RAD-sequencing improves the genetic characterization of a threatened tree peony (Paeonia ludlowii) endemic to China: Implications for conservation

doi:10.1016/j.pld.2022.07.002

Abstract

Abstract: Compared with traditional genetic markers, genomic approaches have proved valuable to the conservation of endangered species. Paeonia ludlowii having rarely and pure yellow flowers, is one of the world's most famous tree peonies. However, only several wild populations remain in the Yarlung Zangbo Valley (Nyingchi and Shannan regions, Xizang) in China due to increasing anthropogenic impact on the natural habitats. We used genome-wide single nucleotide polymorphisms to elucidate the spatial pattern of genetic variation, population structure and demographic history of P. ludlowii from the fragmented region comprising the entire range of this species, aiming to provide a basis for conserving the genetic resources of this species. Unlike genetic uniformity among populations revealed in previous studies, we found low but varied levels of intra-population genetic diversity, in which lower genetic diversity was detected in the population in Shannan region compared to those in Nyingzhi region. These spatial patterns may be likely associated with different population sizes caused by micro-environment differences in these two regions. Additionally, low genetic differentiation among populations (Fst = 0.0037) were detected at the species level. This line of evidence, combined with the result of significant genetic differentiation between the two closest populations and lack of isolation by distance, suggested that shared ancestry among now remnant populations rather than contemporary genetic connectivity resulted in subtle population structure. Demographic inference suggested that P. ludlowii probably experienced a temporal history of sharp population decline during the period of Last Glacial Maximum, and a subsequent bottleneck event resulting from prehistoric human activities on the Qinghai-Tibet Plateau. All these events, together with current habitat fragment and excavation might contribute to the endangered status of P. ludlowii. Our study improved the genetic characterization of the endangered tree peony (P. ludlowii) in China, and these genetic inferences should be considered when making different in situ and ex situ conservation actions for P. ludlowii in this evolutionary hotspot region.

Key words: Conservation, Fragmentation, Genetic structure, Genetic variation, Paeonia, RAD-sequencing

Yu-Juan Zhao, Gen-Shen Yin, Xun Gong. RAD-sequencing improves the genetic characterization of a threatened tree peony (Paeonia ludlowii) endemic to China: Implications for conservation[J]. Plant Diversity, 2023, 45(05): 513-522.

References

[1] Aguilar, R., Quesada, M., Ashworth, L., Herrerias-Diego, Y., and Lobo, J., 2008. Genetic consequences of habitat fragmentation in plant populations: susceptible signals in plant traits and methodological approaches. Mol. Ecol. 17, 5177-5188. https://doi.org/10.1111/j.1365-294X.2008.03971.x.
[2] Ahrens, C.W., Rymer, P.D., Stow, A., Bragg, J., Dillon, S., Umbers, K.D.L., et al., 2018. The search for loci under selection: trends, biases and progress. Mol. Ecol. 27, 1342-1356. https://doi.org/10.1111/mec.14549.
[3] Andrews, S., 2019. "FastQC: a quality control tool for high throughput sequence data", in: https://www.bioinformatics.babraham.ac.uk/projects/fastqc/.
[4] Andrews, K.R., Good, J.M., Miller, M.R., Luikart, G., and Hohenlohe, P.A., 2016. Harnessing the power of RADseq for ecological and evolutionary genomics. Nat. Rev. Genet. 17, 81-92. https://doi.org/10.1038/nrg.2015.28.
[5] Baird, N.A., Etter, P.D., Atwood, T.S., Currey, M.C., Shiver, A.L., Lewis, Z.A., et al., 2008. Rapid SNP discovery and genetic mapping using sequenced RAD markers. PLoS One. 3. https://doi.org/10.1371/journal.pone.0003376.
[6] Banks, S.C., Cary, G.J., Smith, A.L., Davies, I.D., Driscoll, D.A., Gill, A.M., et al., 2013. How does ecological disturbance influence genetic diversity? Trends Ecol. Evol. 28, 670-679. https://doi.org/10.1016/j.tree.2013.08.005.
