| Hippophae is a small genus of Elaeagnaceae,containing 7 species and 11 subspecies,of which 7 species and 7 subspecies were distributed in China.All plants of Hippophae are diploid and dioecious shrub or small trees,with wind-borne pollination and birdmediated seed dispersals.These reproductive characteristics provide convenience for their long-distance migration,and increase the opportunities of hybridization and gene introgression between species and/or subspecies as well.Therefore,Hippophae plants are good materials for studying the mechanisms of homoploid hybrid speciation.H.litangensis and H.goniocarpa are two such hybrids confined to discretely distributed habitats in Qinghai and Sichuan provinces where their putative parental species occur sympatrically.It is proposed that H.litangensis originated from the hybridization between H.rhamnoides ssp.yunnanensis and H.neurocarpa,while H.goniocarpa originated from the hybridization between H.r.ssp.sinensis and H.neurocarpa.However,most of the previous studies were based on a single or a few chloroplast genome fragments and/or nuclear genome fragments.In addition,some studies only sampled few individuals and without sampling on population level.In order to better understand the process of homoploid hybrid speciation of H.litangensis and H.goniocarpa,a total of 78 chloroplast genomes of the two hybrids and their respective parents were sequneced by the Highthroughput sequencing technology,including 60 chloroplast genomes of H.litangensis and its parents in the same region from Litang county and Hongyuan county of Sichuan province,and 18 chloroplast genomes of H.goniocarpa and its parents in the same region from Qilian county of Qinghai province.We comapred the structural characteristics and genetic variation of the chloroplast genomes of the two hybrids and their respective parent species.The phylogenetic relationships were also reconstructed by combining the newly collected chloroplast genomes and those published in the NCBI.In order to increase the reliability of phylogenetic analysis,we also sequenced three samples representing H.gyantsensis,H.tibetana and H.r.ssp turkestanica.The main results we obtained are as follows:（1）We sequenced a total of 78 complete chloroplast genomes（154,944-156,460 bp in size）from five Hippophae species,of which three（H.litangensis,H.goniocarpa,and H.r.ssp.turkestanica）were recorded for the first time.Each of the chloroplast genomes encoded 134 genes,including 88 protein-coding,eight r RNA genes and 38 t RNA genes,and had a typical quardripartite structure with four parts（LSC,SSC and two IRs）.The sizes of LSC,SSC and IRs were 83,047-84,072 bp,18,571-19,073 bp and 26,516-26,679 bp,respectively,while the GC contents ranged from 36.6% to 36.7%.Additionally,the sliding window analysis revealed five higher nucleotide variability sites in non-coding regions: rps16-psb K,rpo B-trn C,psb Z-trnf M,rrn4.5-trn R,trn R-rrn4.5.（2）The chloroplast genome of H.neurocarpa was shorter than the other four taxa by about 1,500 bp.The insertions/deletions,with lengths more than 150 bp,mainly occured in the ycf3-rps4,rbc L-acc D,psb E-pet L fragments in the LSC region,and ndh Frpl32,rpl32-ccs A fragments in the SSC region.In addition,its boundary region was also different from those of the other four taxa.（3）H.litangensis was highly similar to H.r.ssp.yunnanensis in terms of chloroplast genome size,gene composition,GC contents,codons preference and boundary contraction and expansion of IR region.The phylgoenetic analysis showed that H.litangensis was a polyphyletic group,with the individuals from different regions located in different cladeds,indicating that H.litangensis may originate independently from several hybridizations between its parental species in different regions.The analysis based on haplotypes of each gene fragment and intergenic spacer showed that most fragments（80%）were shared by H.litangensis and H.r.ssp.yunnanensis,while the remaining（20%）were shared by the three taxa.It indicated that H.litangensis was more closely related to H.r.ssp.yunnanensis in cholorplast.Together with the previous evidence from nuclear gene marker analysis,H.r.ssp.yunnanensis should be the maternal parent of H.litangensis.（4）H.goniocarpa was highly similar to its parents in terms of chloroplast genome size,gene composition,GC content levels,codons preference and IR region boundary contraction and expansion.H.goniocarpa was not monophyletic.The analysis based on haplotypes of each gene fragment and intergenic spacer showed that most fragments（80%）were shared by H.goniocarpa and H.r.ssp.sinensis,while the remaining（20%）were shared by the three taxa.It indicated that H.goniocarpa was more closely related to H.r.ssp.sinensis in cholorplast.Together with the previous evidence from nuclear gene marker analysis,H.r.ssp.sinensis should be the maternal parent of H.goniocarpa.Our results did not support the propasal of the double maternal origins of H.goniocarpa.（5）The selection pressure analysis shows that the ycf1 gene in the chloroplast genomes of H.litangensis and its parents from Hongyuan county,and the inf A gene in the chloroplast genomes of H.goniocarpa and its parents from Qilian county were showed to be subject to positive selections.The Ka/Ks values for three genes,rps8,atp F and rpo A,were between 0.5 and 1 in all populations from the three studied regions,indicating relaxed selection.To sum up,in the present study we sampled multiple populations representing the two hybrids and their respective parental species.We anlysed the stuctural charcateristics and variation patterns of the complete chloroplast genomes,and reconstructed the phylogenetic relationships of the hybrids and their parents.The study provides insights into the hybrid origin of the two hybrids.The findings highlight the importance of sampling strategy and application of whole genome in studing the mechanisms of speciation.The generated genetic data are also believed to provide useful information for future theoretical and empirical work on Hippophae. |
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