Long-term small effective population size, inbreeding, and a recessive lethal haplotype drive premature death in the endangered Devils Hole pupfish (

As anthropogenic habitat fragmentation and population decline accelerate globally, growing numbers of species face compounding demographic and genetic threats to long-term survival. Many populations are already forced to persist at chronically small sizes, yet the genomic and fitness consequences of this fate remain poorly understood. Here we leverage the demographic history of the Devils Hole pupfish to investigate how long-term small population size and recent bottlenecks have shaped genetic diversity, genetic load, inbreeding, and fitness through comparative population genomics, historical sequencing, and sampling embryos that died prematurely during development. We find that genetic diversity in Devils Hole pupfish is among the lowest recorded in the wild and that fixed load is high, consistent with thousands of generations of isolation at small population size. Even in the face of this low diversity and high fixed load, we show that inbreeding is still strongly associated with premature embryonic death, which affects up to 25% of offspring in the captive refuge and can be identified in advance based on a characteristic elongated heart tube and reduced heart rate. We discovered a recessive lethal haplotype segregating at ~20% frequency that accounts for 50% of embryonic deaths and contains mutations in MIB1 and MMP16, genes associated with cardiomyopathy and atrial fibrillation. Our findings link genotype, phenotype, and fitness in an iconic endangered species to provide a rare comprehensive view into the evolutionary dynamics and consequences of long-term small effective population size, demonstrating that endangered species remain vulnerable to inbreeding depression despite extremely low genetic diversity.