Abstract: Understanding the driving forces of speciation is a central question in evolutionary biology. Cycas bifida (Dyer) K. D. Hill and Cycas micholitzii Dyer are two morphologically similar Cycas (Cycadaceae) species with deeply pectinate megasporophylls and dichotomously divided leaflets. Geographically, they are isolated by the Red River Fault Zone (RRFZ), which acts as a biogeographical barrier for many Cycas species. In this study, we investigated the divergence, genetic diversity, genetic structure, and demographic history of C. bifida and C. micholitzii to uncover the speciation mechanisms for the two endangered species. Results revealed high historical gene flow but low contemporary gene flow between the two species, with a deep divergence occurring in the Late Miocene. The long-term geographical isolation and ecological niche differentiation were identified as important factors in driving the divergence of the two species, supported by significant isolation by distance testing, barrier analysis, and niche consistency detection. Geographical isolation by the RRFZ played a pivotal role in facilitating their divergence by constraining gene flow. Conservation genetic analyses indicated high genetic diversity within both species, coupled with disparate responses to Pleistocene climate fluctuations: C. micholitzii experienced population expansion, while C. bifida displayed a mild bottleneck effect. To ensure the protection and long-term sustainability of two threatened species, relevant conservation guidelines were proposed based on the assessment of their genetic diversity and structure. This study furnishes valuable insights into the mechanisms underpinning species divergence and delivers actionable recommendations for cycad conservation.

Key words: conservation, Cycas bifida, Cycas micholitzii, genetic differentiation, Red River Fault Zone (RRFZ).