[7] Barley, A.J., Cordes, J.E., Walker, J.M., and Thomson, R.C., 2022. Genetic diversity and the origins of parthenogenesis in the teiid lizard Aspidoscelis laredoensis. Mol. Ecol. 31, 266-278. https://doi.org/10.1111/mec.16213.
[8] Barthelmess, E.L., Richards, C.M., and McCauley, D.E., 2006. Relative effects of nocturnal vs diurnal pollinators and distance on gene flow in small Silene alba populations. New Phytol. 169, 689-698. https://doi.org/10.1111/j.1469-8137.2005.01580.x.
[9] Bell, D.A., Robinson, Z.L., Funk, W.C., Fitzpatrick, S.W., Allendorf, F.W., Tallmon, D.A., et al., 2019. The exciting potential and remaining uncertainties of genetic rescue. Trends Ecol. Evol. 34, 1070-1079. https://doi.org/10.1016/j.tree.2019.06.006.
[10] Cai, C., Xiao, J., Ci, X., Conran, J.G., and Li, J., 2021. Genetic diversity of Horsfieldia tetratepala (Myristicaceae), an endangered plant species with extremely small populations to China: implications for its conservation. Plant Syst. Evol. 307, 1-12. https://doi.org/10.1007/s00606-021-01774-z.
[11] Ceballos, G., Ehrlich, P.R., Barnosky, A.D., Garcia, A., Pringle, R.M., and Palmer, T.M., 2015. Accelerated modern human-induced species losses: entering the sixth mass extinction. Sci. Adv. 1. https://doi.org/10.1126/sciadv.1400253.
[12] Chang, C.C., Chow, C.C., Tellier, L., Vattikuti, S., Purcell, S.M., and Lee, J.J., 2015. Second-generation PLINK: rising to the challenge of larger and richer datasets. GigaScience. 4. https://doi.org/10.1186/s13742-015-0047-8.
[13] Chen, S.F., Guo, W., Chen, Z.X., Liao, W.B., and Fan, Q., 2021. Strong genetic structure observed in Primulina danxiaensis, a small herb endemic to Mount Danxia with Extremely Small Populations. Front. Genet. 12. https://doi.org/10.3389/fgene.2021.722149.
[14] Chen, X.L., Hou, G.L., Jin, S.M., Gao, J.Y., and Duan, R.L., 2020. The pollen records of human activities in Qinghai-Tibet Plateau during the Middle and late Holocene. Earth Environ. 48, 643-651.
[15] Cilingir, F.G., Rheindt, F.E., Garg, K.M., Platt, K., Platt, S.G., and Bickford, D.P., 2017. Conservation genomics of the endangered Burmese roofed turtle. Conserv. Biol. 31, 1469-1476. https://doi.org/10.1111/cobi.12921.
[16] Crane, P., 2020. Conserving our global botanical heritage: the PSESP plant conservation program. Plant Diver. 42, 319-322. https://doi.org/10.1016/j.pld.2020.06.007.
[17] D'Aloia, C.C., Andres, J.A., Bogdanowicz, S.M., McCune, A.R., Harrison, R.G., and Buston, P.M., 2020. Unraveling hierarchical genetic structure in a marine metapopulation: a comparison of three high-throughput genotyping approaches. Mol. Ecol. 29, 2189-2203. https://doi.org/10.1111/mec.15405.
[18] Do, C., Waples, R.S., Peel, D., Macbeth, G.M., Tillett, B.J., and Ovenden, J.R., 2014. NeEstimator v2: re-implementation of software for the estimation of contemporary effective population size (Ne) from genetic data. Mol. Ecol. Resour. 14, 209-214. https://doi.org/https://doi.org/10.1111/1755-0998.12157.
[19] Doyle, J., 1991. DNA protocols for plants-CTAB total DNA isolation. In G.M. Hewitt & A. Johnston (Eds), Molecular Techniques in Taxonomy. Berlin, Springer, pp. 283-293.
[20] Dray, S., Dufour, A.B., and Chessel, D., 2007. The ade4 package-II: two-table and K-table methods. R. News. 7, 47-52.
