micrurus laticollaris, was characterized. This protein, named MlatA1, possesses 61 amino acids with 8 conserved cysteine residues, sharing 30-91% sequence identity with other fully sequenced Micrurus toxins. MlatA1 (LD50i.v. = 0.064 mg/kg) antagonizes with both fetal and adult nicotinic acetylcholine receptor (nAChR) as well as α-7 neuronal nAChR in a dose-dependent way. Specific rabbit anti-Mlat serum (titer higher than 18,000) does not show any protective ability against this toxin, nevertheless it was able to recognize protein bands in six out of twelve Micrurus venoms showing the existence of two distinct antigenic groups for α-neurotoxins in North American coral snakes species. The MlatA1 gene was cloned and used to produce recombinant toxin (rMlatA1) that was recognized by rabbit anti-native toxin but was depleted of toxic activity.

Thousands of microsatellite loci from the venomous coralsnake Micrurus fulvius and variability of select loci across populations and related species.

Studies of population genetics increasingly use next-generation DNA sequencing to identify microsatellite loci in nonmodel organisms. There are, however, relatively few studies that validate the feasibility of transitioning from marker development to experimental application across populations and species. North American coralsnakes of the Micrurus fulvius species complex occur in the United States and Mexico, and little is known about their population structure and phylogenetic relationships. This absence of information and population genetics markers is particularly concerning because they are highly venomous and have important implications on human health. To alleviate this problem in coralsnakes, we investigated the feasibility of using 454 shotgun sequences for microsatellite marker development. First, a genomic shotgun library from a single individual was sequenced (approximately 7.74 megabases; 26,831 reads) to identify potentially amplifiable microsatellite loci (PALs). We then hierarchically sampled 76 individuals from throughout the geographic distribution of the species complex and examined whether PALs were amplifiable and polymorphic. Approximately half of the loci tested were readily amplifiable from all individuals, and 80% of the loci tested for variation were variable and thus informative as population genetic markers. To evaluate the repetitive landscape characteristics across multiple snakes, we also compared microsatellite content between the coralsnake and two other previously sampled snakes, the venomous copperhead (Agkistrodon contortrix) and Burmese python (Python molurus).

Rapid range expansion in the Great Plains narrow-mouthed toad (Gastrophryne olivacea) and a revised taxonomy for North American microhylids.

We investigated genetic variation within the Great Plains narrow-mouthed toad, Gastrophryne olivacea, across its geographic range in the United States and Mexico. An analysis of mitochondrial DNA (mtDNA) from 105 frogs revealed remarkably low levels of genetic diversity in individuals inhabiting the central United States and northern Mexico. We found that this widespread matrilineal lineage is divergent (ca. 2% in mtDNA) from haplotypes that originate from the western United States and western coast of Mexico. Using a dataset that included all five species of Gastrophryne and both species of the closely related genus Hypopachus, we investigated the phylogenetic placement of G. olivacea. This analysis recovered strong support that G. olivacea, the tropically distributed G. elegans, and the temperately distributed G. carolinensis constitute a monophyletic assemblage. However, the placement of G. pictiventris and G. usta render Gastrophryne paraphyletic with respect to Hypopachus. To complement our mitochondrial analysis, we examined a small fragment of nuclear DNA and recovered consistent patterns. In light of our findings we recommend (1) the resurrection of the nomen G. mazatlanensisTaylor (1943) for the disjunct western clade of G. olivacea and (2) the tentative placement of G. pictiventris and G. usta in Hypopachus. To explore possible scenarios leading to low levels of genetic diversity in G. olivacea, we used mismatch distributions and Bayesian Skyline plots to examine historic population expansion and demography. Collectively these analyses suggest that G. olivacea rapidly expanded in effective population size and geographic range during the late Pleistocene or early Holocene. This hypothesis is consistent with fossil data from northern localities and contemporary observations that suggest ongoing northern expansion. Given our findings, we suspect that the rapid range expansion of G. olivacea may have been facilitated by ecological associations with open habitats and seasonal water bodies.

