Genetic commonalities in eye and pigment evolution in the Asellus aquaticus species complex extend to the biogeographically and ecologically most divergent cave population, A. infernus.

The Asellus aquaticus species complex, a freshwater isopod crustacean, includes surface and cave ecomorphs that differ markedly in eye and pigmentation phenotypes. Previous genetic studies in Slovenian cave populations revealed that the same genomic regions underlie eye and pigment loss across multiple cave populations, suggesting strong genetic constraints on these traits. To test the generality of this pattern, we examined Asellus infernus, a cave population from Mangalia, Romania, that inhabits thermal, sulfidic waters and is geographically distant from the Slovenian cave populations. Based on its distinct ecology and likely different ancestral surface population, we hypothesized that A. infernus would rely on different genomic regions for similar traits. Using hybrid crosses, we uncovered novel hybrid phenotypes unique to A. infernus, highlighting the presence of additional genetic variation not observed in Slovenian cave populations. However, we found that the same genomic regions previously implicated in Slovenian populations were also associated with similar eye and pigmentation phenotypes in A. infernus. Eye and pigment phenotypes examined were pigmented vs. unpigmented individuals, orange vs. red/brown and red vs. orange/brown eye pigment, stellate vs. diffuse head pigmentation pattern, and presence vs. absence of ommatidia and/or their fragments. These results point to shared genetic mechanisms of eye and pigment reduction between different cave populations. Whether these parallels reflect shared standing genetic variation in ancestral surface populations or convergent evolution through recurrent changes at the same loci due to pleiotropic fitness benefits remains unresolved.