In an attempt to extend the knowledge of the 5S rDNA organization in anurans, the 5S rDNA sequences of Amolops mantzorum were isolated, characterized, and mapped by FISH. Two forms of 5S rDNA, type I (209 bp) and type II (about 870 bp), were found in specimens investigated from various populations. Both of them contained a 118-bp coding sequence, readily differentiated by their non-transcribed spacer (NTS) sizes and compositions. Four probes (the 5S rDNA coding sequences, the type I NTS, the type II NTS, and the entire type II 5S rDNA sequences) were respectively labeled with TAMRA or digoxigenin to hybridize with mitotic chromosomes for samples of all localities. It turned out that all probes showed the same signals that appeared in every centromeric region and in the telomeric regions of chromosome 5, without differences within or between populations. Obviously, both type I and type II of the 5S rDNA arrays arranged in tandem, which was contrasting with other frogs or fishes recorded to date. More interestingly, all the probes detected centromeric regions in all karyotypes, suggesting the presence of a satellite DNA family derived from 5S rDNA.

In an attempt to analyze the organization of repetitive DNAs in the amphibian genome, 7 microsatellite motifs and a 5S rDNA sequence were synthesized and mapped in the karyotypes of 5 Amolops species. The results revealed nonrandom distribution of the microsatellite repeats, usually in the heterochromatic regions, as found in other organisms. These microsatellite repeats showed rapid changes among Amolops species, documenting the recent evolutionary history within this lineage. In contrast, 5S rDNA was localized in chromosomes 5 of all species, suggesting that these chromosomes are homologous within the monophyletic clade. Furthermore, the heteromorphic X and Y sex chromosomes (chromosomes 5) of A.mantzorum, had identical patterns of 5S rDNA, indicating that the subtelocentric Y resulted from a pericentric inversion. Several microsatellite repeats were found in the heteromorphic sex chromosomes, verifying the association of repetitive DNAs with sex chromosome differentiation in A. mantzorum.

Understanding how and why species evolve requires knowledge on intraspecific divergence. In this study, we examined intraspecific divergence in the endangered hotspring snake (Thermophis baileyi), an endemic species on the Qinghai-Tibet Plateau (QTP). Whole-genome resequencing of 58 sampled individuals from 15 populations was performed to identify the drivers of intraspecific divergence and explore the potential roles of genes under selection. Our analyses resolved three groups, with major intergroup admixture occurring in regions of group contact. Divergence probably occurred during the Pleistocene as a result of glacial climatic oscillations, Yadong-Gulu rift, and geothermal fields differentiation, while complex gene flow between group pairs reflected a unique intraspecific divergence pattern on the QTP. Intergroup fixed loci involved selected genes functionally related to divergence and local adaptation, especially adaptation to hot spring microenvironments in different geothermal fields.Analysis of structural variants, genetic diversity, inbreeding, and genetic load indicated that the hot-spring snake population has declined to a low level with decreased diversity, which is important for the conservation management of this endangered species. Our study demonstrated that the integration of demographic history, gene flow, genomic divergence genes, and other information is necessary to distinguish the evolutionary processes involved in speciation.

Advances in next-generation sequencing (NGS) technologies have led to an exponential increase in the number of whole genome sequences (WGS) in databases. This wealth of WGS data has greatly facilitated the recovery of full mitochondrial genomes (mitogenomes), which are vital for phylogenetic, evolutionary and ecological studies. Unfortunately, most existing software cannot easily assemble mitogenome reference sequences conveniently or efficiently. Therefore, we developed a seed-free de novo assembly tool, MEANGS, which applies the trie-search method to extend contigs from self-discovery seeds and assemble a mitogenome from animal WGS data. We then used data from 16 species with different qualities to compare the performance of MEANGS with three other available programs. MEANGS exhibited the best overall performance since it was the only one that completed all tests, and it assembled full or partial mitogenomes for all of the tested samples while the others failed. Furthermore, MEANGS selects superior assembly sequences and annotates protein-coding genes. Thus, MEANGS can be one of the most efficient software for generating high-quality mitogenomes so far, the further use of it will benefit the study on mitogenome based on whole genome NGS data. MEANGS is available at https://github.com/YanCCscu/meangs.

Understanding how and why species evolve often requires knowledge of intraspecific divergence. In this study, we examine intraspecific divergence in the endangered hot spring snake Thermophis baileyi, an endemic species of the Qinghai-Tibet Plateau. Genomic analyses using a hybrid assembly strategy resulted in a revised, high-quality genome. Whole-genome re-sequencing of 31 sampled individuals from 15 sites served to identify drivers of intraspecific divergence, and explore the potential role gene selection plays in divergence. Our analyses resolved three groups, with inter-group admixture occurring in regions of contact. Divergence seems to have occurred during the Pleistocene because of glacial climatic oscillations and geomorphological changes. Highly diverged regions (HDRs) that distinguish the groups most likely owe to gene sorting. Inter-group HDRs involve genes under positive selection that putatively relate functionally to ecological divergence, and especially reproduction. Our findings reveal the need to integrate multiple aspects to distinguish evolutionary processes potentially involved in speciation.

The visual characteristics of animals with different circadian habits, especially colubrid snakes, exhibit highly variable photoreceptor morphology. While studies have reported on the diversity in retinal cell morphology among snakes with different circadian patterns, few studies have examined the expression of genes related to vision. To explore gene expression patterns in the eyes between diurnal and nocturnal snakes, we carried out RNA sequencing of six tissues (eye, heart, liver, lung, kidney, and muscle) in two colubrids with disparate circadian activities, i.e., diurnal Ahaetulla prasina and nocturnal Lycodon flavozonatum, followed by weighted gene co-expression network analysis (WGCNA). The genes in the two most correlated modules were primarily enriched in different functional pathways, thus suggesting different biological functions. Three opsin genes (RH1, LWS, and SWS) were differentially expressed between the two species. Moreover, in the phototransduction pathway, different genes were highly expressed in the eyes of both species, reflecting specific expression patterns in the eyes of snakes with different circadian activity. We also confirmed the dominance of cone- and rod-related genes in diurnal and nocturnal adaptation, respectively. This work provides an important foundation for genetic research on visual adaptation in snakes and provides further insight into the adaptive evolution of such species.

Background Reptiles exhibit a wide variety of skin colors, which serve essential roles in survival and reproduction. However, the molecular basis of these conspicuous colors remains unresolved. Results We investigate color morph-enriched Asian vine snakes (Ahaetulla prasina), to explore the mechanism underpinning color variations. Transmission electron microscopy imaging and metabolomics analysis indicates that chromatophore morphology (mainly iridophores) is the main basis for differences in skin color. Additionally, we assemble a 1.77-Gb high-quality chromosome-anchored genome of the snake. Genome-wide association study and RNA sequencing reveal a conservative amino acid substitution (p.P20S) in SMARCE1, which may be involved in the regulation of chromatophore development initiated from neural crest cells. SMARCE1 knockdown in zebrafish and immunofluorescence verify the interactions among SMARCE1, iridophores, and tfec, which may determine color variations in the Asian vine snake. Conclusions This study reveals the genetic associations of color variation in Asian vine snakes, providing insights and important resources for a deeper understanding of the molecular and genetic mechanisms related to reptilian coloration.