Table_5_Dynamic hybridization between two spleenworts, Asplenium incisum and Asplenium ruprechtii in Korea.docx

Natural hybridization between Asplenium incisum and A. ruprechtii has been observed in Northeast Asia and its allotetraploid species, A. castaneoviride, was reported. However, the hybridization process between the parental species and the origin of the allotetraploid taxon remains obscure. Additionally, the systematic affinities of the recently described hybrid A. bimixtum, considered to have originated from the hybridization of A. ruprechtii, A. trichomanes, and A. incisum, is unresolved owing to its similarity to A. castaneoviride. The goals of this study were to (1) investigate the hybridization between A. ruprechtii and A. incisum; (2) verify the origin of A. castaneoviride occurring in Korea, whether it independently arose from 2x sterile hybrids; and (3) elucidate the reliability of identifying A. bimixtum. Three genotypes, A. incisum, A. ruprechtii, and their hybrid, were identified based on the nuclear gene pgiC sequence and finally divided them into six types by ploidy levels: diploid A. incisum, A. ruprechtii, and four hybrid types (diploid A. × castaneoviride, triploid A. × castaneoviride, allotetraploid A. castaneoviride, and A. bimixtum). In the analyses of plastid DNA, all hybrids had an A. ruprechtii-type rbcL gene. In addition, the four plastomes of A. ruprechtii and the hybrids had high pairwise sequence identities greater than 98.48%. They increased up to 99.88% when a large deletion of A. x castaneoriviride (2x) collected from Buramsan populations was ignored. Notably, this large deletion was also found in triploid A. × castaneoviride and allotetraploid A. castaneoviride in the same populations. Sequence data of the nuclear and plastid genes showed that hybridization is unidirectional, and A. ruprechtii is the maternal parent. The large deletion of rpoC2-rps2 commonly found in the different ploidy hybrids of the Buramsan population suggests that the allotetraploid A. castaneoviride can be created independently from sterile hybrids. We assume that both polyploidization driving allopolyploidy and minority cytotype exclusion took place independently in the population, since A castaenoviride co-occurs with A. ruprechtii in small populations. Furthermore, it was also observed that an enlarged noncoding region in fern organelle (ENRIFO) of the plastome was found in the genus Asplenium.

在东北亚地区已观察到裂叶铁角蕨（Asplenium incisum）与鲁普雷希特铁角蕨（Asplenium ruprechtii）之间的自然杂交事件，且已有研究报道了其异源四倍体衍生物种栗绿铁角蕨（Asplenium castaneoviride）。然而，亲本物种间的杂交过程与该异源四倍体类群的起源仍未明晰。此外，近期报道的杂交物种A. bimixtum被认为由鲁普雷希特铁角蕨、铁线铁角蕨（Asplenium trichomanes）与裂叶铁角蕨杂交产生，但其与栗绿铁角蕨形态相似，因此其系统发育亲缘关系仍未解决。本研究的目标如下：（1）探究鲁普雷希特铁角蕨与裂叶铁角蕨之间的杂交关系；（2）验证韩国境内分布的栗绿铁角蕨的起源，即其是否由二倍体（2x）不育杂种独立演化而来；（3）阐明A. bimixtum物种鉴定的可靠性。本研究基于核基因pgiC序列，鉴定出3个基因型：裂叶铁角蕨、鲁普雷希特铁角蕨及其杂交种；随后根据倍性水平将其划分为6个类型：二倍体裂叶铁角蕨、二倍体鲁普雷希特铁角蕨，以及4个杂交类型（二倍体杂种A. × castaneoviride、三倍体杂种A. × castaneoviride、异源四倍体栗绿铁角蕨与A. bimixtum）。质体DNA分析结果显示，所有杂种均携带鲁普雷希特铁角蕨型rbcL基因。此外，鲁普雷希特铁角蕨与各杂种的4个质体基因组之间的成对序列相似性均高于98.48%；若忽略从Buramsan种群中采集的二倍体杂种A. × castaneoviride的大片段缺失区域，其相似性可提升至99.88%。值得注意的是，在同一种群的三倍体杂种A. × castaneoviride与异源四倍体栗绿铁角蕨中，也发现了该大片段缺失现象。核基因与质体基因的序列数据表明，该杂交过程为单向杂交，且鲁普雷希特铁角蕨为母本。Buramsan种群内不同倍性杂种中普遍存在的rpoC2-rps2区段大片段缺失现象，表明异源四倍体栗绿铁角蕨可由不育杂种独立形成。鉴于栗绿铁角蕨与鲁普雷希特铁角蕨在小型种群中伴生分布，我们推测，驱动异源多倍化的多倍化过程与少数细胞型排除过程在该种群中独立发生。此外，本研究还在铁角蕨属（Asplenium）的质体基因组中发现了蕨类细胞器增大非编码区（enlarged noncoding region in fern organelle，ENRIFO）。