Neuromuscular activity of the venoms of the Colombian coral snakes Micrurus dissoleucus and Micrurus mipartitus: an evolutionary perspective.

The venoms of coral snakes (genus Micrurus) are known to induce a broad spectrum of pharmacological activities. While some studies have investigated their potential human effects, little is known about their mechanism of action in terms of the ecological diversity and evolutionary relationships among the group. In the current study we investigated the neuromuscular blockade of the venom of two sister species Micrurus mipartitus and Micrurus dissoleucus, which exhibit divergent ecological characteristics in Colombia, by using the chick biventer cervicis nerve-muscle preparation. We also undertook a phylogenetic analysis of these species and their congeners, in order to provide an evolutionary framework for the American coral snakes. The venom of M. mipartitus caused a concentration-dependant inhibition (3-10 µg/ml) of nerve-mediated twitches and significantly inhibited contractile responses to exogenous ACh (1 mM), but not KCl (40 mM), indicating a postsynaptic mechanism of action. The inhibition of indirect twitches at the lower venom dose (3 µg/ml) showed to be triphasic and the effect was further attenuated when PLA2 was inhibited. M. dissoleucus venom (10-50 µg/ml) failed to produce a complete blockade of nerve-mediated twitches within a 3 h time period and significantly inhibited contractile responses to exogenous ACh (1 mM) and KCl (40 mM), indicating both postsynaptic and myotoxic mechanisms of action. Myotoxic activity was confirmed by morphological studies of the envenomed tissues. Our results demonstrate a hitherto unsuspected diversity of pharmacological actions in closely related species which exhibit divergent ecological characteristics; these results have important implications for both the clinical management of Coral snake envenomings and the design of Micrurus antivenom.

Molecular systematics of the Middle American genus Hypopachus (Anura: Microhylidae).

We present the first phylogenetic study on the widespread Middle American microhylid frog genus Hypopachus. Partial sequences of mitochondrial (12S and 16S ribosomal RNA) and nuclear (rhodopsin) genes (1275 bp total) were analyzed from 43 samples of Hypopachus, three currently recognized species of Gastrophryne, and seven arthroleptid, brevicipitid and microhylid outgroup taxa. Maximum parsimony (PAUP), maximum likelihood (RAxML) and Bayesian inference (MrBayes) optimality criteria were used for phylogenetic analyses, and BEAST was used to estimate divergence dates of major clades. Population-level analyses were conducted with the programs NETWORK and Arlequin. Results confirm the placement of Hypopachus and Gastrophryne as sister taxa, but the latter genus was strongly supported as paraphyletic. The African phrynomerine genus Phrynomantis was recovered as the sister taxon to a monophyletic Chiasmocleis, rendering our well-supported clade of gastrophrynines paraphyletic. Hypopachus barberi was supported as a disjunctly distributed highland species, and we recovered a basal split in lowland populations of Hypopachus variolosus from the Pacific versant of Mexico and elsewhere in the Mesoamerican lowlands. Dating analyses from BEAST estimate speciation within the genus Hypopachus occurred in the late Miocene/early Pliocene for most clades. Previous studies have not found bioacoustic or morphological differences among these lowland clades, and our molecular data support the continued recognition of two species in the genus Hypopachus.

Complex evolution in the Neotropics: the origin and diversification of the widespread genus Leptodeira (Serpentes: Colubridae).

