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Abstract Understanding and predicting the relationships between genotype and phenotype is often challenging, largely due to the complex nature of eukaryotic gene regulation. A step towards this goal is to map how phenotypic diversity evolves through genomic changes that modify gene regulatory interactions. Using the Prairie Rattlesnake (Crotalus viridis) and related species, we integrate mRNA-seq, proteomic, ATAC-seq and whole genome resequencing data to understand how specific evolutionary modifications to gene regulatory network components produce differences in venom gene expression. Through comparisons within and between species, we find a remarkably high degree of gene expression and regulatory network variation across even a shallow level of evolutionary divergence. We use these data to test hypotheses about the roles of specific trans-factors and cis-regulatory elements, how these roles may vary across venom genes and gene families, and how variation in regulatory systems drive diversity in venom phenotypes. Our results illustrate that differences in chromatin and genotype at regulatory elements play major roles in modulating expression. However, we also find that enhancer deletions, differences in transcription-factor expression, and variation in activity of the insulator protein CTCF also likely impact venom phenotypes. Our findings provide insight into the diversity and gene-specificity of gene regulatory features and highlight the value of comparative studies to link gene regulatory network variation to phenotypic variation. 

Abstract We present genome assemblies for 18 snake species representing 18 families (Serpentes: Caenophidia): Acrochordus granulatus, Aparallactus werneri, Boaedon fuliginosus, Calamaria suluensis, Cerberus rynchops, Grayia smithii, Imantodes cenchoa, Mimophis mahfalensis, Oxyrhabdium leporinum, Pareas carinatus, Psammodynastes pulverulentus, Pseudoxenodon macrops, Pseudoxyrhopus heterurus, Sibynophis collaris, Stegonotus admiraltiensis, Toxicocalamus goodenoughensis, Trimeresurus albolabris, and Tropidonophis doriae. From these new genome assemblies, we extracted thousands of loci commonly used in systematic and phylogenomic studies on snakes, including target-capture datasets composed of ultraconserved elements (UCEs) and anchored hybrid enriched loci (AHEs), as well as traditional Sanger loci. Phylogenies inferred from the two target-capture loci datasets were identical with each other and strongly congruent with previously published snake phylogenies. To show the additional utility of these non-model genomes for investigative evolutionary research, we mined the genome assemblies of two New Guinea island endemics in our dataset (S. admiraltiensis and T. doriae) for the ATP1a3 gene, a thoroughly researched indicator of resistance to toad toxin ingestion by squamates. We find that both these snakes possess the genotype for toad toxin resistance despite their endemism to New Guinea, a region absent of any toads until the human-mediated introduction of Cane Toads in the 1930s. These species possess identical substitutions that suggest the same bufotoxin resistance as their Australian congenerics (Stegonotus australis and Tropidonophis mairii) which forage on invasive Cane Toads. Herein, we show the utility of short-read high-coverage genomes, as well as improving the deficit of available squamate genomes with associated voucher specimens. 

Abstract Divergence dating analyses in systematics provide a framework to develop and test biogeographic hypotheses regarding speciation. However, as molecular datasets grow from multilocus to genomic, sample sizes decrease due to computational burdens, and the testing of fine-scale biogeographic hypotheses becomes difficult. In this study, we use coalescent demographic models to investigate the diversification of poorly known rice paddy snakes from Southeast Asia (Homalopsidae:Hypsiscopus), which have conflicting dates of origin based on previous studies. We use coalescent modeling to test the hypothesis thatHypsiscopusdiversified 2.5 mya during the Khorat Plateau uplift in Thailand. Additionally, we use ecological niche analyses to identify potential differences in the niche space of the two most widely distributed species in the past and present. Our results suggestHypsiscopusdiversified ~ 2.4 mya, supporting that the Khorat Plateau may have initiated the diversification of rice paddy snakes. We also find significant niche differentiation and shifts between species ofHypsiscopus, indicating that environmental differences may have sustained differentiation of this genus after the Khorat Plateau uplift. Our study expands on the diversification history of snakes in Southeast Asia, and highlights how results from smaller multilocus datasets can be useful in developing and testing biogeographic hypotheses alongside genomic datasets. 

Species loss in fragile insular communities can alter the composition and stability of local assemblages. Climate change or anthropogenic pressures are sometimes attributed to the loss of Caribbean bats, but other factors are elusive to document. We studied time-scaled changes in bat assemblage composition from a palaeontological excavation in Cueva Matos, Puerto Rico. Over 800 individual fossils were identified to species, and charcoal was used to develop an AMS¹⁴C chronology. Although three bat species live in the cave today, fossils comprise 10 species. These included five extirpated species from the cave and three no longer present on the island. Losses centred around 2460–4470 kya. Notably, we document the first record ofMormoops megalophyllaas extirpated from Puerto Rico. Nearly 90% of the extirpated bats in Cueva Matos prefer to roost in hot caves where temperatures may reach 40℃. However, these temperatures are currently not held in any cave chamber. Our findings suggest that structural changes in the cave resulted in the loss of heat traps and likely led to a sudden shift in the bat assemblage composition at this cave, which is now void of hot cave specialist bats. 

Characterization of species distributions is a fundamental challenge in biodiversity science, with particular significance for downstream evolutionary studies, conservation efforts, field-based faunal studies and estimates of species diversity. Checklists and phylogenetic studies often focus on poorly known, rare taxa with limited ranges. However, studies of widely distributed, ecologically important species that are abundant in their preferred microhabitats are also important for systematics and local conservation efforts, but less often studied. We collected novel natural history data during fieldwork (2019–2023) for Philippine populations of bockadams (Homalopsidae:Cerberus), one of the most abundant vertebrates in Southeast Asian aquatic systems. Considered a coastal snake, many studies reportCerberusinland. We report the frequency of encounters ofCerberus schneiderii, and the IUCN data-deficient, Philippine-endemicCerberus microlepisduring six expeditions (62 days; 1041 person-hours). We report new occurrence data for 69C. schneideriiand6 C. microlepisfor coastal and inland populations, water measurements and dietary observations. Regression analyses and ecological niche models show the importance of coastal and mangrove habitats forCerberus. Our study is the most comprehensive assessment of PhilippineCerberuspopulations to date and provides critical baseline natural history data for downstream research on widespread and range-restricted species of Southeast Asian snakes. 

Homalopsids (Old World Mud Snakes) include 59 semiaquatic species in Asia and Australasia that display an array of morphological adaptations, behaviors, and microhabitat preferences. These attributes make homalopsids an ideal model system for broader questions in evolutionary biology, but the diversity of this understudied group of snakes is still being described. Recognized species diversity in rice paddy snakes (Hypsiscopus) has recently doubled after nearly 200 years of taxonomic stability. However, the evolutionary distinctiveness of some populations remains in question. In this study, we compare mainland Southeast Asian populations of Hypsiscopus east and west of the Red River Basin in Vietnam, a known biogeographic barrier in Asia, using an iterative approach with molecular phylogenetic reconstruction, machine-learning morphological quantitative statistics, and ecological niche modeling. Our analyses show that populations west of the Red River Basin represent an independent evolutionary lineage that is distinct in genetics, morphospace, and habitat suitability, and so warrants species recognition. The holotype of H. wettsteini, a species originally described in error from Costa Rica, grouped morphometrically with the population at the Red River Basin and eastward, and those west of the Red River Basin are referred to the recently described H. murphyi. The two species may have diversified due to a variety of geological and environmental factors, and their recognition exemplifies the importance of multifaceted approaches in taxonomy for downstream biogeographic studies on speciation scenarios.