Middle Pleistocene climate change influenced northward divergence and cold adaptation in an Asian lady's slipper orchid, Cypripedium macranthos Sw. (Orchidaceae)

The Pleistocene was characterized by climate fluctuations with rapid transitions between glacial and interglacial periods. East Asia's land bridge dynamics and monsoon system during the Middle Pleistocene transition (MPT) further constrained species distributions and dispersals, which promoted local adaptation and species diversification, making East Asia an excellent location to study species biogeography. Cypripedium macranthos Sw., a terrestrial orchid, diverged in the Pleistocene and became widespread in Russia, China, Korea, and Japan. Many populations are now at risk due to illegal harvesting or habitat loss. With comprehensive sampling including 171 individuals among 22 populations across the entire distribution range, together with plastid DNA sequencing, we have explored the origin and impact of palaeoclimate on its spatial genetic structure and historical biogeography. Regression-based analyses commonly used in landscape genetics were also applied to investigate the effects of precipitation, temperature, and wind on gene flow. The results revealed northeastern China as the probable cradle of C. macranthos, with northeastward divergence and range expansion during the MPT, aided by monsoon shifts and wind-driven seed dispersal. Isolation by environment was detected with winter temperature as a driver for north-south divergence controlling for an effect of isolation by distance, revealing cold adaptation of the northern populations or temperature-driven genetic differentiation. We also emphasize the founder speciation of an unique variety on Rebun Island of Japan. The widespread but genetically and ecologically divergent C. macranthos populations highlight the need for more conservation attention on northern populations under global warming.