Lineage diversification in the Neotropics is an interesting topic in evolutionary biology but is also one of the least understood. The abiotic and biotic complexity of the region precludes generalizations that can be drawn regarding the historical evolutionary processes responsible for the diversity observed. The snake genus Leptodeira provides an excellent opportunity to investigate such processes because it spans the entire Neotropical region. In this study, we infer the phylogenetic position of Leptodeira within Dipsadinae, estimate evolutionary relationships among and within Leptodeira species, and estimate the diversification time and biogeography of the genus. Three mitochondrial gene regions were sequenced for individuals representing all the Leptodeira species and most subspecies currently recognized. Additionally, two nuclear protein-coding gene regions were sequenced for representatives of each species and several genera within the Dipsadinae. We infer that several Leptodeira species are either paraphyletic or polyphyletic as currently recognized, and that most recognized subspecies are not monophyletic lineages. Despite the taxonomic discordance with evolutionary relationships, clades appear to correspond very well to major biogeographic regions of Mexico, Central America and South America. Our results thus highlight the important role of the Miocene and Pliocene for lineage diversification in the Neotropics. Additionally, our time estimates suggest that recent intraspecific phylogeographic structure is likely the result of habitat and climatic fluctuations during the Pleistocene. Cumulatively, our inferences of lineage diversification within Leptodeira suggest a complex evolutionary scenario in the Mexican transition zone and a north to south expansion with a final colonization of the tropics in South America.

Cenozoic biogeography and evolution in direct-developing frogs of Central America (Leptodactylidae: Eleutherodactylus) as inferred from a phylogenetic analysis of nuclear and mitochondrial genes.

We report the first phylogenetic analysis of DNA sequence data for the Central American component of the genus Eleutherodactylus (Anura: Leptodactylidae: Eleutherodactylinae), one of the most ubiquitous, diverse, and abundant components of the Neotropical amphibian fauna. We obtained DNA sequence data from 55 specimens representing 45 species. Sampling was focused on Central America, but also included Bolivia, Brazil, Jamaica, and the USA. We sequenced 1460 contiguous base pairs (bp) of the mitochondrial genome containing ND2 and five neighboring tRNA genes, plus 1300 bp of the c-myc nuclear gene. The resulting phylogenetic inferences were broadly concordant between data sets and among analytical methods. The subgenus Craugastor is monophyletic and its initial radiation was potentially rapid and adaptive. Within Craugastor, the earliest splits separate three northern Central American species groups, milesi, augusti, and alfredi, from a clade comprising the rest of Craugastor. Within the latter clade, the rhodopis group as formerly recognized comprises three deeply divergent clades that do not form a monophyletic group; we therefore restrict the content of the rhodopis group to one of two northern clades, and use new names for the other northern (mexicanus group) and one southern clade (bransfordii group). The new rhodopis and bransfordii groups together form the sister taxon to a clade comprising the biporcatus, fitzingeri, mexicanus, and rugulosus groups. We used a Bayesian MCMC approach together with geological and biogeographic assumptions to estimate divergence times from the combined DNA sequence data. Our results corroborated three independent dispersal events for the origins of Central American Eleutherodactylus: (1) an ancestor of Craugastor entered northern Central America from South American in the early Paleocene, (2) an ancestor of the subgenus Syrrhophus entered northern Central America from the Caribbean at the end of the Eocene, and (3) a wave of independent dispersal events from South America coincided with formation of the Isthmus of Panama during the Pliocene. We elevate the subgenus Craugastor to the genus rank.

A new species of Osteocephalus (Anura: Hylidae) from Guyana

A new member of the genus Osteocephalus is described from the Pakaraima mountains of western Guyana. This species is the smallest known member of the genus and is probably closely related to O. subtilis. Both share a small size (less than 40 mm snout-vent length), large and bulgy eyes directed somewhat rostrally, green bones, smooth and brownish dorsal skin, relatively short and truncate snout, small tympanum, subgular and laterally expanded vocal sac, poorly developed subarticular and supernumerary tubercles, a supra-anal glandular ridge, and cream-white venter and subocular region. The new species can be distinguished from O. subtilis by the Buff iris (vs black), smaller overall size (32.7 vs 35.8-38.8 mm snout-vent length), relatively larger toe disks, and less developed foot webbing. The cranium of the new species is well ossified, relatively reduced in width between the orbits, without an exposed frontoparietal fontanelle and with the anterior arm of the squamosal extending to about half the distance to the maxillary. The vocal sac is subgularly poorly developed and possess lateral extensions to the area behind the jaw angles. Well developed supraocular and suprasquamosal cartilages give support to the enlarged eyes of this species.