Proteomic and toxicological characterization of the venoms of the most enigmatic group of rattlesnakes: The long-tailed rattlesnakes.

The most enigmatic group of rattlesnakes is the long-tailed rattlesnake group, consisting of three species: Crotalus ericsmithi, Crotalus lannomi and Crotalus stejnegeri. These species have been the least studied rattlesnakes in all aspects, and no study on the characterization of their venoms has been carried out to date. Our main objective was to investigate the proteomic composition, as well as some of the biochemical and toxic activities of these venoms, and their neutralization by commercial antivenom. The venom proteome of C. ericsmithi mainly contains metalloproteinases (SVMP; 49.3%), phospholipases A2 (PLA2; 26.2%), disintegrins (Dis; 12.6%), and snake venom serine proteases (SVSP; 6.8%), while C. lannomi venom mainly consists of SVMP (47.1%), PLA2 (19.3%), Dis (18.9%), SVSP (6%) and l-amino acid oxidase (LAAO; 2.6%). For these venoms high lethality was recorded in mice, the most potent being that of C. lannomi (LD50 of 0.99 µg/g body weight), followed by C. ericsmithi (1.30 µg/g) and finally C. stejnegeri (1.79 µg/g). The antivenoms Antivipmyn® from SILANES and Fabotherapic polyvalent antiviperin® from BIRMEX neutralized the lethal activity of the three venoms. Although this group of snakes is phylogenetically related to the C. viridis group, no neurotoxic components (crotoxin or crotoxin-like proteins) common in rattlesnakes were found in their venoms. This study expands current knowledge on the venoms of understudied snake species of the Mexican herpetofauna.

Genetic and morphometric analyses of historical type specimens clarify the taxonomy of the Ethiopian Leptopelisgramineus species complex (Anura, Arthroleptidae).

Frogs of the genus Leptopelis have diversified in the Ethiopian Highlands to occupy forests and montane grasslands both east and west of the Great Rift Valley. Genetic studies revealed that the endemic species Leptopelisgramineus (Boulenger, 1898) comprises multiple unnamed taxa. A careful examination of historical type specimens is, however, needed to fully resolve the taxonomy of the group. Here we use mitochondrial DNA and morphological analyses on a large sample of recently-collected Ethiopian Leptopelis, as well as century-old type specimens to demonstrate that the recently resurrected L.montanus Tiutenko & Zinenko, 2021 (previously Pseudocassinaocellata Ahl, 1924) is a junior synonym of L.rugosus (Ahl, 1924) and corresponds to the taxon found west of the Great Rift Valley, not east as previously thought. Our results show that populations inhabiting the mountains and plateaus east of the Rift constitute a distinct and undescribed species. We provide a re-description of L.rugosus and describe two new species inhabiting the Highlands east of the Great Rift Valley. We provide an identification key, as well as a description of the calls of the members of the Leptopelisgramineus species complex.

Mitogenomics of historical type specimens clarifies the taxonomy of Ethiopian Ptychadena Boulenger, 1917 (Anura, Ptychadenidae).

The taxonomy of the Ptychadenaneumanni species complex, a radiation of grass frogs inhabiting the Ethiopian highlands, has puzzled scientists for decades because of the morphological resemblance among its members. Whilst molecular phylogenetic methods allowed the discovery of several species in recent years, assigning pre-existing and new names to clades was challenged by the unavailability of molecular data for century-old type specimens. We used Illumina short reads to sequence the mitochondrial DNA of type specimens in this group, as well as ddRAD-seq analyses to resolve taxonomic uncertainties surrounding the P.neumanni species complex. The phylogenetic reconstruction revealed recurrent confusion between Ptychadenaerlangeri (Ahl, 1924) and P.neumanni (Ahl, 1924) in the literature. The phylogeny also established that P.largeni Perret, 1994 represents a junior synonym of P.erlangeri (Ahl, 1924) and distinguished between two small species, P.nana Perret, 1994, restricted to the Arussi Plateau, and P.robeensis Goutte, Reyes-Velasco, Freilich, Kassie & Boissinot, 2021, which inhabits the Bale Mountains. The phylogenetic analyses of mitochondrial DNA from type specimens also corroborate the validity of seven recently described species within the group. Our study shows how modern molecular tools applied to historical type specimens can help resolve long-standing taxonomic issues in cryptic species complexes.

