| Dysosma, consisting of 7 species, belongs to the subfamily Podophylloideae of Berberidaceae. Their rhizomes or roots contain biologically active components such as podophyllotoxin, which is a precursor of anticancer drugs. Dysosma are geographically restricted to Southeast, South and Southwest of China, of which D. versipellis is widely distributed species, and the rest are local species. D. tsayuensis is located in Eastern Himalayas, and D. pleiantha has a discontinuous distribution in eastern China and Taiwan. Despite approximate 200 years of classification history for Dysosma, its morphological traits variability and incomplete sampling makes it hard to resolve classification revision and systematics of this genus and allied genera. Previous phylogentic and phylogeographic analyses only studied few species, but in the case of origin, differentiation, migration, phylogenetics and divergence time, it is still a gap. Dysosma species also occupy diverse range of habitats across China, whether different species under varied vegetation experienced different evolutionary history is worthy of study. Based nearly 10 years of accumulated materials and extensive sampling strategy, in this study, we used the principles and methods from phylogeny, biogeography and population genetics to perform the molecular phylogeny of the subfamily, biogeography of Dysosma and the time frame and mechnism of Dysosma species differentiation. The main results are as follows:1) Phylogeny and divergence time of Podophylloideae:Through an extensive field sampling for Podophylloideae,63 populations from China, Japan and North America were obtained. The populations from Dysosma geographically covered their distribution region. Based on phyiogcnctic tree of Berberidacene (combined matK and rbcL, Wang et al.,2007), by amplifying additional two chloroplast trnL-trnF, atpH-atpI, nrlTS and nrETS for Dysosma 7 species, we used BEAST’ o estimate the divergence time for Berberidaceae combining two fossil calibration points and a secondary calibration points of Podophylloideae. It is the first to construct the entire phylogenetic tree of Podophylloideae to rev; rved the rclationship among and within gene Podophylloideae is a monophyletic group (PP=1); Diphylleia formed a sister relationship with the rest of Podophylloideae (PP=1); Dysosma is a good monophyletic (PP=1), and except D. versipellis, the rest of Dysosma were consistent with morphological taxonomy. Dysosma and Sinopodophyllum-Podophyllum disjunct in East Asia-North American were a sister relationship. Divergence time estimation indicated that the divergence time of Podophylloideae was in the Pliocene[c.13.57 Ma (11.99-15.12Ma)]; the divergence time of Diphylleia was 8.20 Ma (6.33-10.29 Ma). The split time of Dysosma, Sinododophyllum and Podophyllum (9.59-9.81 Ma) was related to Miocene global climate cooling. BBM analysis revealed the Dysosma originated from the eastern Himalayas, and since the Pliocene migrated from west to east of China. In Dysosma, the split time of D. aurantiocaulis, D, tsayuensis and D. veitchii (8.47-9.81 Ma) was consistent with earlier rapid uplift of the Tibetan Plateau (8-13 Ma), indicating the early vicariance led to the allopatric speciation of the western taxa.2) Phylogeography of Dysosma at the species levelWe selected 46 populations of Dysosma to perform phylogeography analyses to reveal lineages relationship and temporal and spatial distribution pattern of genetic diversity:sequence variation of three chloroplast genes (trnL-trnF, trnL-ndhJ, trnS-trnfM) was used to analyze the genetic diversity, which suggested there were 66 chlorotypes; at the genus level, Dysosma had a relatively high genetic diversity (hT= 0.929, πT= 3.58×10-3) and a significant phylogeographical structure (NST=0.866, GST=0.790, P<0.05); at the species level, widespread species D. versipellis had a higher haplotype diversity and nucleotide diversity (hT=0.314; πT=0.25×10-3) than other Dysosma species; D. versipellis (NST=0.818, GST=0.611, P<0.05), D. difformis (NST=0.865, GST=0.728, P< 0.01) and D. veitchii (NST=0.977, GST= 0.870, P<0.05) had significantly phylogeographical structure. TCS haplotype analysis was consistent with the phylogenetic result, Dysosma were divided into six geographically lineages (Lineage 1-Lineage 