Purifying selection purges harmful variants in the rarest pine

Population genetic theory predicts that severe bottlenecks and extremely small effective population sizes ( N _e ) should reduce the ability of natural selection to eliminate harmful mutations. Under this framework, deleterious alleles are expected to accumulate and even fix, eroding fitness, constraining evolutionary rescue, and potentially precipitating mutational meltdown. Yet, empirical tests of these predictions in species at the extreme lower bound of N _e remain rare. We address this gap using Pinus squamata , one of the rarest tree species on Earth, with only 35 wild individuals remaining. We generated a near-complete reference genome (29.2 Gb) for this species and performed population genomic analyses across nearly all of its extant individuals, together with two closely related species. P. squamata exhibits extraordinarily low nucleotide diversity (π = 3.35 × 10⁻⁵), the lowest reported for any plant. Demographic inference reveals a recent and severe bottleneck (∼20 generations ago) that reduced N _e to ∼2.7 and resulted in intense inbreeding. Contrary to theoretical expectations, we uncover evidence for highly efficient purifying selection: strongly deleterious mutations are markedly depleted, indicating substantial purging despite the extremely small N _e . Genome-wide patterns further implicate selection at linked sites—including background selection and pseudo-overdominance—as dominant forces shaping genomic variation in the species. These results challenge the prevailing view that drift overwhelms selection in extremely small populations. Instead, they suggest that, under certain genomic and demographic conditions, purifying selection can remain unexpectedly effective, potentially mitigating the risk of mutational meltdown in the rarest species.