[21] Eckert, C.G., Samis, K.E., and Lougheed, S.C., 2008. Genetic variation across species' geographical ranges: the central-marginal hypothesis and beyond. Mol. Ecol. 17, 1170-1188. https://doi.org/10.1111/j.1365-294X.2007.03659.x.
[22] Evanno, G., Regnaut, S., and Goudet, J., 2005. Detecting the number of clusters of individuals using the software structure: a simulation study. Mol. Ecol. 14, 2611-2620. https://doi.org/10.1111/j.1365-294X.2005.02553.x.
[23] Excoffier, L., Laval, G., and Schneider, S., 2005. Arlequin (version 3.0): an integrated software package for population genetics data analysis. Evol. Bioinforma. Online. 1, 47-50.
[24] Fan, Y.M., Wang, Q., Dong, Z.J., Yin, Y.J., da Silva, J.A.T., and Yu, X.N., 2020. Advances in molecular biology of Paeonia L. Planta. 251, 1-47. https://doi.org/10.1007/s00425-019-03299-9.
[25] Farwig, N., Braun, C., and Bohning-Gaese, K., 2008. Human disturbance reduces genetic diversity of an endangered tropical tree, Prunus africana (Rosaceae). Conserv. Genet. 9, 317-326.
[26] Foll, M., and Gaggiotti, O., 2008. A genome-scan method to identify selected loci appropriate for both dominant and codominant markers: a bayesian perspective. Genetics. 180, 977-993. https://doi.org/10.1534/genetics.108.092221.
[27] Frankham, R., 1995. Effective population-size adult-population size ratios in wildlife - a review. Genet. Res. 66, 95-107. https://doi.org/10.1017/s0016672300034455.
[28] Frankham, R., 2010. Challenges and opportunities of genetic approaches to biological conservation. Biol. Conserv. 143, 1919-1927. https://doi.org/10.1016/j.biocon.2010.05.011.
[29] Frankham, R., Ballou, J.D., Eldridge, M.D.B., Lacy, R.C., Ralls, K., Dudash, M.R., et al., 2011. Predicting the probability of outbreeding depression. Conserv. Biol. 25, 465-475. https://doi.org/10.1111/j.1523-1739.2011.01662.x.
[30] Fraser, D.J., and Bernatchez, L., 2001. Adaptive evolutionary conservation: towards a unified concept for defining conservation units. Mol. Ecol. 10, 2741-2752. https://doi.org/10.1046/j.1365-294X.2001.t01-1-01411.x.
[31] Funk, W.C., McKay, J.K., Hohenlohe, P.A., and Allendorf, F.W., 2012. Harnessing genomics for delineating conservation units. Trends Ecol. Evol. 27, 489-496. https://doi.org/10.1016/j.tree.2012.05.012.
[32] Gutenkunst, R.N., Hernandez, R.D., Williamson, S.H., and Bustamante, C.D., 2009. Inferring the joint demographic history of multiple populations from multidimensional SNP frequency data. PLoS Genet. 5. https://doi.org/10.1371/journal.pgen.1000695.
[33] Hampe, A., and Petit, R.J., 2005. Conserving biodiversity under climate change: the rear edge matters. Ecol. Lett. 8, 461-467. https://doi.org/10.1111/j.1461-0248.2005.00739.x.
[34] Hamrick, J.L., and Godt, M.J.W., 1996. Effects of life history traits on genetic diversity in plant species. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 351, 1291-1298. https://doi.org/10.1098/rstb.1996.0112.
[35] Hao, H.P., He, Z., Li, H., Shi, L., and Tang, Y.D., 2014. Effect of root length on epicotyl dormancy release in seeds of Paeonia ludlowii, Tibetan peony. Ann. Bot. 113, 443-452. https://doi.org/10.1093/aob/mct273.
[36] Hewitt, G., 2000. The genetic legacy of the Quaternary ice ages. Nature. 405, 907-913. https://doi.org/10.1038/35016000.
[37] Hong, D.Y., 1997. Paeonia (Paeoniaceae) in Xizang (Tibet). Novon. 156-161. https://doi.org/10.2307/3392188.
