DNA Barcoding Reveals Cryptic Diversity and Informs Conservation Priorities in Chinese Firmiana Species (Malvaceae) Using Genome Skimming Data

Abstract

Accurate species delineation is crucial for biodiversity conservation. The genus Firmiana Marsili (Malvaceae) comprises deciduous trees and shrubs, many of which are rare and endangered in China, underscoring the urgent need for effective conservation measures. However, morphological similarities among closely related Firmiana species complicate taxonomic identification, and traditional morphology-based approaches are often insufficient for taxa with complicated evolutionary histories. To address this, we conducted genome skimming on 62 Firmiana samples representing all 10 recognized Chinese species and two unidentified taxa, assembling plastome and nuclear ribosomal DNA sequences. We evaluated the effectiveness of super-barcodes (plastid genomes and nrDNA), plastid hyper-variable barcodes, and four universal barcodes. Our results show that nrITS exhibited the highest discriminatory power, successfully identifying all 10 recognized Firmiana species, and is thus recommended as the primary barcode for the genus. In addition, two cryptic lineages were discovered within the Firmiana major complex. These results provide critical insights for the conservation, management, and sustainable use of endangered Firmiana species. This study underscores the urgent need to revise species boundaries within the F. major complex, and highlights the potential of DNA barcoding as an efficient tool for species identification and conservation of the genus Firmiana.

Keywords

conservation / DNA barcode / identification / phylogeny / plastome

References

[1] Abdullah, I. Shahzadi, F. Mehmood, et al. 2019. “Comparative Analyses of Chloroplast Genomes Among Three Firmiana Species: Identification of Mutational Hotspots and Phylogenetic Relationship With Other Species of Malvaceae.” Plant Gene 19: 100199. https://doi.org/10.1016/j.plgene.2019.100199.

[2] Agapow, P. M., O. R. P. Bininda-Emonds, K. A. Crandall, et al. 2004. “The Impact of Species Concept on Biodiversity Studies.” Quarterly Review of Biology 79, no. 2: 161–179. https://doi.org/10.1086/383542.

[3] Alfaro, M. E. 2003. “Bayes or Bootstrap? A Simulation Study Comparing the Performance of Bayesian Markov Chain Monte Carlo Sampling and Bootstrapping in Assessing Phylogenetic Confidence.” Molecular Biology and Evolution 20, no. 2: 255–266. https://doi.org/10.1093/molbev/msg028.

[4] Allendorf, F. W., W. C. Funk, S. N. Aitken, M. Byrne, G. Luikart, and A. Antunes. 2022. Conservation and the Genomics of Populations. Oxford University Press.

[5] Bickford, D., D. J. Lohman, N. S. Sodhi, et al. 2007. “Cryptic Species as a Window on Diversity and Conservation.” Trends in Ecology & Evolution 22, no. 3: 148–155. https://doi.org/10.1016/j.tree.2006.11.004.

[6] Boehm, M. M. A., and Q. C. B. Cronk. 2021. “Dark Extinction: The Problem of Unknown Historical Extinctions.” Biology Letters 17, no. 3: rsbl.2021.0007. https://doi.org/10.1098/rsbl.2021.0007.

[7] Carolan, J. C., T. E. Murray, Ú. Fitzpatrick, et al. 2012. “Colour Patterns Do Not Diagnose Species: Quantitative Evaluation of a DNA Barcoded Cryptic Bumblebee Complex.” PLoS One 7: e29251. https://doi.org/10.1371/journal.pone.0029251.

[8] Cha, W., M. Shi, F. Du, Y. Wang, and R. Dai. 2023. “Community Characteristics of Tiny Population Firmiana major.” Journal of Southwest Forestry University 43, no. 3: 71–78. https://doi.org/10.11929/j.swfu.202201008.

[9] Chen, S., X. Yin, J. Han, et al. 2023. “DNA Barcoding in Herbal Medicine: Retrospective and Prospective.” Journal of Pharmaceutical Analysis 13, no. 5: 431–441. https://doi.org/10.1016/j.jpha.2023.03.008.

