Deciphering the evolution and biogeography of ant-ferns Lecanopteris s.s

Southeast Asia is a biodiversity hotspot characterized by a complex paleogeography, and its Polypodiopsida flora is particularly diverse. While hybridization is recognized as common in ferns, further research is needed to investigate the relationship between hybridization events and fern diversity. Lecanopteris s.s., an ant-associated fern, has been subject to debate regarding species delimitations primarily due to limited DNA markers and species sampling. Our study integrates 22 newly generated plastomes, 22 transcriptomes, and flow cytometry of all native species along with two cultivated hybrids. Our objective is to elucidate the reticulate evolutionary history within Lecanopteris s.s. through the integration of phylobiogeographic reconstruction, gene flow inference, and genome size estimation. Key findings of our study include: (1) An enlarged plastome size (178–187 Kb) in Lecanopteris s.s., attributed to extreme expansion of the Inverted Repeat (IR) regions; (2) The traditional ‘pumila’ and ‘crustacea’ groups are paraphyletic; (3) Significant cytonuclear discordance attributed to gene flow; (4) Natural hybridization and introgression in the ‘pumila’ and ‘darnaedii’ groups; (5) L. luzonensis is the maternal parent of L. ‘Yellow Tip’, with L. pumila suggested as a possible paternal parent; (6) L. ‘Tatsuta’ is a hybrid between L. luzonensis and L. crustacea; (7) Lecanopteris s.s. first diverged during the Neogene and then during the middle Miocene climatic optimum in the Indochina and Sundaic regions. In conclusion, the biogeographic history and speciation of Lecanopteris have been profoundly shaped by past climate changes and geodynamics of Southeast Asia. Dispersals, hybridization and introgression between species act as pivotal factors in the evolutionary trajectory of Lecanopteris s.s.. This research provides a robust framework for further exploration and understanding of the complex dynamics driving the diversification and distribution patterns within Polypodiaceae subfamily Microsoroideae.