[38] Hong, D.Y., Zhou, S.L., He, X.J., Yuan, J.H., Zhang, Y.L., Cheng, F.Y., et al., 2017. Current status of wild tree peony species with special reference to conservation. Biodivers. Sci. 25, 781-793. https://doi.org/10.17520/biods.2017129.
[39] Hutchison, Delbert W., Templeton, Alan R., 1999. Correlation of pairwise genetic and geographic distance measures: inferring the relative influences of gene flow and drift on the distribution of genetic variability. Evolution 53, 1898–1914, https://doi.org/https://doi.org/10.1111/j.1558-5646.1999.tb04571.x.
[40] Iannucci, A., Benazzo, A., Natali, C., Arida, E.A., Zein, M.S.A., Jessop, T.S., et al., 2021. Population structure, genomic diversity and demographic history of Komodo dragons inferred from whole-genome sequencing. Mol. Ecol. 30, 6309-6324. https://doi.org/10.1111/mec.16121.
[41] Jakobsson, M., and Rosenberg, N.A., 2007. CLUMPP: a cluster matching and permutation program for dealing with label switching and multimodality in analysis of population structure. Bioinformatics. 23, 1801-1806. https://doi.org/10.1093/bioinformatics/btm233.
[42] Jensen, E.L., Edwards, D.L., Garrick, R.C., Miller, J.M., Gibbs, J.P., Cayot, L.J., et al., 2018. Population genomics through time provides insights into the consequences of decline and rapid demographic recovery through head-starting in a Galapagos giant tortoise. Evol. Appl. 11, 1811-1821. https://doi.org/10.1111/eva.12682.
[43] Kumar, S., Stecher, G., and Tamura, K., 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870-1874. https://doi.org/10.1093/molbev/msw054.
[44] Li, J.L., Milne, R.I., Ru, D.F., Miao, J.B., Tao, W.J., Zhang, L., et al., 2020. Allopatric divergence and hybridization within Cupressus chengiana (Cupressaceae), a threatened conifer in the northern Hengduan Mountains of western China. Mol. Ecol. 29, 1250-1266. https://doi.org/10.1111/mec.15407.
[45] Li, J., and Wang, Z.H., 2019. Nutrients, fatty acid composition and antioxidant activity of the flowers and seed oils in wild populations of Paeonia ludlowii. Emir. J. Food Agric. 31, 206-213. https://doi.org/10.9755/ejfa.2019.v31.i3.1922.
[46] Li, S.M., Lv, S.Z., Yu, K., Wang, Z.Y., Li, Y.F., Ni, X.M., et al., 2019. Construction of a high-density genetic map of tree peony (Paeonia suffruticosa Andr. Moutan) using restriction site associated DNA sequencing (RADseq) approach. Tree Genet. Genomes 15. https://doi.org/10.1007/s11295-019-1367-0.
[47] Lee, K.M., Ranta, P., Saarikivi, J., Kutnar, L., Vres, B., Dzhus, M., et al., 2020. Using genomic information for management planning of an endangered perennial, Viola uliginosa. Ecol. Evol. 10, 2638-2649. https://doi.org/10.1002/ece3.6093.
[48] Lischer, H.E.L., and Excoffier, L., 2012. PGDSpider: an automated data conversion tool for connecting population genetics and genomics programs. Bioinformatics. 28, 298-299. https://doi.org/10.1093/bioinformatics/btr642.
[49] Liu, X.M., and Fu, Y.X., 2015. Exploring population size changes using SNP frequency spectra. Nat. Genet. 47, 555-559. https://doi.org/10.1038/ng.3254.
[50] Liu, J.G., Ouyang, Z.Y., Pimm, S.L., Raven, P.H., Wang, X.K., Miao, H., et al., 2003. Protecting China's biodiversity. Science. 300, 1240-1241. https://doi.org/10.1126/science.1078868.
[51] Liu, D.T., Zhang, L., Wang, J.H., and Ma, Y.P., 2020. Conservation genomics of a threatened Rhododendron: contrasting patterns of population structure revealed from neutral and selected SNPs. Front. Genet. 11. https://doi.org/10.3389/fgene.2020.00757.