[10] Chen, S.-F., W.-Y. Zhao, Y.-S. Huang, et al. 2023. “The Origin and Dispersal of Firmiana danxiaensis Among Isolated Specific Landscapes.” Journal of Systematics and Evolution 62: 102–119. https://doi.org/10.1111/jse.12954.

[11] Coissac, E., P. M. Hollingsworth, S. Lavergne, and P. Taberlet. 2016. “From Barcodes to Genomes: Extending the Concept of DNA Barcoding.” Molecular Ecology 25, no. 7: 1423–1428. https://doi.org/10.1111/mec.13549.

[12] Corlett, R. T. 2023. “Achieving Zero Extinction for Land Plants.” Trends in Plant Science 28, no. 8: 913–923. https://doi.org/10.1016/j.tplants.2023.03.019.

[13] Dittrich-Reed, D. R., and B. M. Fitzpatrick. 2013. “Transgressive Hybrids as Hopeful Monsters.” Evolutionary Biology 40, no. 2: 310–315. https://doi.org/10.1007/s11692-012-9209-0.

[14] Doyle, J. J., and J. L. Doyle. 1987. “A Rapid DNA Isolation Procedure for Small Quantities of Fresh Leaf Tissue.” Phytochemistry 19: 11–15.

[15] Erixon, P., B. Svennblad, T. Britton, and B. Oxelman. 2003. “Reliability of Bayesian Posterior Probabilities and Bootstrap Frequencies in Phylogenetics.” Systematic Biology 52, no. 5: 665–673. https://doi.org/10.1080/10635150390235485.

[16] Fazekas, A. J., K. S. Burgess, P. R. Kesanakurti, et al. 2008. "Multiple Multilocus DNA Barcodes From the Plastid Genome Discriminate Plant Species Equally Well." PLOS ONE 3: e2802.

[17] Ferri, E., M. Barbuto, O. Bain, et al. 2009. “Integrated Taxonomy: Traditional Approach and DNA Barcoding for the Identification of Filarioid Worms and Related Parasites (Nematoda).” Frontiers in Zoology 6, no. 1: 1. https://doi.org/10.1186/1742-9994-6-1.

[18] Funk, W. C., M. Caminer, and S. R. Ron. 2012. “High Levels of Cryptic Species Diversity Uncovered in Amazonian Frogs.” Proceedings of the Royal Society B: Biological Sciences 279, no. 1734: 1806–1814. https://doi.org/10.1098/rspb.2011.1653.

[19] Funk, W. C., J. K. McKay, P. A. Hohenlohe, and F. W. Allendorf. 2012. “Harnessing Genomics for Delineating Conservation Units.” Trends in Ecology & Evolution 27, no. 9: 489–496. https://doi.org/10.1016/j.tree.2012.05.012.

[20] Grant, P. R., and B. R. Grant. 2019. “Hybridization Increases Population Variation During Adaptive Radiation.” Proceedings of the National Academy of Sciences 116, no. 46: 23216–23224. https://doi.org/10.1073/pnas.1913534116.

[21] Hamilton, J. A., and J. M. Miller. 2015. “Adaptive Introgression as a Resource for Management and Genetic Conservation in a Changing Climate.” Conservation Biology 30, no. 1: 33–41. https://doi.org/10.1111/cobi.12574.

[22] Hausdorf, B. 2021. “A Holistic Perspective on Species Conservation.” Biological Conservation 264: 109375. https://doi.org/10.1016/j.biocon.2021.109375.

[23] Hebert, P. D. N., A. Cywinska, S. L. Ball, and J. R. deWaard. 2003. “Biological Identifications Through DNA Barcodes.” Proceedings of the Royal Society of London. Series B: Biological Sciences 270, no. 1512: 313–321. https://doi.org/10.1098/rspb.2002.2218.