Taxonomic revision of grass frogs (Ptychadenidae, Ptychadena) endemic to the Ethiopian highlands.

Frogs of the genus Ptychadena that inhabit the Ethiopian highlands serve as a model system to understand biogeography, diversification, and adaptations to high elevations. Despite recent studies focusing on the systematics of this group, the taxonomy of the Ptychadena neumanni species complex remains only partially resolved, owing largely to the morphological resemblance of its members. Here, the taxonomy of this historically problematic group of frogs is revised by integrating morphological and molecular analyses on both century-old type specimens and more recently collected material. Based on these multiple lines of evidence, the P. neumanni species complex is shown to be more speciose than previously thought and four new species are described. With the aim of clarifying and stabilizing the taxonomy of the group, six species are also re-described and morphological and acoustic identification keys are provided. This study also establishes species distribution maps and reveals important differences in range size between the members of the P. neumanni complex, calling for adapted conservation measures across the Ethiopian highlands.

A New Species of Mud Turtle of Genus Kinosternon (Testudines: Kinosternidae) from the Pacific Coastal Plain of Northwestern Mexico.

We describe a new species of mud turtle of the genus Kinosternon from the Pacific Coastal Plain of the Mexican states Sinaloa and Nayarit. The new species shares morphological characters with the recently described Kinosternon vogti, which are unique to these two turtles and separate them from the other species of the genus. The new species differs from K. vogti by skin coloration, size, and the scutellation of both carapace and plastron. We also present a molecular phylogeny of the family Kinosternidae based on two mitochondrial and four nuclear loci. Our results show that the new species is most closely related to K. vogti, and together they form the sister group to the K. hirtipes and K. integrum species groups of Kinosternon.

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.

A new species of puddle frog from an unexplored mountain in southwestern Ethiopia (Anura, Phrynobatrachidae, Phrynobatrachus).

A new species of Phrynobatrachus is described from the unexplored and isolated Bibita Mountain, southwestern Ethiopia, based on morphological characters and sequences of the mitochondrial rRNA16s. The new species can be distinguished from all its congeners by a small size (SVL = 16.8 ± 0.1 mm for males, 20.3 ± 0.9 mm for females), a slender body with long legs and elongated fingers and toes, a golden coloration, a completely hidden tympanum, and a marked canthus rostralis. The phylogenetic hypothesis based on 16s sequences places the new species as sister to the species group that includes P.natalensis, although it is morphologically more similar to other dwarf Phrynobatrachus species, such as the Ethiopian P.minutus.

Molecular Adaptations for Sensing and Securing Prey and Insight into Amniote Genome Diversity from the Garter Snake Genome.

Colubridae represents the most phenotypically diverse and speciose family of snakes, yet no well-assembled and annotated genome exists for this lineage. Here, we report and analyze the genome of the garter snake, Thamnophis sirtalis, a colubrid snake that is an important model species for research in evolutionary biology, physiology, genomics, behavior, and the evolution of toxin resistance. Using the garter snake genome, we show how snakes have evolved numerous adaptations for sensing and securing prey, and identify features of snake genome structure that provide insight into the evolution of amniote genomes. Analyses of the garter snake and other squamate reptile genomes highlight shifts in repeat element abundance and expansion within snakes, uncover evidence of genes under positive selection, and provide revised neutral substitution rate estimates for squamates. Our identification of Z and W sex chromosome-specific scaffolds provides evidence for multiple origins of sex chromosome systems in snakes and demonstrates the value of this genome for studying sex chromosome evolution. Analysis of gene duplication and loss in visual and olfactory gene families supports a dim-light ancestral condition in snakes and indicates that olfactory receptor repertoires underwent an expansion early in snake evolution. Additionally, we provide some of the first links between secreted venom proteins, the genes that encode them, and their evolutionary origins in a rear-fanged colubrid snake, together with new genomic insight into the coevolutionary arms race between garter snakes and highly toxic newt prey that led to toxin resistance in garter snakes.