[52] Llorens, T.M., Ayre, D.J., and Whelan, R.J., 2018. Anthropogenic fragmentation may not alter pre-existing patterns of genetic diversity and differentiation in perennial shrubs. Mol. Ecol. 27, 1541-1555. https://doi.org/10.1111/mec.14552.
[53] Lv, S.Z., Cheng, S., Wang, Z.Y., Li, S.M., Jin, X., Lan, L., et al., 2020. Draft genome of the famous ornamental plant Paeonia suffruticosa. Ecol. Evol. 10, 4518-4530. https://doi.org/10.1002/ece3.5965.
[54] Ma, Y.P., Chen, G., Grumbine, R.E., Dao, Z.L., Sun, W.B., and Guo, H.J., 2013. Conserving plant species with extremely small populations (PSESP) in China. Biodivers. Conserv. 22, 803-809. https://doi.org/10.1007/s10531-013-0434-3.
[55] Marques, D.A., Meier, J.I., and Seehausen, O., 2019. A combinatorial view on speciation and adaptive radiation. Trends Ecol. Evol. 34, 531-544. https://doi.org/10.1016/j.tree.2019.02.008.
[56] Nei, M., and Kumar, S., 2000. Molecular Evolution and Phylogenetics. New York: Oxford University.
[57] Ni, S.W., and Wang, L.Y., 2009. Introduction and Ex-Situ Conservation of Paeonia delavayi, Paeonia lutea, Paeonia ludlowii. Beijing Forestry University.
[58] Nielsen, E.S., Beger, M., Henriques, R., and von der Heyden, S., 2020. A comparison of genetic and genomic approaches to represent evolutionary potential in conservation planning. Biol. Conserv. 251. https://doi.org/10.1016/j.biocon.2020.108770.
[59] Ouborg, N.J., Pertoldi, C., Loeschcke, V., Bijlsma, R., and Hedrick, P.W., 2010. Conservation genetics in transition to conservation genomics. Trends Genet. 26, 177-187. https://doi.org/10.1016/j.tig.2010.01.001.
[60] Papuga, G., Gauthier, P., Pons, V., Farris, E., and Thompson, J.D., 2018. Ecological niche differentiation in peripheral populations: a comparative analysis of eleven Mediterranean plant species. Ecography. 41, 1650-1664. https://doi.org/10.1111/ecog.03331.
[61] Peakall, R., and Smouse, P.E., 2006. Genalex 6: genetic analysis in Excel. Population genetic software for teaching and research. Mol. Ecol. Notes 6, 288-295. https://doi.org/10.1111/j.1471-8286.2005.01155.x.
[62] Pimm, S.L., Jenkins, C.N., Abell, R., Brooks, T.M., Gittleman, J.L., Joppa, L.N., et al., 2014. The biodiversity of species and their rates of extinction, distribution, and protection. Science. 344. https://doi.org/10.1126/science.1246752.
[63] Pritchard, J.K., Stephens, M., and Donnelly, P., 2000. Inference of population structure using multilocus genotype data. Genetics. 155, 945-959. https://doi.org/doi: 10.1111/j.1471-8286.2007.01758.x.
[64] Puritz, J.B., Addison, J.A., and Toonen, R.J., 2012. Next-generation phylogeography: a targeted approach for multilocus sequencing of non-model organisms. PLoS One. 7. https://doi.org/10.1371/journal.pone.0034241.
[65] Rochette, N.C., Rivera-Colon, A.G., and Catchen, J.M., 2019. Stacks 2: analytical methods for paired-end sequencing improve RADseq-based population genomics. Mol. Ecol. 28, 4737-4754. https://doi.org/10.1111/mec.15253.
[66] Rosenberg, N.A., 2004. DISTRUCT: a program for the graphical display of population structure. Mol. Ecol. Notes 4, 137-138. https://doi.org/10.1046/j.1471-8286.2003.00566.x.
[67] Shuai, Y.T., and Zang, J.C., 2016. Paeonid ludlowii and Paeonia delavayi flower characteristics and change of flower-visiting insects and phenotypic selection. Southwest China J. Agric. Sci. 29, 2714-2719.