[24] Hernández-Triana, L. M., V. A. Brugman, N. I. Nikolova, et al. 2019. “DNA Barcoding of British Mosquitoes (Diptera, Culicidae) to Support Species Identification, Discovery of Cryptic Genetic Diversity and Monitoring Invasive Species.” ZooKeys 832: 57–76. https://doi.org/10.3897/zookeys.832.32257.

[25] Hillis, D. M., and J. J. Bull. 1993. “An Empirical Test of Bootstrapping as a Method for Assessing Confidence in Phylogenetic Analysis.” Systematic Biology 42, no. 2: 182–192. https://doi.org/10.1093/sysbio/42.2.182.

[26] Hollingsworth, P. M., L. L. Forrest, J. L. Spouge, et al. 2009. “A DNA Barcode for Land Plants.” Proceedings of the National Academy of Sciences of the United States of America 106, no. 31: 12794–12797. https://doi.org/10.1073/pnas.0905845106.

[27] Hollingsworth, P. M., D.-Z. Li, M. van der Bank, and A. D. Twyford. 2016. “Telling Plant Species Apart With DNA: From Barcodes to Genomes.” Philosophical Transactions of the Royal Society, B: Biological Sciences 371, no. 1702: 20150338. https://doi.org/10.1098/rstb.2015.0338.

[28] Hong, D.-Y. 2016. “Biodiversity Pursuits Need a Scientific and Operative Species Concept.” Biodiversity Science 24, no. 9: 979–999. https://doi.org/10.17520/biods.2016203.

[29] Hu, J.-L., X.-Q. Ci, Z.-F. Liu, et al. 2022. “Assessing Candidate DNA Barcodes for Chinese and Internationally Traded Timber Species.” Molecular Ecology Resources 22, no. 4: 1478–1492. https://doi.org/10.1111/1755-0998.13546.

[30] Huang, Y.-S., W.-H. Wu, W.-B. Xu, and Y. Liu. 2011. “Firmiana calcarea sp. nov. (Malvaceae) From Limestone Areas in Guangxi, China.” Nordic Journal of Botany 29: 608–610. https://doi.org/10.1111/j.1756-1051.2011.01278.x.

[31] Huang, Z.-H., W.-H. Luo, S.-X. Huang, and S.-Q. Huang. 2018. “Sunbirds Serve as Major Pollinators for Various Populations of Firmiana Kwangsiensis, a Tree Endemic to South China.” Journal of Systematics and Evolution 56, no. 3: 243–249. https://doi.org/10.1111/jse.12311.

[32] Jin, J.-J., W.-B. Yu, J.-B. Yang, et al. 2020. “Getorganelle: A Fast and Versatile Toolkit for Accurate De Novo Assembly of Organelle Genomes.” Genome Biology 21: 241. https://doi.org/10.1186/s13059-020-02154-5.

[33] Katoh, K., and D. M. Standley. 2013. “MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability.” Molecular Biology and Evolution 30, no. 4: 772–780. https://doi.org/10.1093/molbev/mst010.

[34] Kearse, M., R. Moir, A. Wilson, et al. 2012. “Geneious Basic: An Integrated and Extendable Desktop Software Platform for the Organization and Analysis of Sequence Data.” Bioinformatics 28, no. 12: 1647–1649. https://doi.org/10.1093/bioinformatics/bts199.

[35] Kolaczkowski, B., and J. W. Thornton. 2007. “Effects of Branch Length Uncertainty on Bayesian Posterior Probabilities for Phylogenetic Hypotheses.” Molecular Biology and Evolution 24, no. 9: 2108–2118. https://doi.org/10.1093/molbev/msm141.

[36] Kostermans, A. J. G. H. 1954. “A Note on Some African Sterculiaceae.” Bulletin du Jardin botanique de l'État a Bruxelles 24: 335–338.

[37] Kostermans, A. J. G. H. 1957. “The Genus Firmiana Marsili (Sterculiaceae).” Reinwardtia 4, no. 2: 281–310.