Squamate reptiles challenge paradigms of genomic repeat element evolution set by birds and mammals.

Broad paradigms of vertebrate genomic repeat element evolution have been largely shaped by analyses of mammalian and avian genomes. Here, based on analyses of genomes sequenced from over 60 squamate reptiles (lizards and snakes), we show that patterns of genomic repeat landscape evolution in squamates challenge such paradigms. Despite low variance in genome size, squamate genomes exhibit surprisingly high variation among species in abundance (ca. 25-73% of the genome) and composition of identifiable repeat elements. We also demonstrate that snake genomes have experienced microsatellite seeding by transposable elements at a scale unparalleled among eukaryotes, leading to some snake genomes containing the highest microsatellite content of any known eukaryote. Our analyses of transposable element evolution across squamates also suggest that lineage-specific variation in mechanisms of transposable element activity and silencing, rather than variation in species-specific demography, may play a dominant role in driving variation in repeat element landscapes across squamate phylogeny.

Diversification of African tree frogs (genus Leptopelis) in the highlands of Ethiopia.

The frog genus Leptopelis is composed of ~50 species that occur across sub-Saharan Africa. The majority of these frogs are typically arboreal; however, a few species have evolved a fossorial lifestyle. Most species inhabit lowland forests, but a few species have adapted to high elevations. Five species of Leptopelis occupy the Ethiopian highlands and provide a good opportunity to study the evolutionary transition from an arboreal to a fossorial lifestyle, as well as the diversification in this biodiversity hot spot. We sequenced 14 nuclear and three mitochondrial genes, and generated thousands of SNPs from ddRAD sequencing to study the evolutionary relationships of Ethiopian Leptopelis. The five species of highland Leptopelis form a monophyletic group, which diversified during the late Miocene and Pliocene. We found strong population structure in the fossorial species L. gramineus, with levels of genetic differentiation between populations similar to those found between arboreal species. This could indicate that L. gramineus is a complex of cryptic species. We propose that after the original colonization of the Ethiopian highlands by the ancestor of the L. gramineus group, episodes of vicariance fragmented the ancestral populations of this group. We also report the re-evolution of arboreality in L. susanae, which evolved from a fossorial ancestor, a rare ecological switch in frogs that had previously been reported only once.

Revisiting the phylogeography, demography and taxonomy of the frog genus Ptychadena in the Ethiopian highlands with the use of genome-wide SNP data.

Understanding the diversification of biological lineages is central to evolutionary studies. To properly study the process of speciation, it is necessary to link micro-evolutionary studies with macro-evolutionary mechanisms. Micro-evolutionary studies require proper sampling across a taxon's range to adequately infer genetic diversity. Here we use the grass frogs of the genus Ptychadena from the Ethiopian highlands as a model to study the process of lineage diversification in this unique biodiversity hotspot. We used thousands of genome-wide SNPs obtained from double digest restriction site associated DNA sequencing (ddRAD-seq) in populations of the Ptychadena neumanni species complex from the Ethiopian highlands in order to infer their phylogenetic relationships and genetic structure, as well as to study their demographic history. Our genome-wide phylogenetic study supports the existence of approximately 13 lineages clustered into 3 species groups. Our phylogenetic and phylogeographic reconstructions suggest that those endemic lineages diversified in allopatry, and subsequently specialized to different habitats and elevations. Demographic analyses point to a continuous decrease in the population size across the majority of lineages and populations during the Pleistocene, which is consistent with a continuous period of aridification that East Africa experienced since the Pliocene. We discuss the taxonomic implications of our analyses and, in particular, we warn against the recent practice to solely use Bayesian species delimitation methods when proposing taxonomic changes.

