Natural Hybridization In Three Sympatric Ligularia Species

A key process in speciation concerns the formation and maintenance of reproductive isolating barriers between diverging lineages.Although species boundaries are frequently investigated between pairs of species across many taxa,reproductive isolating barriers among multiple species(>2),which may represent the most common phenomenon in the nature,remain to be clarified.Ligularia(Asteraceae)is a highly diversified genus in the eastern Qinghai-Tibetan Plateau(QTP)and adjacent areas,moreover allopatric speciation during the uplifts of the QTP and interspecific hybridization caused by secondary sympatry are suggested to give rise to its high species diversity.Many Ligularia species occur sympatrically with overlapping flowering periods and natural hybridization is common in Ligularia.In this study,we assessed the natural hybridization in sympatric L.cyathiceps,L.duciformis and L.yunnanensis to reveal whether the species boundaries can be maintained in sympatric Ligularia species in the eastern QTP and adjacent areas when hybridization occurs.We used three low-copy nuclear loci to identify if natural hybridization occurred in three Ligularia species at a sympatric site,and we assessed the direction of natural hybridization based on chloroplast DNA fragments once hybridization is confirmed.Then we used the SNPs obtained from next-generation double digest restriction-site associated DNA sequencing(ddRAD-seq)to reveal the genetic structure of the hybrid zone.The main results are as follows.1.The confirmation of natural hybridizationIn this study,three low-copy nuclear loci(A12,B14 and D30)and three chloroplast intergenic spacers(psbA-trnH,trnL-rpl32 and trnQ-5'rpsl6)were used to test the natural hybridization hypothesis in L.cyathiceps,L.duciformis and L.yunnanensis and to detect the direction of natural hybridization.The haplotype network analysis based on the haplotypes for all the investigated individual at three nuclear loci showed that Type A and Type B had haplotypes from L.cyathiceps and L.duciformis,while Type C and Type D had haplotypes from L.duciformis and L.yunnanensis.The results were also supported by the phylogenetic trees for haplotypes of all the individuals at three nuclear loci based on the Bayesian Inference(BI)and Maximum Parsimony(MP)methods.The results provide strong evidence for the natural hybridization hypothesis,i.e.,morphologically intermediate individuals Type A and Type B are the hybrids of L.cyathiceps and L.duciformis,while morphologically intermediate individuals Type C and Type D are the hybrids of L.duciformis and L,yunnanensis.Moreover,the results suggested that there were no hybrids between L.cyathiceps and L.yunnanensis.The cpDNA results suggested that two hybridization groups L.cyathiceps × L.duciformis and L.duciformis × L.yunnanensis both showed bidirectional but asymmetric hybridization.Ligularia duciformis(70.83%)was the primary maternal parent in the L.cyathiceps × L.duciformis hybridization group,and L.yunnanensis(82.81%)for the L.duciformis × L.yunnanensis hybridization group.The shared pollinators' preference and the relative abundance of parental species may lead to the asymmetry of two hybridization groups,respectively.In the present study,most hybrid individuals in two hybridization groups show chromatogram additivity at three nuclear loci,suggesting they might be F1s.In addition,although there are each two morphology-differential types of hybrids(Type A and Type B,Type C and Type D)produced by two hybridization groups respectively,it is noteworthy that different types in the same hybridization group showed similar nuclear and chloroplast haplotype composition,suggesting that there are no significant differences in the paternal/maternal species and the genetic components for the hybrids in the same hybridization group.2.The genetic structure of the hybrid zoneWe obtained three datasets through the ddRAD sequencing of 68 individuals of L.cyathiceps,L.duciforis,L.yunnanensis and four types of hybrids(68 individuals,2150 SNPs;L.cyathiceps × L.duciformis,38 individuals,156 SNPs;L.duciformis ×L.yunnanensis,40 individuals,1447 SNPs).The results of STRUCTURE analysis,maximum-likelihood(ML)tree and principle coordinate analysis(PCoA)based on 2150 SNPs confirmed the results based on three nuclear loci and proved the natural hybridization of L.cyathiceps X L.duciformis and L.duciformis x L.yunnanensis.The results of NewHybrids analysis suggested the hybrid individuals of L.cyathiceps x L.duciformis(Type A and Type B)were all F1s,and most hybrid individuals of L.cyathiceps ×L.duciformis(Type C and Type D)were F1s,with three backcross individuals.STRUCTURE analysis suggested that there were no gene introgression in three parental species L.cyathiceps,L.duciformis and L.yunnanensis.Moreover,hybrid swarm simulation using HYBRIDLAB indicated that the SNP datasets used in this study had sufficient discriminatory power for accurate hybrid detection.Hybridization L.cyathiceps × L.duciformis and L.duciformis x L.yunnanensis were both restricted to F1s plus a few first-generation backcrosses,and there were no later-generation hybrids,implying· reproductive isolation between hybridizing species.Although natural hybridization takes place,three Ligularia species show strong reproductive isolation and maintain species boundaries even under the circumstances of human disturbance,which may be a key mechanism to maintain species diversity of Ligualria in the eastern QTP and adjacent areas.Besides,two FST-based outlier approaches BAYESCAN and LOSITAN showed 39 outliers under divergent selection.BLAST2GO annotation indicated one locus was related to chromatin remodeling,which may be associated to chromosomal rearrangements in the hybrids and may provide indirect evidence for hybrid sterility.While another locus was related to phytochrome photoreceptor,suggesting that light may play an important role in the ecological divergence of three Ligularia species and hybrids.