| The subtropical region of China,which preserved many rare and endangered plant taxa,is a crucial refugium for Tertiary relict plant species.However,little is known about how global climate change since Tertiary and human activities have influenced the formation of genetic diversity and adaptive potential of these relict species.Dipteronia Oliv.is an ancient relict woody genus that widely distributed in both East Asia and North America during the Palaeogene.It contains only two extant species,i.e.,D.sinensis Oliv.and D.dyeriana Henry,both endemic to East Asia.Dipteronia sinensis occupies a relatively extensive range in Central and Southwestern China,while D.dyeriana is restricted to small isolated areas in Southeastern Yunnan.In this study,we employed phylogenomics,comparative genomics,and population genomics approaches to reconstruct the phylogenetic relationship of Acer and Dipteronia,elucidate the nucleotide diversity,lineage divergence,demographic history,inbreeding,and genetic load of the two Dipteronia species.The main results are as follows:(1)Reconstruction of phylogenetic relationship of Acer and Dipteronia.In this study,we present large-scale,phylogenomic data sets,incorporating complete chloroplast(cp)genome sequences and transcriptome data from 13Sapindaceae species,to resolve the phylogenetic relationship between Dipteronia and Acer.Corroborating classical morphology-based classifications,both cp genome and nuclear datasets(2,466 co-orthologous genes and 273 co-SCNGs)recovered Dipteronia and Acer as mutually monophyletic groups.Besides,our fossil-calibrated molecular phylogenies dated the crown of the two extant Dipteronia species to the Paleocene/Eocene boundary,implying that these morphologically highly similar taxa are amongst the oldest‘living fossils'of the East Asian Flora.(2)Comparative genomics analysis of the two Dipteronia species.The assembled genome sequences were 711.47 Mbp(95.95%were anchored to10 pseudo-chromosomes)and 914.69 Mbp(99.5%were anchored to 9 pseudo-chromosomes)in D.sinensis and D.dyeriana,respectively.The genome assemblies of D.sinensis and D.dyeriana comprised 437.41 Mbp(61.48%)and 601.08 Mbp(65.71%)repeat sequences,33,282 and 32,837 protein-coding genes,respectively.Colinearity analysis of D.sinensis,D.dyeriana,A.yangbiense,and A.truncatum indicated that large-scale chromosome rearrangement occurred in the genomes of Dipteronia species.Comparative genomics analysis indicated the Dipteronia species underwent only one ancient triplication event,and no recent whole-genome duplication events were detected in this genus.Reconstruction of phylogenetic relationships for 16 Rosids species including the two Dipteronia species and the two Acer species based on 196single-copy orthologous genes corroborated our transcriptome phylogeny.Gene family evolution analysis based on this phylogeny revealed larger numbers of expanded gene families in widely distributed species(A.truncatum:1,055;D.sinensis:761)than endangered species(A.yangbiense:536;D.dyeriana:470).(3)Lineage divergence and demographic history of the two Dipteronia speciesBased on whole-genome resequencing data including 54 individuals from 16populations of D.sinensis and 25 individuals from 7 populations of D.dyeriana,we identified 5,231,915 and 2,079,413 high-quality single nucleotide polymorphisms.Nucleotide diversity analysis revealed lower genetic diversity of D.dyeriana(?=5.02×10–4)than D.sinensis(?=7.08×10–4).Population structure analysis using ADMIXTURE suggested the best number of genetic clusters for D.sinensis and D.dyeriana is K=2 and K=3,respectively.Lineage divergence,demographic history inference and simulation using PSMC and FASTSIMCOAL2 suggested relatively recent lineage divergence for both Dipteroina species.Dipteronia sinensis experienced population contraction since Late Miocene,and diverged into the South and North lineages which are geographically isolated by the Yangtze River(Phylogenomics:?4.56 Ma;FASTSIMCOAL2:?3.91 Ma).Lineage diversification of D.dyeriana was dated back to Early-to Mid-Pleistocene(Phylogenomics:?1.38 Ma;FASTSIMCOAL2:?1.57 Ma)when the MLP lineage diverged from the WS-LZ lineage,and the latter lineage diverged in Mid-Pleistocene(Phylogenomics:?0.89 Ma;FASTSIMCOAL2:?0.70 Ma).These results indicated that the global cooling in Late-Miocene/Early-Pliocene and climate fluctuations in Pleistocene are main drivers of the continuous fragmentation and lineage diversification of these East Asia relict plant species.The climate changes since the Late-Pleistocene and Holocene caused population contraction and fragmentation of the two Dipteronia species,and human activities have exacerbated this process.(4)Evaluation of inbreeding and mutation loadEstimation of ROH for each individual and calculation of inbreeding coefficient(FIS)for each lineage suggested extensive inbreeding in populations of the two Dipteronia species.A higher level of inbreeding was found in D.dyeriana than D.sinensis(FROH:0.33 vs.0.23),asexual reproduction was also found in D.dyeriana.The ratio of nonsynonymous nucleotide diversity and synonymous nucleotide diversity revealed higher selection efficacy in D.sinensis than D.dyeriana(?N/?S=0.38 vs.?N/?S=0.50).Evaluation of mutation load based on SIFT score,Grantham score,and annotation of loss of function mutations revealed a significant negative relationship between the proportion of deleterious mutation and effective population size(R=–0.86,P<2.2e–16).Dipteronia dyeriana had a significantly higher proportion of deleterious mutations than D.sinensis(P<0.0001).The South lineage of D.sinensis accumulated more deleterious mutations than the North lineage,while the former also showed a certain purging of extremely deleterious mutations.Dipteronia dyeriana was generally weak in the purging of deleterious mutations.The MLP lineage showed the highest accumulation of deleterious mutations and the least purging of extremely deleterious mutations.Meanwhile,the distribution of fitness effects of new mutations of this lineage has shifted far away from the fitness optimum,indicating a great risk of extinction.In conclusion,our phylogenomics analysis resolved the monophyly of Dipteronia.The Early Tertiary divergence of the two Dipteronia species corroborated the ancientness of the East Asia Flora.The global climate change since the Late-Miocene drove the lineage divergence of both Dipteronia species,while climate fluctuations since Late-Pleistocene and recent human activity caused the continuous population contraction and fragmentation.Long-term isolation and population decline in D.dyeriana had reduced the efficacy of natural selection,caused extensive inbreeding and increased accumulation of deleterious mutations,which further reduced the adaptive potential of D.dyeriana.The decline of effective population size also increased the mutation load in D.sinensis,meanwhile,this process also allowed the purging of extremely deleterious mutations.Finally,we proposed conservation strategies for the two Dipteronia species and Tertiary relict species with similar distribution patterns. |
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