Microsatellite landscape evolutionary dynamics across 450 million years of vertebrate genome evolution.

The evolutionary dynamics of simple sequence repeats (SSRs or microsatellites) across the vertebrate tree of life remain largely undocumented and poorly understood. In this study, we analyzed patterns of genomic microsatellite abundance and evolution across 71 vertebrate genomes. The highest abundances of microsatellites exist in the genomes of ray-finned fishes, squamate reptiles, and mammals, while crocodilian, turtle, and avian genomes exhibit reduced microsatellite landscapes. We used comparative methods to infer evolutionary rates of change in microsatellite abundance across vertebrates and to highlight particular lineages that have experienced unusually high or low rates of change in genomic microsatellite abundance. Overall, most variation in microsatellite content, abundance, and evolutionary rate is observed among major lineages of reptiles, yet we found that several deeply divergent clades (i.e., squamate reptiles and mammals) contained relatively similar genomic microsatellite compositions. Archosauromorph reptiles (turtles, crocodilians, and birds) exhibit reduced genomic microsatellite content and the slowest rates of microsatellite evolution, in contrast to squamate reptile genomes that have among the highest rates of microsatellite evolution. Substantial branch-specific shifts in SSR content in primates, monotremes, rodents, snakes, and fish are also evident. Collectively, our results support multiple major shifts in microsatellite genomic landscapes among vertebrates.

Two new species of Eleutherodactylus (subgenus Syrrhophus) from western Mexico.

We describe two new species of Eleutherodactylus, subgenus Syrrhophus, from two separate mountain ranges in western Mexico. Eleutherodactylus grunwaldi sp. nov. inhabits the Sierra de Manantlán in Colima and Jalisco from 1300 to 2200 m, whereas E. wixarika sp. nov. is known from a single locality in the Sierra Huichola of northern Jalisco at 2400 m, but is probably more widespread. Eleutherodactylus grunwaldi is readily distinguishable from most members of mainland Syrrhophus by a combination of its large size, broad, truncate digital pads more than three times the narrowest part of the digit, and a black and green marbled color pattern. This species is saxicolous, inhabiting limestone outcrops, and has been found in caves during the dry season. Eleutherodactylus wixarika is a moderate sized species, most similar to E. teretistes, E. pallidus and E. modestus. It is distinguished from all other members of the subgenus by the combination a tuberculate, reddish dorsum, lack of compact lumbar glands, and expanded digital pads less than twice the width of the narrowest part of the digit. This species inhabits areas with secondary vegetation in pine forest. Males of both species call at night during the rainy season. The advertisement call of both species consists of a short, narrow band, pure-tone note organized into a discrete train at a rate of about six times per minute. Spectral and temporal acoustic properties differ between species. The subgenus Syrrhophus of the genus Eleutherodactylus is one of the most poorly studied groups of frogs in Mexico but probably one of the most diverse.

Expression of venom gene homologs in diverse python tissues suggests a new model for the evolution of snake venom.

Snake venom gene evolution has been studied intensively over the past several decades, yet most previous studies have lacked the context of complete snake genomes and the full context of gene expression across diverse snake tissues. We took a novel approach to studying snake venom evolution by leveraging the complete genome of the Burmese python, including information from tissue-specific patterns of gene expression. We identified the orthologs of snake venom genes in the python genome, and conducted detailed analysis of gene expression of these venom homologs to identify patterns that differ between snake venom gene families and all other genes. We found that venom gene homologs in the python are expressed in many different tissues outside of oral glands, which illustrates the pitfalls of using transcriptomic data alone to define "venom toxins." We hypothesize that the python may represent an ancestral state prior to major venom development, which is supported by our finding that the expansion of venom gene families is largely restricted to highly venomous caenophidian snakes. Therefore, the python provides insight into biases in which genes were recruited for snake venom systems. Python venom homologs are generally expressed at lower levels, have higher variance among tissues, and are expressed in fewer organs compared with all other python genes. We propose a model for the evolution of snake venoms in which venom genes are recruited preferentially from genes with particular expression profile characteristics, which facilitate a nearly neutral transition toward specialized venom system expression.

