Genomic Insights into Microrefugial Persistence of two Narrow Montane Endemics in the eastern Northern Limestone Alps

Endemic species associated with alpine areas offer important insights into how mountain flora evolved throughout Pleistocene climatic oscillations. The eastern Northern Limestone Alps (eNLA) area region of elevated endemic richness, yet the evolution of their endemics remains poorly understood. Callianthemum anemonoides and Pulmonaria kerneri are two narrow montane endemics of the eNLA. By disentangling their evolutionary histories we aim to illuminate how the interplay of range expansions and range contractions to microrefugia shaped the present genetic diversity. To do so, we generated RADseq-derived SNP data from a range-wide sampling, and analyzed these data in the framework of phylogeography and population genomics. We find geographically structured intraspecific variation within both species in the form of three major groups that align along an east–west gradient. Interestingly, this structure did not match geographic features such as major rivers, or the Last Glacial Maximum (LGM; 21 ka) glaciation, but rather reflects the extent of glaciers during the Penultimate Glacial Maximum (PGM; 140 ka). We interpret this finding as support for the existence of multiple forested microrefugia in the area that persisted throughout the Last Glacial Period (LGP). Intraspecific divergence was more pronounced in C. anemonoides, and the age of initial diversification (144–80 ka) clearly predated the LGM. While divergence times could not be estimated for P. kerneri, the species’ shallower intraspecific structure may reflect a more recent fragmentation. Taken together, these findings show that the repeated Pleistocene glacial cycles shaped the population structure, and that long-term persistence in spatially and climatically stable microrefugia is mirrored in deep genetic differentiation. Such a scenario is also supported by the distribution of nucleotide diversity and private alleles reflecting long-term stability in some populations, allowing identifying local hotspots of genetic diversity and high conservation value. We also evaluate how well the distribution of genetic diversity is represented by the network of protected areas, and discuss implications for future conservation management. In conclusion, our results underscore the evolutionary importance of microrefugia and their protection for maintaining the evolutionary heritage of the eNLA flora.