Cryptic diversity in the Mexican highlands: Thousands of UCE loci help illuminate phylogenetic relationships, species limits and divergence times of montane rattlesnakes (Viperidae: Crotalus).

With the continued adoption of genome-scale data in evolutionary biology comes the challenge of adequately harnessing the information to make accurate phylogenetic inferences. Coalescent-based methods of species tree inference have become common, and concatenation has been shown in simulation to perform well, particularly when levels of incomplete lineage sorting are low. However, simulation conditions are often overly simplistic, leaving empiricists with uncertainty regarding analytical tools. We use a large ultraconserved element data set (>3,000 loci) from rattlesnakes of the Crotalus triseriatus group to delimit lineages and estimate species trees using concatenation and several coalescent-based methods. Unpartitioned and partitioned maximum likelihood and Bayesian analysis of the concatenated matrix yield a topology identical to coalescent analysis of a subset of the data in bpp. ASTRAL analysis on a subset of the more variable loci also results in a tree consistent with concatenation and bpp, whereas the SVDquartets phylogeny differs at additional nodes. The size of the concatenated matrix has a strong effect on species tree inference using SVDquartets, warranting additional investigation on optimal data characteristics for this method. Species delimitation analyses suggest up to 16 unique lineages may be present within the C. triseriatus group, with divergences occurring during the Neogene and Quaternary. Network analyses suggest hybridization within the group is relatively rare. Altogether, our results reaffirm the Mexican highlands as a biodiversity hotspot and suggest that coalescent-based species tree inference on data subsets can provide a strongly supported species tree consistent with concatenation of all loci with a large amount of missing data.

In vitro effects of Crotalus atrox snake venom on chick and mouse neuromuscular preparations.

The neuromuscular effect of venoms is not a major clinical manifestation shared between rattlesnakes native to the Americas, which showed two different venom phenotypes. Taking into account this dichotomy, nerve muscle preparations from mice and chicks were used to investigate the ability of Crotalus atrox venom to induce in vitro neurotoxicity and myotoxicity. Unlike crotalic venoms of South America, low concentrations of C. atrox venom did not result in significant effects on mouse neuromuscular preparations. The venom was more active on avian nerve-muscle, showing reduction of twitch heights after 120 min of incubation with 10, 30 and 100 µg/mL of venom with diminished responses to agonists and KCl. Histological analysis highlighted that C. atrox was myotoxic in both species of experimental animals; as evidenced by degenerative events, including edematous cells, delta lesions, hypercontracted fibers and muscle necrosis, which can lead to neurotoxic action. These results provide key insights into the myotoxicity and low neurotoxicity of C. atrox in two animal models, corroborating with previous genomic and proteomic findings and would be useful for a deeper understanding of venom evolution in snakes belonging to the genus Crotalus.

Rattling the border wall: Pathophysiological implications of functional and proteomic venom variation between Mexican and US subspecies of the desert rattlesnake Crotalus scutulatus.

While some US populations of the Mohave rattlesnake (Crotalus scutulatus scutulatus) are infamous for being potently neurotoxic, the Mexican subspecies C. s. salvini (Huamantlan rattlesnake) has been largely unstudied beyond crude lethality testing upon mice. In this study we show that at least some populations of this snake are as potently neurotoxic as its northern cousin. Testing of the Mexican antivenom Antivipmyn showed a complete lack of neutralisation for the neurotoxic effects of C. s. salvini venom, while the neurotoxic effects of the US subspecies C. s. scutulatus were time-delayed but ultimately not eliminated. These results document unrecognised potent neurological effects of a Mexican snake and highlight the medical importance of this subspecies, a finding augmented by the ineffectiveness of the Antivipmyn antivenom. These results also influence our understanding of the venom evolution of Crotalus scutulatus, suggesting that neurotoxicity is the ancestral feature of this species, with the US populations which lack neurotoxicity being derived states.

Integrated Venomics and Venom Gland Transcriptome Analysis of Juvenile and Adult Mexican Rattlesnakes Crotalus simus, C. tzabcan, and C. culminatus Revealed miRNA-modulated Ontogenetic Shifts.

