Severe inbreeding and gene loss in the historical and extant population of the critically endangered Devils Hole pupfish

Small populations with limited geographic distributions are predicted to be threatened by inbreeding and lack of genetic diversity, both of which may negatively impact fitness and exacerbate population decline. One of the most extreme natural examples is the Devils Hole pupfish (Cyprinodon diabolis), an iconic and critically endangered species with the smallest known habitat range of any vertebrate. This imperiled species has experienced severe declines in population size over the last thirty years and suffered major, repeated bottlenecks in 2007 and 2013, when the population sunk to 38 and 35 individuals, respectively. Here we sequenced contemporary and historical genomes of Devils Hole and neighboring Death Valley and Ash Meadows desert pupfishes to examine the genomic consequences of small population size. We found extreme inbreeding (FROH = 0.71 - 0.82) and increased genetic load in the Devils Hole pupfish. We also document unique fixed loss-of-function (LOF) alleles and deletions in genes associated with sperm motility, stress, and hypoxia within the extant Devils Hole pupfish population that likely reduce fitness. Comparisons between contemporary samples (2008 - 2012) and a genome sequenced from a 1980 formalin-fixed museum specimen suggest that inbreeding has increased 6% as the population has declined, but that many putatively deleterious variants have been segregating in the population since at least 1980. This includes a fixed early stop codon in cfap43 (n = 8/8 samples), which is associated with sperm flagellum defects and causes infertility in humans and mice. Out of ninety-four unique deletions, fifteen were detected within 2 kb of annotated genes. Five have roles in physiological responses to hypoxia and mitochondrial activity, such as redd1 (n = 7/7 samples), suggesting impaired hypoxia tolerance in this species despite the low oxygen concentrations of Devils Hole. We thus document one of the most extreme inbreeding events in a natural population and a set of candidate deleterious variants to inform management and potential genetic rescue in this conservation icon.