[38] Kress, W. J. 2017. “Plant DNA Barcodes: Applications Today and in the Future.” Journal of Systematics and Evolution 55, no. 4: 291–307. https://doi.org/10.1111/jse.12254.

[39] Kress, W. J., C. García-Robledo, M. Uriarte, and D. L. Erickson. 2015. “DNA Barcodes for Ecology, Evolution, and Conservation.” Trends in Ecology & Evolution 30, no. 1: 25–35. https://doi.org/10.1016/j.tree.2014.10.008.

[40] Kress, W. J., K. J. Wurdack, E. A. Zimmer, L. A. Weigt, and D. H. Janzen. 2005. “Use of DNA Barcodes to Identify Flowering Plants.” Proceedings of the National Academy of Sciences of the United States of America 102, no. 23: 8369–8374. https://doi.org/10.1073/pnas.0503123102.

[41] Kumar, S., G. Stecher, M. Li, C. Knyaz, and K. Tamura. 2018. “MEGA X: Molecular Evolutionary Genetics Analysis Across Computing Platforms.” Molecular Biology and Evolution 35, no. 6: 1547–1549. https://doi.org/10.1093/molbev/msy096.

[42] Le, D. T., Y. Q. Zhang, Y. Xu, et al. 2020. “The Utility of DNA Barcodes to Confirm the Identification of Palm Collections in Botanical Gardens.” PLoS One 15, no. 7: e0235569. https://doi.org/10.1371/journal.pone.0235569.

[43] Levin, D. A., J. Francisco-Ortega, and R. K. Jansen. 1996. “Hybridization and the Extinction of Rare Plant Species.” Conservation Biology 10, no. 1: 10–16. https://doi.org/10.1046/j.1523-1739.1996.10010010.x.

[44] Li, C., Y. Chen, F. Yang, D. Wang, and J. Yang. 2020. “Population Structure and Regeneration Dynamics of Firmiana major, a Dominant but Endangered Tree Species.” Forest Ecology and Management 462: 117993. https://doi.org/10.1016/j.foreco.2020.117993.

[45] D. Z. Li, ed. 2018. A Dictionary of the Families and Genera of Chinese Vascular Plants, 211. Science Press.

[46] Li, D. Z., L. M. Gao, H. T. Li, et al. 2011. “Comparative Analysis of a Large Data Set Indicates That Internal Transcribed Spacer (Its) Should Be Incorporated Into the Core Barcode for Seed Plants.” Proceedings of the National Academy of Sciences 108, no. 49: 19641–19646. https://doi.org/10.1073/pnas.1104551108.

[47] Li, D. Z., J. Q. Liu, Z. D. Chen, et al. 2011. “Plant DNA Barcoding in China.” Journal of Systematics and Evolution 49, no. 3: 165–168. https://doi.org/10.1111/j.1759-6831.2011.00137.x.

[48] Li, J., R. I. Milne, D. Ru, et al. 2020. “Allopatric Divergence and Hybridization Within Cupressus chengiana (Cupressaceae), a Threatened Conifer in the Northern Hengduan Mountains of Western China.” Molecular Ecology 29, no. 7: 1250–1266. https://doi.org/10.1111/mec.15407.

[49] Li, R. Z., J. Cai, J. B. Yang, Z. R. Zhang, D. Z. Li, and W. B. Yu. 2022. “Plastid Phylogenomics Resolving Phylogenetic Placementand Genera Phylogeny of Sterculioideae.” Guihaia 42, no. 1: 25–38. https://doi.org/10.11931/guihaia.gxzw202103060.

[50] Li, Y., L. Nie, S. Deng, et al. 2024. “Characterization of Firmiana danxiaensis Plastomes and Comparative Analysis of Firmiana: Insight Into Its Phylogeny and Evolution.” BMC Genomics 25, no. 1: 203. https://doi.org/10.1186/s12864-024-10046-2.

