Phylogenomics of Fresh and Formalin Specimens Resolves the Systematics of Old World Mud Snakes (Serpentes: Homalopsidae) and Expands Biogeographic Inference

Our knowledge of the biodiversity of Asia and Australasia continues to expand with more focused studies on systematics of various groups and their biogeography. Historically, fluctuating sea levels and cyclic connection and separation of now-disjunct landmasses have been invoked to explain the accumulation of biodiversity via species pump mechanisms. However, recent research has shown that geological shifts of the mainland and species dispersal events may be better explanations of the biodiversity in these regions. We investigate these processes using the poorly studied and geographically widespread Mud Snakes (Serpentes: Homalopsidae) using a target capture approach of ~4,800 nuclear loci from fresh tissues and supplemental mitochondrial data from formalin tissues from museum specimens. We use these datasets to reconstruct the first resolved phylogeny of the group, identify their biogeographic origins, and test hypotheses regarding the roles of sea-level change and habitat selection on their diversification. Divergence dating and ancestral range estimation yielded support for an Oligocene origin and diversification from mainland Southeast Asia and Sundaland in the rear-fanged group ~20 million years ago, followed by eastward and westward dispersal. GeoHiSSE models indicate that niche expansion of ancestral, rear-fanged lineages into aquatic environments did not impact their diversification rates. Our results highlight that Pleistocene sea-level changes and habitat specificity did not primarily lead to the extant species richness of Homalopsidae and that, alternatively, geological shifts in mainland Southeast Asia may have been a major driver of diversity in this group. We also emphasize the importance of using fresh and degraded tissues, and both nuclear and mitochondrial DNA, for filling knowledge gaps in poorly known but highly diverse and conceptually important groups. Here, Homalopsidae represents a non-traditional but effective model study system for understanding transitions between terrestrial, marine, and freshwater environments.