Abstract
Despite a long-standing interest since Darwin's time, the genomic implications of obligate self-fertilization remain elusive. Complete cleistogamy-the obligate production of closed, self-pollinating flowers-represents an extreme reproductive strategy. Here, we present the genomic profiles and evolutionary history of two lineages of the mycoheterotrophic orchid Gastrodia, both of which independently acquired complete cleistogamy, based on detailed sampling and a combination of simple sequence repeat (SSR), multiplexed ISSR genotyping by sequencing (MIG-seq) and RNA-seq data. Our analysis reveals clear species delimitation, with no evidence of introgression between the completely cleistogamous species and their co-occurring allogamous sisters. Intriguingly, all analyses indicate that both the completely cleistogamous Gastrodia species and their allogamous sisters exhibit genetic profiles typical of self-pollinating plants. This pattern suggests that their ancestors, probably bearing allogamous flowers, had already evolved mechanisms to mitigate the deleterious effects of selfing, potentially facilitating the emergence of complete cleistogamy through benefits such as reproductive assurance, enhanced colonization ability and species reinforcement. Meanwhile, further analyses suggest that complete cleistogamy evolved very recently (possibly within the last 1000-2000 years) in these two Gastrodia lineages. Combined with the scant evidence of complete cleistogamy outside Gastrodia, our findings imply a limited and ephemeral role for complete cleistogamy in plant speciation.