6):D. aurantiocaulis (Lineage 6), D. tsayuensis (Lineage 5) and D. veitchii (Lineage 4), which distributed in the western China, were split one by one. D. difformis, D. majorensis, D. versipellis and D. pleiantha, which distributed in central and southeastern China formed a monophyly, of which D. versipellis were split into two lineages (northwest lineage Lineage 2 vs. southeast lineage Lineage 1) and formed a paraphyly with monophylic D. pleiantha (Lineage 3); D. majorensis and D. difformis were not an independent lineage, D. difformis was nested in Lineage 1 and shared H1; D. majorensis and D. difformis shared H55. Based on ITS data, no ribotypes were shared between Dysosma species. The cytoplasmic-nuclear discordance of haplotypes was detected D. versipellis, D. difformis and D. majorensis, indicating there was introgression among these species.Mismatch analysis showed Lineage 1 and Lineage 3 has experienced significant population expansion, but there was no expansion signature detected in Lin2 and Lin4. The expansion time of Lineagel was in the early Pleistocene [c.1.78 Ma (95% CI: 1.01-3.03 Ma)], which indicats that climate cooling drives the expansion of D. versipellis of warm-deciduous forest. The expansion time of Lineage 3 was dated in the late Pleistocene [c.0.18 Ma (95% CI:0.00072-0.75 Ma)], which suggests that climate warming drives the expansion of D. pleiantha of evergreen forest.3) DNA barcodingWe evaluated the nine candidate DNA barcoding (ITS, matK, rbcL, trnL-trnF, trnL-ndhJ, trnS-trnfM, atpH-atpl, rpl32-trnL, rps18-clpp) in the identification of Podophylloideae on assessing their amplification success rate, intraspecific and interspecific variation and barcoding gap. Meanwhile, we used BLAST, DISTANCE and phylogenetic tree-based analysis to evaluate their ability in identifying Podophylloideae species. The results showed that except rp.rpS18-clpp, the remaining borcodes had 100% of transferability, of which ITS had the highest variation of intra-and interspecific, the largest barcodina;:ap and the highest identification success rate (83.3%) than other borcodes. Therefore, ITS was selected as the best barcode for Podophylloideae.4) Comparative transeriptomicsWo sequenced the transcriptomes of high altitude D. aurantiocaulis and low all ude D. versipellis using Hiseq 2000 sequencing platform. By de novo assembly, 53,929 and 44,855 unigenes were obtained. Based on sequence similarity, we found 4,593 pairs of orthologs, of which 3,126 pairs of orthologous genes had Ka/Ks ratio and there was no one pairs of ortholog showed significantly greater than 1, while 1,273 pairs showed significant purifying selection (Ka/Ks< 0.5, P< 0.05). In addition, we examined the transferability of 51 pairs of EST-SSR primers, the result showed there were 26 pairs (51%) and 41 pairs (80.4%) tested cross-species transferability within Podophylloideae and Dysosma. Finally, we conducted the molecular evolution of the CYP719A genes of Podophylloideae. The result illustrated CYP719A gene in Podophylloideaes experienced a strong selective constrains and in Dysosma, there was a relaxed purifying selection for high altitude species or more positive selection. Comparative transcriptomics has generated a wealth of molecular resources potentially useful for pharmacogenetic and evolutionary studies in Dysosma and allied taxa.Taken together, the phylogenetic analysis showed that Podophylloideae was a well-supported monophyly. Diphylleia was a fosal group, of which Diphylleia grayi and Diphylleia cymosa were a sister relationship. Dysosma was a well-supported monophyly. Vicariance driven by the uplift of QTP led to the allopatric speciation of western Dysosma species. The climate changes of Plio-Pleistocene were the primary reason for divergence and introgrcssion of species of central/east Dysosma, and D. pleiantha was divergence until early Pleistocene. ITS was estimated as the best DNA barcode for identifying Podophylloideae species. A wealth of EST-SSR and single copy genes were developed by NGS technology to fill a gap in lack of genetic resources of Dysosma. The molecular evolution of CYP719A genes in Dysosma showed that there was a relaxed purifying selection for high altitude species or more positive selection. |
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