Phylogeny and Underground Adaptation of Eulipotyphla Revealed by Whole Genome Comparison Phylogeny and Adaptation of Eulipotyphla

Background: Phylogenetic relationships within Eulipotyphla have long been debated due to their complex evolutionary history and the frequent inconsistency among phylogenetic trees inferred from different data sources. This order comprises both above-ground and subterranean mammals, providing an opportunity to investigate their adaptation to hypoxic, hypercapnic, and dark environments. Methods: In this study, we reconstructed the phylogeny of Eulipotyphla based on whole-genome comparisons and explored the causes of phylogenetic incongruence as well as the genetic basis of underground adaptation. We analyzed the genomes of ten species, including four above-ground species and six subterranean species. We also identified homologous coding sequences through whole-genome alignment and inferred phylogenetic trees based on genome-wide windows of 1000 bases. Divergence times among major lineages were estimated using MCMCtree, and the causes of inconsistent tree topologies were examined using QuIBL to distinguish incomplete lineage sorting from introgression. Finally, we designated the six subterranean species as foreground branches and applied branch-site models to identify genes under positive and negative selection. Results: Whole-genome analyses recovered a clear clustering pattern, in which the six subterranean species formed a monophyletic group, whereas the four above-ground species clustered into a distinct clade. Divergence time estimation suggested that the split between above-ground and subterranean lineages occurred approximately 53.51 to 68.78 million years ago. Gene tree analyses revealed substantial variation in tree topologies at several internal nodes, and QuIBL results indicated that introgression contributed to phylogenetic discordance in addition to incomplete lineage sorting. Positive selection analyses identified genes associated with heart regulation, blood circulation, oxidative stress response, and erythrocyte differentiation, while negatively selected genes were linked to cardiac septum and chamber development. Conclusions: These results clarify the phylogenetic relationships within Eulipotyphla and provide insights into the genomic basis of adaptation to underground environments.