Adult rattlesnakes within genus Crotalus express one of two distinct venom phenotypes, type I (hemorrhagic) and type II (neurotoxic). In Costa Rican Central American rattlesnake, ontogenetic changes in the concentration of miRNAs modulate venom type II to type I transition. Venomics and venom gland transcriptome analyses showed that adult C. simus and C. tzabcan expressed intermediate patterns between type II and type I venoms, whereas C. culminatus had a canonical type I venom. Neonate/juvenile and adult Mexican rattlesnakes showed notable inter- and intraspecific variability in the number, type, abundance and ontogenetic shifts of the transcriptional and translational venom gland activities. These results support a role for miRNAs in the ontogenetic venom compositional changes in the three congeneric Mexican rattlesnakes. It is worth noting the finding of dual-action miRNAs, which silence the translation of neurotoxic heterodimeric PLA2 crotoxin and acidic PLA2 mRNAs while simultaneously up-regulating SVMP-targeting mRNAs. Dual transcriptional regulation potentially explains the existence of mutually exclusive crotoxin-rich (type-II) and SVMP-rich (type-I) venom phenotypic dichotomy among rattlesnakes. Our results support the hypothesis that alterations of the distribution of miRNAs, modulating the translational activity of venom gland toxin-encoding mRNAs in response to an external cue, may contribute to the mechanism generating adaptive venom variability.

Ablation of the ability to control the right-to-left cardiac shunt does not affect oxygen uptake, specific dynamic action or growth in the rattlesnake Crotalus durissus.

The morphologically undivided ventricle of the heart in non-crocodilian reptiles permits the mixing of oxygen-rich blood returning from the lungs and oxygen-poor blood from the systemic circulation. A possible functional significance for this intra-cardiac shunt has been debated for almost a century. Unilateral left vagotomy rendered the single effective pulmonary artery of the South American rattlesnake, Crotalus durissus, unable to adjust the magnitude of blood flow to the lung. The higher constant perfusion of the lung circulation and the incapability of adjusting the right-left shunt in left-denervated snakes persisted over time, providing a unique model for investigation of the long-term consequences of cardiac shunting in a squamate. Oxygen uptake recorded at rest and during spontaneous and forced activity was not affected by removing control of the cardiac shunt. Furthermore, metabolic rate and energetic balance during the post-prandial metabolic increment, plus the food conversion efficiency and growth rate, were all similarly unaffected. These results show that control of cardiac shunting is not associated with a clear functional advantage in adjusting metabolic rate, effectiveness of digestion or growth rates.

A cascavel neotropical Crotalus durissus: uma abordagem morfológica e da historia natural em populações do Brasil / The neotropical rattlesnake Crotalus durissus: a morphological and natural history approach in populations from Brazil

A variação de cunho geográfico nas populações naturais está intimamente ligada às características ecológicas do ambiente como também aos processos históricos que resultaram no estabelecimento de linhagens distintas. Poucas espécies de serpentes têm sido estudadas adequadamente para determinar se existe variação geográfica na morfologia associada ao seu ciclo reprodutivo e dieta. As serpentes do gênero Crotalus são consideradas um bom modelo para estudos relacionados à variação geográfica devido à sua ampla distribuição, que abrange diversos tipos de hábitats. A cascavel C. durissus está restrita a América do Sul. No Brasil ocorre em todos os estados, exceto no Acre e Espírito Santo. São reconhecidas algumas populações isoladas na Floresta Amazônica. Algumas das suas populações exibem um nível considerável de variação morfológica e ecológica, sendo que populações adjacentes podem diferir drasticamente. De um modo geral, são escassos os estudos com esta espécie no Brasil, requerendo urgentes investigações para estabelecer se a variabilidade observada está associada a algum fenômeno específico. Neste estudo, foi examinado um total de 870 exemplares de C. durissus no Brasil. Em termos gerais, de acordo com o dimorfismo sexual, os resultados indicaram que as fêmeas apresentaram um maior número de escamas no ventre, enquanto que os machos apresentaram um número maior de escamas associadas à cauda e de losângulos. Em poucos casos as fêmeas exibiram comprimentos rostro-cloacais maiores, no entanto os maiores tamanhos da cabeça foram evidenciados neste sexo. Por outro lado, na maioria dos casos, os machos foram mais compridos do que as fêmeas, apresentando caudas mais longas. Similarmente as listras paravertebrais foram significativamente maiores nos machos. As análises discriminantes indicaram que as populações apresentaram padrões de variabilidade morfológica altamente complexos, ainda que a segregação de algumas das populações possa refletir fortes tendências evolutivas próprias dentro de algumas linhagens. A análise de variação geográfica indicou que variáveis ambientais influenciaram parcialmente a variabilidade morfológica nas populações de acordo com a sua distribuição. De acordo com os parâmetros relacionados a historia natural, o ciclo reprodutivo das fêmeas foi bem conservador, refletindo um padrão bienal e sazonal. Nos machos o ciclo espermatogênico mostra também um padrão sazonal exibindo variações anuais entre as populações. A associação entre os processos históricos, aspectos fisiológicos e condições climáticas são provavelmente os principais fatores que influenciaram as mudanças nestes padrões reprodutivos em fêmeas e machos. A dieta foi constituída principalmente por roedores, fato possivelmente associado à maior abundância e disponibilidade deste tipo de presa ao longo do ano nas áreas de ocorrência da espécie. No entanto, lagartos do grupo dos teiídeos podem ser considerados como itens alimentares eventualmente importantes na dieta de C. durissus do Brasil