[68] Stankowski, S., and Ravinet, M., 2021. Defining the speciation continuum. Evolution. 75, 1256-1273. https://doi.org/10.1111/evo.14215.
[69] Tamura, K., and Nei, M., 1993. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol. Biol. Evol. 10, 512-526. https://doi.org/10.1093/oxfordjournals.molbev.a040023.
[70] Tang, Y., Yuan, T., and Chen, T.Q., 2021. Flowering characteristics and breeding system of Paeonia ludlowii. Acta Bot. Boreal.-Occident. Sin. 41, 782-794.
[71] Terhorst, J., and Song, Y.S., 2015. The sample frequency spectrum. Proc. Natl. Acad. Sci. U. S. A. 112, 7677-7682. https://doi.org/10.1073/pnas.1503717112.
[72] Thompson, J.D., Gaudeul, M., and Debussche, M.A.X., 2010. Conservation value of sites of hybridization in peripheral populations of rare plant species. Conserv. Biol. 24, 236-245. https://doi.org/10.1111/j.1523-1739.2009.01304.x.
[73] Vranckx, G., Jacquemyn, H., Muys, B., and Honnay, O., 2012. Meta-analysis of susceptibility of woody plants to loss of genetic diversity through habitat fragmentation. Conserv. Biol. 26, 228-237. https://doi.org/10.1111/j.1523-1739.2011.01778.x.
[74] Whiteley, A.R., Fitzpatrick, S.W., Funk, W.C., and Tallmon, D.A., 2015. Genetic rescue to the rescue. Trends Ecol. Evol. 30, 42-49. https://doi.org/10.1016/j.tree.2014.10.009.
[75] Wikstrom, N., Savolainen, V., and Chase, M.W., 2001. Evolution of the angiosperms: calibrating the family tree. Proc. R. Soc. B-Biol. Sci. 268, 2211-2220. https://doi.org/10.1098/rspb.2001.1782.
[76] Xu, X.X., Cheng, F.Y., Peng, L.P., Sun, Y.Q., Hu, X.G., Li, S.Y., et al., 2019. Late Pleistocene speciation of three closely related tree peonies endemic to the Qinling-Daba Mountains, a major glacial refugium in Central China. Ecol. Evol. 9, 7528-7548. https://doi.org/10.1002/ece3.5284.
[77] Xiao, J.H., Ding, X., Li, L., Ma, H., Ci, X.Q., van der Merwe, M., et al., 2020. Miocene diversification of a golden-thread nanmu tree species (Phoebe zhennan, Lauraceae) around the Sichuan Basin shaped by the East Asian monsoon. Ecol. Evol. 10, 10543-10557. https://doi.org/10.1002/ece3.6710.
[78] Xu, T.T., Wang, Q., Olson, M.S., Li, Z.H., Miao, N., and Mao, K.S., 2017. Allopatric divergence, demographic history, and conservation implications of an endangered conifer Cupressus chengiana in the eastern Qinghai-Tibet Plateau. Tree Genet. Genomes 13. https://doi.org/10.1007/s11295-017-1183-3.
[79] Xue, Y.Q., Liu, R., Xue, J.Q., Wang, S.L., and Zhang, X.X., 2021. Genetic diversity and relatedness analysis of nine wild species of tree peony based on simple sequence repeats markers. Hortic. Plant J. 7, 579-588. https://doi.org/10.1016/j.hpj.2021.05.004.
[80] Yang, J., Cai, L., Liu, D., Chen, G., Gratzfeld, J., and Sun, W., 2020a. China's conservation program on plant species with extremely small populations (PSESP): progress and perspectives. Biol. Conserv. 244, 108535. https://doi.org/https://doi.org/10.1016/j.biocon.2020.108535.
[81] Yang, Y., Ma, T., Wang, Z., Lu, Z., Li, Y., Fu, C., et al., 2018. Genomic effects of population collapse in a critically endangered ironwood tree Ostrya rehderiana. Nat. Commun. 9, 5449. https://doi.org/10.1038/s41467-018-07913-4.