Incipient speciation with biased gene flow between two lineages of the Western Diamondback Rattlesnake (Crotalus atrox).

We used mitochondrial DNA sequence data from 151 individuals to estimate population genetic structure across the range of the Western Diamondback Rattlesnake (Crotalus atrox), a widely distributed North American pit viper. We also tested hypotheses of population structure using double-digest restriction site associated DNA (ddRADseq) data, incorporating thousands of nuclear genome-wide SNPs from 42 individuals. We found strong mitochondrial support for a deep divergence between eastern and western C. atrox populations, and subsequent intermixing of these populations in the Inter-Pecos region of the United States and Mexico. Our nuclear RADseq data also identify these two distinct lineages of C. atrox, and provide evidence for nuclear admixture of eastern and western alleles across a broad geographic region. We identified contrasting patterns of mitochondrial and nuclear genetic variation across this genetic fusion zone that indicate partially restricted patterns of gene flow, which may be due to either pre- or post-zygotic isolating mechanisms. The failure of these two lineages to maintain complete genetic isolation, and evidence for partially-restricted gene flow, imply that these lineages were in the early stages of speciation prior to secondary contact.

Two low coverage bird genomes and a comparison of reference-guided versus de novo genome assemblies.

As a greater number and diversity of high-quality vertebrate reference genomes become available, it is increasingly feasible to use these references to guide new draft assemblies for related species. Reference-guided assembly approaches may substantially increase the contiguity and completeness of a new genome using only low levels of genome coverage that might otherwise be insufficient for de novo genome assembly. We used low-coverage (∼3.5-5.5x) Illumina paired-end sequencing to assemble draft genomes of two bird species (the Gunnison Sage-Grouse, Centrocercus minimus, and the Clark's Nutcracker, Nucifraga columbiana). We used these data to estimate de novo genome assemblies and reference-guided assemblies, and compared the information content and completeness of these assemblies by comparing CEGMA gene set representation, repeat element content, simple sequence repeat content, and GC isochore structure among assemblies. Our results demonstrate that even lower-coverage genome sequencing projects are capable of producing informative and useful genomic resources, particularly through the use of reference-guided assemblies.

The Burmese python genome reveals the molecular basis for extreme adaptation in snakes.

Snakes possess many extreme morphological and physiological adaptations. Identification of the molecular basis of these traits can provide novel understanding for vertebrate biology and medicine. Here, we study snake biology using the genome sequence of the Burmese python (Python molurus bivittatus), a model of extreme physiological and metabolic adaptation. We compare the python and king cobra genomes along with genomic samples from other snakes and perform transcriptome analysis to gain insights into the extreme phenotypes of the python. We discovered rapid and massive transcriptional responses in multiple organ systems that occur on feeding and coordinate major changes in organ size and function. Intriguingly, the homologs of these genes in humans are associated with metabolism, development, and pathology. We also found that many snake metabolic genes have undergone positive selection, which together with the rapid evolution of mitochondrial proteins, provides evidence for extensive adaptive redesign of snake metabolic pathways. Additional evidence for molecular adaptation and gene family expansions and contractions is associated with major physiological and phenotypic adaptations in snakes; genes involved are related to cell cycle, development, lungs, eyes, heart, intestine, and skeletal structure, including GRB2-associated binding protein 1, SSH, WNT16, and bone morphogenetic protein 7. Finally, changes in repetitive DNA content, guanine-cytosine isochore structure, and nucleotide substitution rates indicate major shifts in the structure and evolution of snake genomes compared with other amniotes. Phenotypic and physiological novelty in snakes seems to be driven by system-wide coordination of protein adaptation, gene expression, and changes in the structure of the genome.

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.