Hidden in the dark: complex evolutionary history revealed by genome-wide analyses of cave amphipods.

Subterranean environments provide unique habitats and maintain high biodiversity. However, their underlying evolutionary processes remain poorly understood, and especially complex evolutionary hypotheses have not been tested or documented well. In this study, we investigate the evolutionary origin of spatially isolated groups of the subterranean amphipod Pseudocrangonyx (Crustacea: Amphipoda: Pseudocrangonyctidae) within a single cave (Fusa Cave) in northeastern Japan. To archieve this goal, we conducted phylogenetic and population genetic analyses using mitochondrial and nuclear markers, as well as genome-wide single nucleotide polymorphisms (SNPs), with enhanced taxon sampling primarily from northeastern Japan. Both analyses revealed that the isolated group was genetically distinct from another group within Fusa Cave, and that both populations were genetically related to another group of an adjacent cave (Kuma-ana Cave). In addition, migration analysis estimated limited gene flow among these populations. Demographic simulations based on SNPs suggested that the isolated population diverged first, followed by the differentiation of the two cave populations. Then, a secondary contact-likely involving hybridisation-occurred within Fusa Cave, resulting in the observed genetic similarity. Our findings suggest that complex evolutionary histories, including secondary contact are more likely than apparent sympatric or micro-allopatric diversification. This study emphasises the importance of considering complex evolutionary hypotheses in subterranean diversification and highlights the value of genome-wide approaches combined with multiple types of analyses for uncovering hidden evolutionary scenarios.