Unexpected excess heterozygosity in a threatened, mycoheterotrophic, self-pollinating orchid

Self-fertilization results in reduction of heterozygosity over time, accelerated by small population sizes, restricted ranges, and fragmented habitats. Yet some species maintain heterozygosity despite these factors. One is Corallorhiza bentleyi , a threatened, mycoheterotrophic, self-pollinating orchid restricted to Virginia and West Virginia, USA. Reduced representation sequencing was employed across five of the ∼12 known localities of C . bentleyi , including representatives of sister species C. involuta from Mexico, to quantify patterns of population structure and genomic diversity. Surveys of C. bentleyi revealed a predominance of the cleistogamous (closed-flowered) growth form. Differentiation was observed between the two species but with weak population structure in C. bentleyi . Surprisingly, excess heterozygosity and negative inbreeding were recovered in both species, counter to expectations. Analyses provided evidence against bioinformatic artifacts (collapsed paralogs), small-sample bias (Robertson effect), and accumulation of mutations along chromosomes due to low recombination associated with selfing (Meselson effect) as explanations for excess heterozygosity. Demographic analyses favored a model of post-glacial divergence among C. bentleyi and C. involuta lacking bottlenecks. Finally, simulations under alternative selection scenarios favored models of overdominance vs. those of negative frequency dependent selection. It was concluded that overdominance, namely selection against selfed homozygotes, is the most likely explanation for elevated heterozygosity in C. bentleyi . Conservation concerns are discussed in light of these findings and the threats of extinction for one of North America's most vulnerable orchid species.