[51] Librado, P., and J. Rozas. 2009. “DnaSP v5: A Software for Comprehensive Analysis of DNA Polymorphism Data.” Bioinformatics 25, no. 11: 1451–1452. https://doi.org/10.1093/bioinformatics/btp187.

[52] Lu, Q., Z. Huang, and W. Luo. 2021. “Characterization of Complete Chloroplast Genome in Firmiana danxiaensis and F. kwangsiensis With Extremely Small Populations.” Biodiversity Science 29, no. 5: 586–595. https://doi.org/10.17520/biods.2020263.

[53] Lu, Q., and W. Luo. 2022. “The Complete Chloroplast Genome of Two Firmiana Species and Comparative Analysis With Other Related Species.” Genetica 150, no. 6: 395–405. https://doi.org/10.1007/s10709-022-00169-3.

[54] Lu, Q., W. Luo, and Z. Huang. 2019. “The Complete Chloroplast Genome of Firmiana danxiaensis, an Endangered Species Endemic to Danxia Landform in Southern China.” Mitochondrial DNA Part B 4, no. 2: 4071–4072. https://doi.org/10.1080/23802359.2019.1689861.

[55] Lu, Z. L., H. N. Qin, X. H. Jin, et al. 2021. “On the Necessity, Principle and Process of Updating the List of National Key Protected Wild Plants.” Biodiversity Science 29: 1577–1582. https://doi.org/10.17520/biods.2021394.

[56] Ma, Y., C. Li, J. Jin, C. Liao, J. Yang, and W. Sun. 2022. “Conservation Genetics of Firmiana major, a Threatened Tree Species With Potential for Afforestation of Hot, Arid Climates.” Global Ecology and Conservation 36: e02136. https://doi.org/10.1016/j.gecco.2022.e02136.

[57] Ministry of Ecology and Environment of the People's Republic of China, & Chinese Academy of Sciences. 2013. “Red List of Chinese Biodiversity: Higher Plants.” https://www.mee.gov.cn/gkml/hbb/bgg/201309/t20130912_260061.htm.

[58] Miu, Y. S., H. Z. Huang, Y. Q. Li, et al. 2020. “Resource Survey and Protection of the Key National Protected Species Firmiana danxiaensis Endemic to Guangdong, China.” Subtropical Plant Science 49, no. 1: 71–75. https://doi.org/10.3969/j.issn.1009-7791.2020.01.013.

[59] Nic Lughadha, E., S. P. Bachman, T. C. C. Leão, et al. 2020. “Extinction Risk and Threats to Plants and Fungi.” Plants, People, Planet 2, no. 5: 389–408. https://doi.org/10.1002/ppp3.10146.

[60] Patel, R. K., and M. Jain. 2012. “NGS QC Toolkit: A Toolkit for Quality Control of Next Generation Sequencing Data.” PLoS One 7, no. 2: e30619. https://doi.org/10.1371/journal.pone.0030619.

[61] POWO. 2024. “Plants of the World Online. Facilitated by the Royal Botanic Gardens, Kew.” http://www.plantsoftheworldonline.org/.

[62] R Core Team. 2023. “R: A Language and Environment for Statistical Computing.” R Foundation for Statistical Computing. https://www.R-project.org/.

[63] Ragupathy, S., S. G. Newmaster, M. Murugesan, and V. Balasubramaniam. 2009. “Dna Barcoding Discriminates a New Cryptic Grass Species Revealed in an Ethnobotany Study by the Hill Tribes of the Western Ghats in Southern India.” Molecular Ecology Resources 9, no. Suppl s1: 164–171. https://doi.org/10.1111/j.1755-0998.2009.02641.x.

[64] Reydon, T. A. C. 2019. “ Are Species Good Units for Biodiversity Studies and Conservation Efforts?” In From Assessing to Conserving Biodiversity, History, Philosophy and Theory of the Life Sciences, edited by E. Casetta, J. Marques da Silva, and D. Vecchi, 24, 167–193. Springer.

[65] Ridley, H. N. 1934. “Firmiana and Erythropsis.” Bulletin of Miscellaneous Information (Royal Gardens, Kew) 1934, no. 5: 214–217.

