Exploring natural hybridizations among Asplenium ruprechtii and related taxa in Korea

The purported four hybrid origins of Asplenium in Korea were tested based on morphological, cytological and DNA sequence data. Asplenium castaneo-viride, A. ${\times}$ uiryeongse, A. ${\times}$ montanus, and A. ${\times}$ kitazawae share several morphological characteristics with the Asian walking fern A. ruprechtii and related taxa as parents and show a sympatric distribution with the putative parents, raising the possibility of hybrid origins: A. castaneo-viride (A. ruprechtii and A. incisum), A. ${\times}$ uiryeongse (A. ruprechtii and A. pekinense), A. ${\times}$ montanus (A. ruprechtii, A. trichomanes, and A. incisum), and A. ${\times}$ kitazawae (A. ruprechtii and A. sarelii). We investigated flow cytometry and chloroplast DNA sequence data (rbcL, rps4-trnS, and rps4-trnS intergenic spacer) to clarify the hybridization and origin of each hybrid. In the flow cytometry analyses, A. ruprechtii shows diploid (2x) only, whereas A. castaneo-viride (3x, 4x), A. ${\times}$ uiryeongse (3x), A. ${\times}$ montanus (3x, 4x), and A. ${\times}$ kitazawae (2x, 4x) exhibit polyploidy, suggesting hybrid events along speciation. The rbcL and rps4-trnS and rps4-trnS intergenic spacer data suggest that A. ruprechtii is one the maternal ancestors of all four hybrids. In addition, the rps4-trnS and rps4-trnS intergenic spacer data indicate that A. incisum is also the maternal ancestor of A. ${\times}$ kitazawae and A. ${\times}$ montanus, proposing multiple hybridization events for these two hybrids. In A. ${\times}$ montanus, morphological features such as the leaf forms and sympatric distributions of the species also support the multimaternal hypothesis, but the morphological features of A. ${\times}$ kitazawae must be examined with consideration of hybrid events. To clarify the complex hybrid evolutionary lineages of the four Asplenium hybrids, further research with taxon sampling and molecular markers should be conducted.