Life phases integration uncovers long-lived gametophytes mediated reticulate evolution in Haplopteris (Pteridaceae)

Accumulating evidence suggests that speciation often resembles a reticulate network rather than a bifurcating process. In ferns, which possess independent gametophyte and sporophyte phases, reticulate evolution is common, especially where sporophytes of congeneric species co-occur. However, the role of long-lived gametophytes that distribute beyond their sporophytic counterparts in facilitating reticulate evolution remains underexplored. To address this gap, we investigated two species complexes of Haplopteris, a genus within one of the five lineages known to have evolved long-lived gametophytes. We integrated evidence from morphology, chromosome numbers, genome sizes, plastid DNA, and nuclear DNA, sampling comprehensively both gametophyte and sporophyte phases. We identified ploidy levels and genomic formulas of sampled individuals and reconstructed their reticulate evolutionary histories. Our results confirmed the reticulate evolution of Haplopteris for the first time and demonstrated that independent gametophyte populations, despite not producing their own sporophytes, play a critical role by contributing sperm to form hybrids. This study offers new insights into fern reproductive biology and gametophyte biogeography by reporting the first documented case of fern reticulate evolution involving both gametophyte and sporophyte samples, and the second known instance of hybridization involving independent gametophytes. Furthermore, a tetraploid species with a hybrid-origin is newly described.