[66] Rius, M., and J. A. Darling. 2014. “How Important Is Intraspecific Genetic Admixture to the Success of Colonising Populations?” Trends in Ecology & Evolution 29, no. 4: 233–242. https://doi.org/10.1016/j.tree.2014.02.003.

[67] Ronquist, F., M. Teslenko, P. van der Mark, et al. 2012. “MrBayes 3.2: Efficient Bayesian Phylogenetic Inference and Model Choice Across a Large Model Space.” Systematic Biology 61, no. 3: 539–542. https://doi.org/10.1093/sysbio/sys029.

[68] Saitoh, T., N. Sugita, S. Someya, et al. 2014. “DNA Barcoding Reveals 24 Distinct Lineages as Cryptic Bird Species Candidates in and Around the Japanese Archipelago.” Molecular Ecology Resources 15: 177–186. https://doi.org/10.1111/1755-0998.12282.

[69] Solomon Raju, A. J., S. P. Rao, and V. Ezradanam. 2004. “Bird-Pollination in Sterculia colorata Roxb. (Sterculiaceae), a Rare Tree Species in the Eastern Ghats of Visakhapatnam and East Godavari Districts of Andhra Pradesh.” Current Science 87, no. 1: 28–31.

[70] Sommer, R. J. 2020. “Phenotypic Plasticity: From Theory and Genetics to Current and Future Challenges.” Genetics 215, no. 1: 1–13. https://doi.org/10.1534/genetics.120.303163.

[71] Stamatakis, A. 2014. “RAxML Version 8: A Tool for Phylogenetic Analysis and Post-Analysis of Large Phylogenies.” Bioinformatics (Oxford, England) 30, no. 9: 1312–1313. https://doi.org/10.1093/bioinformatics/btu033.

[72] Straub, S. C. K., M. Parks, K. Weitemier, M. Fishbein, R. C. Cronn, and A. Liston. 2012. “Navigating the Tip of the Genomic Iceberg: Next-Generation Sequencing for Plant Systematics.” American Journal of Botany 99, no. 2: 349–364. https://doi.org/10.3732/ajb.1100335.

[73] Sun, W. 1998. “Firmiana major. IUCN Red List of Threatened Species 1998: e.T32349A9700122.” Yunnan Science and Technology Press.

[74] Sun, W. B. 2021. List of Yunnan Protected Plant Species With Extremely Small Populations. Yunnan Science and Technology Press.

[75] Sutherland, W. J., R. P. Freckleton, H. C. J. Godfray, et al. 2013. “Identification of 100 Fundamental Ecological Questions.” Journal of Ecology 101, no. 1: 58–67. https://doi.org/10.1111/1365-2745.12025.

[76] Tan, F., A. K. Banerjee, J. Deng, et al. 2023. “Characterization of the Complete Chloroplast Genome of Firmiana Hainanensis (Malvaceae), an Endemic and Vulnerable Tree Species of China.” Mitochondrial DNA Part B 8, no. 1: 57–60. https://doi.org/10.1080/23802359.2022.2160669.

[77] Tang, Y., M. G. Gilbert, and L. J. Dorr. 2007. “ Sterculiaceae.” In Flora of China, vol. 12, edited by C. Y. Wu, P. H. Raven, and D. Y. Hong, 310–312. Science Press, Beijing & Missouri Botanical Garden Press.

[78] Taylor, S. A., and E. L. Larson. 2019. “Insights From Genomes Into the Evolutionary Importance and Prevalence of Hybridization in Nature.” Nature Ecology & Evolution 3, no. 2: 170–177. https://doi.org/10.1038/s41559-018-0777-y.

[79] Todesco, M., M. A. Pascual, G. L. Owens, et al. 2016. “Hybridization and Extinction.” Evolutionary Applications 9, no. 7: 892–908. https://doi.org/10.1111/eva.12367.

