Phylogeography of the endangered orchids Cypripedium japonicum and Cypripedium formosanum in East Asia: Deep divergence at infra- and interspecific levels

To date, little is known about the past evolutionary trajectories of rare and endangered orchids native to mainland China, Japan, and Korea (the CJK region). In this study, we focus on two endangered orchids, Cypripedium japonicum (present in the three countries) and C. formosanum (endemic to Taiwan), to understand the divergence/speciation models that would have been operating in this group, including genetic diversity, geographic structure, and colonization pathways across the region. Using a combination of five cpDNA regions, we reconstructed phylogenetic trees and investigated the genetic diversity/structure of 20 populations. Ecological niche modeling was used to gain insight into the paleodistribution and dispersal corridors at the Last Glacial Maximum and to survey climatic niche differences. Populations from mainland China + Korea, Japan, and Taiwan formed three distinct monophyletic lineages and were placed into separate genetic clusters, agreeing with geographic barriers and species boundaries. Populations of C. japonicum in mainland China harbored the highest diversity, suggesting the presence of multiple glacial refugia. The Korean populations would have originated from either western/central or eastern China, probably using a dispersal corridor across the East China Sea shelf. The divergence of C. formosanum is proposed under an allopatric speciation model, also highly influenced by a climate niche shift. In the context of previous studies, a deep divergence in cpDNA sequences between Chinese + Korean and Japanese populations of C. japonicum may be taken as an example of the speciation events of the CJK flora since the late Neogene that have led to its current species richness.This study was supported by the Biodiversity Survey, Observation and Assessment Program of the Ministry of Ecology and Environment of China to HZT and by Basic Science Program through the National Research Foundation of Korea (NRF-2017R1A2B4012215) to MGC, and funded by the Ministry of Science and ICT of the Republic of Korea (NRF-2020R1I1A3074635) to MYC.INTRODUCTION MATERIALS AND METHODS Study species Population sampling DNA extraction cpDNA-PCR optimum primer selection cpDNA sequence alignment and assembly Haplotype distribution, phylogenetic analyses, and genetic diversity Genetic differentiation and structure Mismatch distribution analysis, neutrality detection, and demographic history ENM and population connectivity Niche comparisons in E-space RESULTS Haplotype distribution and phylogeny Genetic diversity Genetic differentiation and structure Mismatch distribution analysis, neutrality detection, and demographic history ENM and population connectivity Niche comparisons in E-space DISCUSSION Deep genetic and climatic divergence of Cypripedium sect. Flabellinervia in the CJK region: taxonomic considerations Haplotype and nucleotide diversity in Cypripedium sect. Flabellinervia: inference of glacial refugia and demographic history Origin of Korean populations of Cypripedium japonicum Origin of Cypripedium formosanum CONCLUSIONS AUTHOR CONTRIBUTIONS ACKNOWLEDGEMENTS Appendix 1: The cpDNA sequence information of Cypripedium sect. Flabellinervia deposited in the GenBank databas