Phylogeny And Phylogography Of Epilithic Sister Genera Oresitrophe And Mukdenia

Oresitrophe Bunge and Mukdenia Koidz.,usually grown on cliffs or rocks,are epilithic sister genera in the family Saxifragaceae.Oresitrophe Bunge is a monotypic genus,and the only species of the genus,O.rupifraga Bunge,is distributed in northern to central China.M.rossii(Oliv.)and M.acanthifolia Nakai,which are distributed in northeastern China to Korea.Currently,there has been controversy about the systematic taxonomy of the two genera,especially the phylogenetic position of M.acanthifolia within Mukdenia.Moreover,previous phylogeographic studies of temperate East Asian plants have often involved only single forest species and were dominated by woody plants.The geographical distribution pattern of O.rupifraga provides an ideal model for understanding the population dynamics and genealogical differentiation of temperate species in East Asia and the response to the climate change.In this study,nine individuals of three species within Oresitrophe and Mukdenia were sequenced and analyzed using genome skimming sequencing technology,the phylogenetic relationships between the two genera and the phylogeography of O.rupifraga were investigated based on the data obtained above.The main results are as follows:(1)Chloroplast genome,mitochondrial genome and nuclear ITS/ETS assembly and analysis The chloroplast genome,partial mitochondrial genome and ITS/ETS sequences were assembled based on the raw data obtained from the sequencing company.The results showed that the two chloroplast genomes of O.rupifraga were 156,738 bp and 156,775 bp,respectively,while the chloroplast genome sizes of M.rossii ranged from 156,420 bp-157,018 bp and M.acanthifolia ranged from 156,341 bp-156,935 bp.All the genomes have a majority angiosperm tetrad structure and together encoded 131 genes,including 86 proteincoding genes,37 t RNAs and 8 r RNAs.In the mitochondrial genome assembly,a partial mitochondrial genome was assembled,ranging from 259,355 bp to 263,180 bp in size.There were 63 genes annotated in the partial genomes,including 36 protein-coding genes,20 t RNA genes,6 r RNA genes and one pseudogene.The nuclear ETS and ITS sequences were also successfully assembled and the size was ranged from 457-458 bp and 638 bp-639 bp for ETS and ITS,respectively.(2)Comparative analysis of the chloroplast genomes for Oresitrophe and MukdeniaComparative analysis of the chloroplast genomes of all sequenced individuals of Oresitrophe and Mukdenia revealed that the chloroplast genomes of the two genera were relatively conservative in terms of gene content and gene order,and no structural variation such as gene rearrangements,translocations,or inversions were detected.In addition,the two genera also showed greater similarity in chloroplast genome codon usage,with a total of 64 different codons detected in all individuals,61 of which encoded 20 different amino acids and three encoded stop codons.The codon encoding leucine had the largest proportion of all codons and had the largest codon usage preference.(3)Genetic resources development for Oresitrophe and MukdeniaGenetic diversity was calculated for different chloroplast regions including genomic coding regions,intron regions,and spacer regions using Dna SP software.Six regions with high genetic diversity were successfully screened between Oresitrophe and Mukdenia,including trn H-psb A,trn K-atp A,pet N-psb M,psb C-trn S,pet Apsb J,ccs A-ndh D;Five regions were screened within Mukdenia,namely trn H-psb A,psb C-trn S,pet N-psb M,pet A-psb J,ccs A-ndh D.There only one region was detected(rpl33-rps8)within Oresitrophe.In addition,six regions(trn H-psb A,pet N-psb M,trn M-atp E,rps16-trn Q,ycf1 and ndh F)contained a higher number of species-specific parsimony-informative sites that may serve as potential DNA barcodes for Mukdenia species identification.(4)Phylogenetic relationships between Oresitrophe and MukdeniaThe phylogenetic relationships of Oresitrophe and Mukdenia were successfully resolved by reconstructing phylogenetic trees based on chloroplast,mitochondrial and nuclear ETS/ITS sequences.The results showed that the different individuals of the same species firstly formed a clade,and the inferred topology((M.rossii,M.acanthifolia),O.rupifraga)are well supported in trees reconstructed using the complete chloroplast genome sequences,chloroplast CDS regions and partial mitochondrial genome sequences.M.acanthifolia and M.rossii did not form a separate clade in the trees based on ETS+ITS data,while the mitochondrial CDS trees are not well-resolved.Hybridization or incomplete lineage sorting may be the cause of discordance between trees reconstructed from organellar and nuclear data.(5)Phylogeographic study of Oresitrophe rupifragaThe phylogeography of Oresitrophe rupifraga was investigated using two chloroplast fragments including ccs A-ndh D and rbc L.The results showed that the O.rupifraga populations could be divided into three distinct genetic lineages,named the north of Taihang Mountain and Yanshan lineage,the Wuling Mountain lineage,and the south of Taihang Mountain lineage.There was a high-level genetic diversity in O.rupifraga populations,with the highest value in the north of Taihang Mountain and Yanshan Mountain population.The Oresitrophe populations were more differentiated between populations than within populations at the chloroplast level and had a strong genealogical geographic structure.Mismatch distribution analysis showed that the Oresitrophe population did not experience rapid expansion.