Adaptation to a wide range of pathogenic environments is a major aspect of the ecological adaptations of vertebrates during evolution. Toll-like receptors (TLRs) are ancient membrane-bound sensors in animals and are best known for their roles in detecting and defense against invading pathogenic microorganisms. To understand the evolutionary history of the vertebrate TLR gene family, we first traced the origin of single-cysteine cluster TLRs that share the same protein architecture with vertebrate TLRs in early-branching animals and then analyzed all members of the TLR family in over 200 species covering all major vertebrate clades. Our results indicate that although the emergence of single-cysteine cluster TLRs predates the separation of bilaterians and cnidarians, most vertebrate TLR members originated shortly after vertebrate emergence. Phylogenetic analyses divided 1,726 vertebrate TLRs into 8 subfamilies, and TLR3 may represent the most ancient subfamily that emerged before the branching of deuterostomes. Our analysis reveals that purifying selection predominated in the evolution of all vertebrate TLRs, with mean dN/dS (ω) values ranging from 0.082 for TLR21 in birds to 0.434 for TLR11 in mammals. However, we did observe patterns of positive selection acting on specific codons (527 of 60,294 codons across all vertebrate TLRs, 8.7‰), which are significantly concentrated in ligand-binding extracellular domains and suggest host–pathogen coevolutionary interactions. Additionally, we found stronger positive selection acting on nonviral compared with viral TLRs, indicating the more essential nonredundant function of viral TLRs in host immunity. Taken together, our findings provide comprehensive insight into the complex evolutionary processes of the vertebrate TLR gene family, involving gene duplication, pseudogenization, purification, and positive selection.

Next Generation Sequencing has been widely used to characterize the prevalence of fecal bacteria in many different species. In this study, we attempted to employ a low-cost and high-throughput sequencing model to discern information pertaining to the wolf microbiota. It is hoped that this model will allow researchers to elucidate potential protective factors in relation to endangered wolf species. We propose three high-throughput sequencing models to reveal information pertaining to the micro-ecology of the wolf. Our analyses advised that, among the three models, more than 100,000 sequences are more appropriate to retrieve the communities’ richness and diversity of micro-ecology. In addition, the top five wolf microbiome OTUs (99%) were members of the following five phyla: Bacteroidetes, Fusobacteria, Firmicutes, Proteobacteria, and Actinobacteria. While Alloprevotella, Clostridium_sensu_stricto_1, Anaerobiospirillum, Faecalibactreium and Streptococcus were shared by all samples, their relative abundances were differentially represented between domestic dogs and other wolves. Our findings suggest that altitude, human interference, age, and climate all contribute towards the micro-ecology of the wolf. Specifically, we observed that genera Succinivibrio and Turicibacter are significantly related to altitude and human interference (including hunting practices).

The gut microbiota is a complex and essential system organ that plays an integrative role in balancing key vital functions in the host. Knowledge of the impact of altitude on the gut microbiota of European mouflon (Ovis orientalis musimon) and blue sheep (Pseudois nayaur) is currently limited. In this study, we compared the characteristics of gut microbiota in 5 mouflon at low altitude (K group), 4 mouflon at high altitude (L group), 4 blue sheep at low altitude (M group), and 4 blue sheep at high altitude (N group). The V3–V4 region of the 16S rRNA gene was analyzed using high-throughput sequencing. Analyses based on the operational taxonomic units showed significant changes in the gut microbial communities between groups at different altitudes. At the phylum level, groups at the high altitudes had a higher relative abundance of Firmicutes and a lower relative abundance of Bacteroidetes than those at the low altitudes. A higher Firmicutes:Bacteroidetes ratio is beneficial to animals in terms of the gut microbiota-mediated energy harvest. The relative abundance of Proteobacteria was significantly higher in the gut microbiota of mouflon sheep at high altitudes. At the genus level, the Bacteroides:Prevotella ratio was significantly higher in the low-altitude group (than the high-altitude group) of mouflon sheep and the ratio was significantly higher in the high-altitude group (than the low-altitude group) in blue sheep. In addition, the Ruminococcaceae_UCG-005 related to cellulose and starch digestion was the predominant genus in blue sheep and the relative abundance of the genus was significant higher in the high-altitude group than the low-altitude group of blue sheep (P < 0.01). In conclusion, our results suggested that the gut microbiota of high-altitude groups of sheep had stronger abilities related to energy metabolism and the decomposition of substances, e.g., fiber and cellulose, and that such abilities are associated with high-altitude adaptation.

Convergent evolution is an important sector of evolutionary biology. High-altitude environments are one of the extreme environments for animals, especially in the Qinghai Tibet Plateau, driving the inquiry of whether, under broader phylogeny, high-altitude factors drive the convergent evolution of Artiodactyla and Perissodactyla gut microbiomes. Therefore, we profiled the gut microbiome of Artiodactyla and Perissodactyla at high and low altitudes using 16S rRNA gene sequencing. According to cluster analyses, the gut microbiome compositions of high-altitude Artiodactyla and Perissodactyla were not grouped together and were far from those of low-altitude Artiodactyla and Perissodactyla. The Wilcoxon’s test in high-altitude ungulates showed significantly higher Sobs and Shannon indices than in low-altitude ungulates. At the phylum level, Firmicutes and Patescibacteria were significantly enriched in the gut microbiomes of high-altitude ungulates, which also displayed a higher Firmicutes/Bacteroidetes value than low-altitude ungulates. At the family level, Ruminococcaceae, Christensenellaceae, and Saccharimonadaceae were significantly enriched in the gut microbiomes of high-altitude ungulates. Our results also indicated that the OH and FH groups shared two significantly enriched genera, Christensenellaceae_R_7_group and Candidatus_Saccharimonas. These findings indicated that a high altitude cannot surpass the order level to drive the convergent evolution of ungulate gut microbiome composition but can drive the convergent evolution of alpha diversity and indicator microbiota in the gut microbiome of ungulates. Overall, this study provides a novel perspective for understanding the adaptation of ungulates to high-altitude environments.