Population genetics, trait mapping and fungal pathogen surveillance using untargeted sequencing in timber rattlesnakes (Crotalus horridus).

Genomic tools are increasingly important for conservation and wildlife health surveillance, yet their use is often constrained by limited resources, sample quality, and the need for minimally invasive approaches. Here we show that untargeted low-coverage sequencing applied to scale-clip DNA can jointly address population structure, genotype-phenotype associations, and pathogen surveillance in timber rattlesnakes (Crotalus horridus), a species threatened by habitat fragmentation, human encroachment, and the emerging fungal pathogen Ophidiomyces ophidiicola. We sequenced 107 snakes sampled from eight populations in the Appalachian Mountains of the northeastern United States to a mean depth of 0.94x per individual. Population differentiation is pronounced and only weakly correlated with geographic distance, and inbreeding coefficients in some populations are high enough to suggest fitness consequences. Genome-wide association analysis identifies a locus for black-to-yellow color morph containing ALDH4A1, a member of a gene family implicated in vertebrate pigmentation but distinct from melanin pathway pigmentation genes. Our untargeted sequencing method also captures host-associated microbes: O. ophidiicola loads are higher in snakes with clinical signs of snake fungal disease, though some asymptomatic individuals carry substantial loads, and skin-associated microbial communities are disrupted in snakes with higher fungal burdens. That all of these inferences derive from the same minimally invasive sampling and sequencing workflow highlights the scalability of this approach for biodiversity conservation.