Ancestral gene flow shaped the singular origin of the Amazon molly.

The evolutionary origins of asexuality remain poorly understood, despite extensive research on its ecological and evolutionary consequences. Asexuality often arises through hybridization between species with intermediate genomic divergence, implying that hybrid-induced asexuality may be partly repeatable. The Amazon molly (Poecilia formosa), the first asexual vertebrate known to science, challenges this view: repeated experimental crosses between its extant parental species have failed to recreate a stable Amazon molly-like lineage. This apparent paradox gave rise to the Rare Formation Hypothesis, which proposes that stable asexuality requires an exceptionally specific genomic combination. Here, we combine experimental crosses, molecular cytogenetics, and population genomics to test whether ancestral introgression before the hybrid speciation event set the stage for the singular origin of the Amazon molly. We show that most experimental hybrids are viable but sexual, but that a subset of F1 hybrids produce unreduced eggs through a mechanism distinct from that of the Amazon molly. Population genomic analyses reveal that introgression between parental species likely predated the formation of the Amazon molly, and shared homozygous tracts across Amazon molly genomes support inheritance from admixed progenitors. Together, our findings reconcile the repeatable and contingent views of the origin of asexuality, suggesting that ancestral introgression may be the missing mechanism assembling the rare genomic combinations required for seemingly unrepeatable evolutionary innovations, including the emergence of asexual species.