Male sterility in natural, domesticated and feral populations of the gynodioecious European chestnut (Castanea sativa)

European chestnut (Castanea sativa Mill.) has undergone centuries of selection for nut production, yet its domestication syndrome remains poorly characterised. This study investigates evolutionary shifts in sexual expression, genetic ancestry, and morphological traits across a human-impact gradient in Northwest Iberia: natural (Wild Atlantic), domesticated, and feral (Wild Inland) chestnut germplasm. We analysed 292 accessions using nuclear microsatellites and morphological characterisation of male catkin types, alongside several traits potentially related to female advantage and domestication. Genetic structure analysis highlighted a stark contrast between Wild Atlantic populations—dominated by North Iberian (Nib) ancestry and male-fertile (longistaminate) individuals—and Wild Inland/Domesticated groups, predominantly comprising intraspecific hybrids between Nib and Mediterranean demes exhibiting a high sex ratio (up to 89%). Our results reveal a clear functional signal associated with male sterility: male-sterile accessions exhibit significantly higher individual heterozygosity, higher fruit set (0.82 vs. 0.74), and 88% more female inflorescences per bisexual catkin (3.61 vs. 1.92) compared to fertile ones. Furthermore, we identified significant differences in male catkin length across sexual types, a finding that emerges when analysed across a diverse collection of wild trees of a similar age. These results provide robust evidence for the female advantage as a driver of chestnut domestication, indicating that selection for nut yield favoured male-sterile intraspecific hybrids stabilised through grafting. This study clarifies the anthropogenic evolutionary history of C. sativa and underscores the role of feral populations—genetic mosaics of Mediterranean and indigenous demes—as a source of new varieties. Domestication triggered male sterility through intraspecific hybridisation and female advantage selection. This transformed germplasm into an evolutionary laboratory, contrasting the male-sterile intraspecific hybrids of Inland Galicia with the predominantly male-fertile ancestral populations preserved in the Wild Atlantic region.