The geographical variation is intimately associated to the ecological characteristics such as the historical process of a species. A few species of snakes have been studied adequately to determine an existence of geographical variation in morphology, reproduction and diet. Rattlesnakes (Crotalus) are considered a good model for geographical variation studies, due to their wide distribution that include several habitat types. The rattlesnake, Crotalus durissus is restricted to South America. In Brazil, this group is present throughout the country, except in states of Acre and Espírito Santo. In addition, there are isolated populations in open areas in Amazonia, in the Amazonian savannas in the states of Amazonas, Roraima, Pará and Amapá. Some populations of C. durissus show considerable variation in the morphology, reproduction and diet composition even with neighboring populations differing drastically from each other. Generally, there has been little research conducted of C. durissus in Brazil needing urgent investigation to this respect. A total of 870 specimens of C. durissus were examined. In general, according to the sexual dimorphism, the results indicated that the females have a higher number of ventral scales, while the males showed a greater number of scales associated to the tail and the lozanges. In a few cases, the females exhibited larger snout-vent length but larger head sizes were shown in this sex. Overall, males were longer than females, exhibiting tails and paravertebral stripes which were relatively longer. The discriminant analysis showed in general populations with patterns of highly complex morphological variability, even though the segregation of some populations may reflect strong evolutionary tendencies of their own within some groups. The analysis of geographic variation indicated that environmental variables partially influenced the morphological variability in populations according to their distribution. In accordance with parameters related to natural history, the reproductive cycle of females, was extremely conservative, reflecting a biennial seasonal pattern. In males, the spermatogenic cycle also shows a seasonal pattern, which demonstrated annual variations between populations. The association between the historical processes, physiological aspects and climatic conditions is probably the main factors that influenced these reproductive patterns in females and males. The diet was composed mainly of rodents, habit related to the abundance and availability of the prey, however teid lizards can also be considered as an important food item of the diet of C. durissus from Brazil

Structural and biological characterization of two crotamine isoforms IV-2 and IV-3 isolated from the Crotalus durissus cumanensis venom.

In this work, we isolated the two new crotamine isoforms from the Crotalus durissus cumanensis rattlesnake venom and its "in vitro" neurotoxic, myotoxic and lethality (DL(50)) intracerebroventricular (i.c.v.) effects were characterized. These proteins were named IV-2 and IV-3 and were purified by combination of two chromatographic steps on molecular exclusion chromatography on Superdex 75 and reverse phase HPLC (mu-Bondapack C18). The molecular mass of the crotamine isoforms was 4905.96 Da for isoform IV-2 and 4956.97 Da for IV-3 and, as determined by mass spectrometry, and both contained six Cys residues. Enzymatic hydrolysis followed by de novo sequencing by tandem mass spectrometry was used to determine the primary structure of both isoforms. The positions of five sequenced tryptic peptides, including the N-terminal of the isoform IV-2 and four from isoform IV-3 were deduced by comparison with a homologous protein from the crotamine family. The isoforms IV-2 and IV-3 had a sequence of amino acids of 42 amino acid residues IV-2: YKRCHIKGGH CFPKEKLICI PPSSDIGKMD CPWKRKCCKK RS and pI value 9.54 and IV-3: YKQCHKKGGH CFPKEVLICI PPSSDFGKMD CRWKRKCCKK RS with a pI value of 9.54. This protein showed high molecular amino acid sequence identity with other crotamine-like proteins from Crotalus durissus terrificus. These new crotamine isoforms induced potent blockade of neuromuscular transmission in young chicken biventer cervicis preparation and potent myotoxic effect. In mice, both isoforms induced myonecrosis, upon intramuscular or subcutaneous injections. These activities were modulated by the presence of positively charged amino acid residues. The LD(50) of isoform IV-2 was 0.07 mg/kg and isoform IV-3 was 0.06 mg/kg the animal weight, by i.c.v. route.