Speciation, endangerment and adaptation in limestone rocky environment of Urophysa (Ranunculaceae)

Urophysa (Ranunculaceae) are plants endemic to the limestone regions of southern China, representing an attractive model for studying species differentiation, endangerment, and adaptive evolution in karst environments. Here, we assembled a chromosome-level genome of U. rockii, and re-sequenced 97 individuals of two Urophysa species. The final genome size was 303.21 Mb with a heterozygosity level of approximately 0.26%. Demographic analyses revealed that U. rockii diverged from U. henryi around 5.95 million years ago, dramatic orogenies and climatic shifts in the Hengduan Mountain (HDM) during the Late Miocene to Pleistocene enhanced population divergence and accelerated allopatric speciation. Extremely low genetic diversity was detected in U. rockii, particularly in the populations CXS and PZ (8.7 × 10^-5 and 9.6 × 10^-5). Long-term bottleneck effects, limited gene flow, habitat specialization and severe inbreeding collectively contributed to its endangerment status. Genomic scan (top 5% F _ST and π ratio) of Urophysa species identified 257 selected genes linked to karst adaptation. Complementary physiological experiments and transcriptome analyses further detected multiple candidate genes under positive selection and expansion (e.g., TPC1, PTR/POT, and CAX3). Maintaining cell wall integrity, regulating ion absorption and excretion, and sequestering excess ions into organelles (e.g., vacuole and mitochondrion) are key survival strategies for Urophysa species in harsh limestone rocky environments. Our findings elucidate the evolutionary patterns and mechanisms underlying species adaptation to karst ecosystems, and identify key drivers of endangerment within such ecologically specialized habitats.