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Recent studies have found that geographical fragmentation and recurrent colonization result in complex genetic structures in refugial areas. This phenomenon, known as ‘refugia within refugium’, has been identified from many geographical locations. In Japan, the high-elevation mountains of central Honshu provided an interglacial refugium for alpine plants. Here we focused on the Geranium yesoense complex, which exhibits increased morphological variation in the refugial area, to determine whether this variation was shaped by recurrent colonization, range fragmentation or phenotypic changes independent of population history. We analysed single nucleotide polymorphism data and chloroplast genome sequences. Diversification in the G. yesoense species complex occurred in the mid-Pleistocene. The varieties are distinct entities and suggest the presence of a genetic cluster with highly disjunct distributions, occurring both in northern Japan and in southern refugial areas in central Honshu. Demographic analysis suggests that a single ancestral variety (var. nipponicum) evolved in the alpine region of central Honshu, and that subsequent migration from one of the two diverged northern varieties (var. pseudopratense) led to secondary contact with var. nipponicum during the last glacial period. Recolonization into refugial populations in central Honshu and hybridization between diverged populations have resulted in complex genetic structures among refugial populations.

Nuclear microsatellite markers were developed for the threatened plant Geranium soboliferum var. kiusianum, which has decreased its population size as a result of loss of its wetland habitat in Kyushu, Japan. Utilizing RNA-seq data obtained by next-generation sequencing techniques, 10 polymorphic microsatellite markers with 3-16 alleles in a nuclear genome were developed and characterized. Two to 15 alleles were observed in G. soboliferum. These markers will be used to investigate the genetic circumstance of remnant populations of G. soboliferum var. kiusianum and their phylogenetic relationship with G. soboliferum.

Premise of the study:Microsatellite markers were developed for Callitris sulcata (Cupressaceae), an endangered conifer species in New Caledonia.Methods and Results:Using sequencing by synthesis (SBS) of an RNA-Seq library, 15 polymorphic nuclear and chloroplast microsatellite markers were developed. When evaluated with 48 individuals, these markers showed genetic variations ranging from two to 15 alleles and expected heterozygosity ranging from 0 to 0.881.Conclusions:These markers will be useful for examining the genetic diversity and structure of remaining wild populations and improving the genetic status of ex situ populations.