The Phylogenomic Of Aurantioideae And Phylogeography Of Wild Citron(Citrus Medica)and Ichang Papeda(Citrus Ichangensis)

China is one of the origin centers for the fruit trees in the world,and one of the most important origin centers for Citrus.Aurantioideae,as one of the large subfamilies of Rutaceae family,comprises two tribes,Clauseneae and Citreae.According to the Swingle and Reece classification largely adopted by the citrus research community,Aurantioideae subfamily comprise 33 genera,203 species and 33 varieties.During the last two centuries,international and domestic academics have dedicated to studying the phylogeny of Aurantioideae,especially paying much attention to the origin and evolutionary history in Citrus.But,unfortunately,the deeply phylogenetic relationship of Aurantioideae was still highly controversial.What is more,the taxonomy of Citrus and the origin of some hybrid citrus were full of mystery without consensus opinion.Plenty of wild germplasms in Citrus were characterized by the existence of wild and natural populations in mountainous regions in southwestern and middle-west China,such as citron(Citrus medica)and Ichang papeda(Citrus ichangensis).Study of the investigation,collection and utilization of wild citrus germplasms in these regions was just in the infancy stage,as a result relatively little information about these species' distribution range,genetic diversity,population structure and demographic history was available.In the present study,the nSSR,cpSSR,single copy nuclear gene and chloroplast gene were analyzed systematically in attempt to clarify the genetic diversity,population differentiation of citron and Ichang papeda.We also put forward the hypothesis about the demographic history of Ichang papeda.Furthermore,with the help of whole genome reseuqence datasets,we presented new phylogenetic analyses of the nuclear and chloroplast genome of 73 different species which constituted the most comprehensive and detailed study to date the evolution and variability of the subfamily Aurantioideae.The phylogenetic framework of the genus Citrus was also reconstructed and we identified the origin of some hybrid citrus.The main results are as follows:1.Genetic diversity and phylogenetic relationships of citron and its relativesIn south China,wild and semi-wide citron is extensively distributed across a large area,such as Yunnan Province and Tibet Autonomous Region.The diversity pattern of the 56 accessions for the 77 nSSR loci revealed a highly variable number of alleles.A total of 387 alleles were detected,and the number of alleles ranging from 2 to 12.Across the accessions,the average of observed heterozygosity and expected heterozygosity was 0.36 and 0.49,respectively.The results of both PCoA and UPGMA clustering were relatively congruent and revealed a clear delimitation among all the analyzed accessions.There was two different gene pools that shaped the genetic structure of the populations of citron.Analysis of the geographic origin of each individual revealed that one gene pool mainly included fingered citrons(Citrus medica var.sarcodactylis)and wild citrons collected from Tibet,while the other gene pool included most of the citrons presumed to be from Yunnan.Finger citron had a close relationship with citron and might evolve with citron from the common ancestor.The genetic contributors of some hybrid species,such as Citrus limonia,Citrus aurantifolia,and Citrus limon,were also discussed,and citron appeared to be a primary contributor to the speciation of these secondary citrus species.2.Molecular phylogeography and population evolution analysis of Ichang papedaIchang papeda,a wild and endemic perennial fruit tree,is mainly distributed in southwestern and middle-west China.A total of 47 chloroplast haplotypes were identified in sixteen sampled populations,231 accessions,based on the combination of different alleles produced through 15 cpSSR markers.The number of haplotypes per population varied from 1 to 9.In addition,only three populations had no private haplotypes and all of them were fixed for a single haplotype.The distribution of chloroplast haplotypes of Ichang papeda also showed obvious geographic features.Sixty-six nSSR markers,from which a total of 338 alleles are amplified,revealed an average of 5.121 alleles per locus and an average of 1.703 effective alleles per population.The average of expected heterozygosity and observed heterozygosity is 0.313 and 0.267,respectively.The analyses of neighbor-joint tree,PCoA and population structure got the similar results that three distinct clusters could be identified in Ichang papeda populations,with one cluster spanning the potential refugia in Wuling Mountains and Ta-pa Mountains,and other two clusters covering a more limited distribution range.The nSSR-derived Analysis of Molecular Variance(AMOVA)produced consistent results,which showed that the variation was mostly within populations(44.14% for the Fst and 46.26% for the Rst)and among groups(44.89% for Fst and 43.56% for Rst).A total of 9 unlinked nuclear loci were sequenced for 88 individuals representing 16 populations of Ichang papeda and relatively low genetic diversity could be found in Ichang papeda.The mean values of Tajima's D and D* and F* of Fu and Li were negative for most populations of Ichang papeda,indicating population size expansion and/or purifying(negative)selection.Clear signals of recent bottlenecks and strong patterns of isolation by distance were detected among different subpopulations,indicating a low extent of historical gene flow for this species and that genetic drift would occur after population differentiation.Owing to the complicated topography,the mountainous regions and the Yangtze River have provided long-term stable habitats for Ichang papeda and acted as main barriers for its expansion,which might facilitate the process of speciation.Restricted and asymmetric gene flow,bottlenecks,and genetic drift played important roles in the evolution of Ichang papeda by shuffling the genetic variation among different populations,which led to the current population structure and distribution pattern.3.Phylogenomics elucidate the complex origin and evolution history of AurantioideaeA total of 73 different species in Aurantioideae were selected and chloroplast gene tree,nuclear gene tree and nuclear species tree were reconstructed with each best evolution model to elucidate the complex origin and evolution history,respectively.It was clear that the Clauseneae was totally different from the Citreae,which could be confirmed by both chloroplast and nuclear phylogenomic analyses.Plants in Triphasiinae could be one of most primitive species in Citreae.Our results strongly supported the fact that Atalantia belongs to the near citrus,but Citropsis was more likely to be the member of primitive citrus instead that of the near citrus.Our results showed that the true citrus group was monophyletic,which contained Poncirus,Microcitrus,Eremocitrus,Clymenia,Fortunella,and Citrus.Poncirus was the first genus separated from other five genera in this group and Fortunella might have a close relationship with Citrus.Except for Citrus medica,Citrus reticulata and Citrus grandis,Citrus ichangensis and Citrus nobilis might be the other two basic species in Citrus and other genotypes were derived from hybridizations between these basic species.Citrus aurantifolia would be a direct interspecific hybrid between Citrus medica and Citrus micrantha or Citrus hystrix,with the former as the male parent and the later as the female parent.Both Citrus reticulata and Citrus medica participated in the formation of Citrus limonia,with the former as the female and the later as the male parent.The chloroplast of Citrus limon was derived from sour orange or sweet orange and the male parent might be Citrus medica.Citrus indica was a hybrid and Citrus mdeica might be its female parent and Citrus reticulata might be its male parent.Citrus sinensis might act as a female parent to participate in the origin of Citrus junos and a male parent to participate in the origin of Citrus paradisi.Citrus ichangensis might be the male parent of Citrus junos,and Citrus grandis might be the female parent of Citrus paradisi.According to the estimation about divergence time from the chronogram,the oldest speciation event was the speciation between the Clauseneae and Citreae,which occurred approximately 24.96 Ma(million years ago).The divergence time between Triphasiinae and other two subtribes,Citrinae and Balsamocitrinae,was estimated at 22.01 Ma.The true citrus in Citrinae diverged with the Balsamocitrinae,near citrus and primitive citrus in Citrinae about 19.21 Ma.Atalantia and Hesperethusa diverged at 10.04 Ma.Poncirus was one of the first genera to split from the true citrus at 14.19 Ma.Citrus medica,Citrus grandis and Citrus reticulata in Citrus,occurred from 3.43 to 4.72 Ma.