Life phases integration uncovers long-lived gametophytes mediated reticulate evolution in ferns
Abstract
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 of 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.
Methodology and Implementation Steps
Comprehensive sampling was a critical foundation of this study. Through international collaboration, we obtained material from all currently known localities of the target species. This broad sampling enabled comparisons across populations, geographic regions, and life-history stages, providing a robust basis for reconstructing their evolutionary history. To disentangle their reticulate evolution, we integrated multiple lines of evidence. In addition to examining external morphology, we measured genome size, determined chromosome numbers, and analysed plastid and nuclear DNA data generated using third-generation sequencing. By combining these datasets, we identified lineages that likely originated through hybridization and polyploidization, reconstructed their evolutionary relationships, and evaluated how these processes may have contributed to the formation of new species.
Innovation and Cross-Disciplinary Collaboration
Fern systematics has traditionally focused on the sporophyte, whereas the morphologically simple and often difficult-to-identify gametophyte generation has remained comparatively understudied, leaving a major gap in our understanding of fern diversity and evolution. This study addresses that gap by systematically integrating gametophyte data into molecular and population-level analyses. The results show that long-lived gametophytes can play important roles in both speciation and population persistence. By moving beyond the conventional sporophyte-centred framework, this work introduces a new methodological perspective and broadens our understanding of the processes shaping fern evolution. Its conceptual and methodological originality provides a valuable framework that can be widely applied and further developed in fern systematics and evolutionary biology.
Expected Results and Contributions
Haplopteris yakushimensis is a globally rare fern known only from Japan, Taiwan, and Vietnam. By integrating data from both the gametophyte and sporophyte generations, this study shows that its long-lived gametophytes may contribute not only to interspecific hybridization but also to population persistence and recovery. When environmental conditions are unsuitable for sporophyte formation, gametophytes can survive for extended periods, forming a potential “cryptic population reservoir” from which sporophytes may re-emerge when conditions improve. These findings suggest that fern conservation should not focus solely on the distribution and abundance of sporophytes, but should also incorporate gametophytes into field surveys and conservation planning. More broadly, the study provides new insights into how ferns may persist under climate change and habitat disturbance.
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