[82] Yang, Y., Sun, M., Li, S., Chen, Q., Teixeira da Silva, J.A., Wang, A., et al., 2020b. Germplasm resources and genetic breeding of Paeonia: a systematic review. Hortic. Res. 7, 107. https://doi.org/10.1038/s41438-020-0332-2.
[83] Yang, X.L., Wang, Q.J., Lan, X.Z., and Li, C.Y., 2007. Numeric dynamics of the endangered plant population of Paeonia ludlowii. Acta Ecol. Sin. 27, 1242-1247.
[84] Yao, X.H., Ye, Q.G., Kang, M., and Huang, H.W., 2007. Microsatellite analysis reveals interpopulation differentiation and gene flow in the endangered tree Changiostyrax dolichocarpa (Styracaceae) with fragmented distribution in central China. New Phytol. 176, 472-480. https://doi.org/10.1111/j.1469-8137.2007.02175.x.
[85] Young, A., Boyle, T., Brown, T., 1996. The population genetic consequences of habitat fragmentation for plants. Trends Ecol. Evol. 11, 413–418, https://doi.org/https://doi.org/10.1016/0169-5347(96)10045-8.
[86] Younger, J.L., Clucas, G.V., Kao, D.M., Rogers, A.D., Gharbi, K., Hart, T., et al., 2017. The challenges of detecting subtle population structure and its importance for the conservation of emperor penguins. Mol. Ecol. 26, 3883-3897. https://doi.org/10.1111/mec.14172.
[87] Yu, H.B., Deane, D.C., Sui, X.H., Fang, S.Q., Chu, C.J., Liu, Y., et al., 2019a. Testing multiple hypotheses for the high endemic plant diversity of the Tibetan Plateau. Glob. Ecol. Biogeogr. 28, 131-144. https://doi.org/10.1111/geb.12827.
[88] Yu, H.B., Favre, A., Sui, X.H., Chen, Z., Qi, W., and Xie, G.W., 2019b. Mapping the genetic patterns of plants in the region of the Qinghai-Tibet Plateau: implications for conservation strategies. Divers. Distrib. 25, 310-324. https://doi.org/10.1111/ddi.12847.
[89] Zhang, L., 2008. Population Characteristics and Seed's Biology of Paeonia ludlowii. Master, Beijing Forestry University.
[90] Zhang, J.M., Lopez-Pujol, J., Gong, X., Wang, H.F., Vilatersana, R., and Zhou, S.L., 2018. Population genetic dynamics of Himalayan-Hengduan tree peonies, Paeonia subsect. Delavayanae. Mol. Phylogenet. Evol. 125, 62-77. https://doi.org/10.1016/j.ympev.2018.03.003.
[91] Zhang, D.J., Shen, X.K., Cheng, T., Xia, H., Liu, W., Gao, X., et al., 2020. New advances in the study of prehistoric human activity on the Tibetan Plateau. Chin. Sci. Bull. 65, 475-482.
[92] Zhang, X., Zhai, Y., Yuan, J., and Hu, Y., 2019. New insights into Paeoniaceae used as medicinal plants in China. Sci. Rep. 9, 18469. https://doi.org/10.1038/s41598-019-54863-y.
[93] Zhao, Y.J., Yin, G.S., Pan, Y.Z., Tian, B., and Gong, X., 2021. Climatic refugia and geographical isolation contribute to the speciation and genetic divergence in Himalayan-Hengduan tree peonies (Paeonia delavayi and Paeonia ludlowii). Front. Genet. 11. https://doi.org/10.3389/fgene.2020.595334.
[94] Zheng, B.X., Xu, Q.Q., and Shen, Y.Q., 2002. The relationship between climate change and Quaternary glacial cycles on the Qinghai-Tibetan Plateau: review and speculation. Quat. Int. 97-98, 93-101. https://doi.org/10.1016/S1040-6182(02)00054-X.
[95] Zhou, S.L., Xu, C., Liu, J., Yu, Y., Wu, P., Cheng, T., et al., 2021. Out of the pan-himalaya: evolutionary history of the Paeoniaceae revealed by phylogenomics. J. Syst. Evol. 59, 1170-1182. https://doi.org/10.1111/jse.12688.