[80] vonHoldt, B. M., K. E. Brzeski, D. S. Wilcove, and L. Y. Rutledge. 2018. “Redefining the Role of Admixture and Genomics in Species Conservation.” Conservation Letters 11, no. 2: e12371. https://doi.org/10.1111/conl.12371.

[81] Wang, D. 2001. “Discovery of a Wild Population of Firmiana major in Panzhihua City, Sichuan Province.” Plant 03: 4–5.

[82] Wang, J., C. N. Fu, Z. Q. Mo, et al. 2022. “Testing the Complete Plastome for Species Discrimination, Cryptic Species Discovery and Phylogenetic Resolution in Cephalotaxus (Cephalotaxaceae).” Frontiers in Plant Science 13: 768810. https://doi.org/10.3389/fpls.2022.768810.

[83] Wang, J.-H., M. J. Moore, H. Wang, Z.-X. Zhu, and H.-F. Wang. 2021. “Plastome Evolution and Phylogenetic Relationships Among Malvaceae Subfamilies.” Gene 765: 145103. https://doi.org/10.1016/j.gene.2020.145103.

[84] Wang, S., and Y. Xie. 2004. China Species Red List. Higher Education Press.

[85] Wang, W. G., X. D. Ma, C. L. Yan, J. P. Shi, H. Lai, and J. Y. Shen. 2019. “Firmiana kerrii (Craib) Kosterm., a Newly Recorded Species of Malvaceae From China.” Acta Botanica Boreali-Occidentalia Sinica 39, no. 12: 2278–2280. https://doi.org/10.7606/i.issn.1000-4025.2019.12.2278.

[86] Wilkie, P., A. Clark, R. T. Pennington, M. Cheek, C. Bayer, and C. C. Wilcock. 2006. “Phylogenetic Relationships Within the Subfamily Sterculioideae (Malvaceae/Sterculiaceae-Sterculieae) Using the Chloroplast Gene ndhF.” Systematic Botany 31, no. 1: 160–170. https://doi.org/10.1600/036364406775971714.

[87] Wu, X., L. Chen, M. Wang, et al. 2018. “Population Structure and Genetic Divergence in Firmiana danxiaensis.” Biodiversity Science 26, no. 11: 1168–1179.

[88] Xie, J., Y. Chen, G. Cai, R. Cai, Z. Hu, and H. Wang. 2023. “Tree Visualization by One Table (tvBOT): A Web Application for Visualizing, Modifying and Annotating Phylogenetic Trees.” Nucleic Acids Research 51, no. W1: W587–W592. https://doi.org/10.1093/nar/gkad359.

[89] Xu, S. J., and X. H. Xu. 2000. “Comments on the Taxonomic Position of Some Genera in Sterculiaceae.” Journal of tropical and Subtropical Botany 1: 11–16.

[90] Ya, J.-D., Z.-X. Yu, Y.-Q. Yang, et al. 2018. “Complete Chloroplast Genome of Firmiana major (Malvaceae), a Critically Endangered Species Endemic to Southwest China.” Conservation Genetics Resources 10, no. 4: 713–715. https://doi.org/10.1007/s12686-017-0915-x.

[91] Zhang, D., F. Gao, I. Jakovlić, et al. 2020. “Phylosuite: An Integrated and Scalable Desktop Platform for Streamlined Molecular Sequence Data Management and Evolutionary Phylogenetics Studies.” Molecular Ecology Resources 20, no. 1: 348–355. https://doi.org/10.1111/1755-0998.13096.

[92] Zhang, G. L., L. Cai, J. Q. Duan, T. Wang, and J. Y. Xiang. 2020. “Firmiana daweishanensis sp. nov. (Malvaceae) From Southeast Yunnan, China.” Phytotaxa 456, no. 2: 215–218. https://doi.org/10.11646/phytotaxa.456.2.10.

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2025 The Author(s). Integrative Conservation published by John Wiley & Sons Australia, Ltd on behalf of Xishuangbanna Tropical Botanical Garden (XTBG).