Genetic Diversity in the Wrinkled Frog, Glandirana rugosa, Evaluated Using Microsatellite Markers Identified by Nanopore Sequencing

Wrinkled frogs (Glandirana rugosa) are a common species found in various freshwater habitats across Japan. Due to multiple sex chromosome turnovers and the discovery of cryptic species, understanding the historical population dynamics of this species is crucial for studying its genomic evolution and speciation. On the other hand, in some populations, urbanization has led to a decline in both population size and their distribution range. To better understand the historical population dynamics and recent population decline of G. rugosa, it can be helpful to conduct novel genetic analyses. As a crucial first step, we primarily focused on developing 14 microsatellite markers using nanopore sequencing. These markers were then validated by assessing the genetic diversity of G. rugosa in four populations: three from mainland Japan (two from Shimane and one from Gunma Prefectures) and one from an island (Oki Islands, Shimane Prefecture). The total number of alleles per locus ranged from 4 to 36, and the observed heterozygosity ranged from 0 to 0.950. Population genetic analyses using these markers revealed substantial genetic diversity among populations and a weak correlation between intra-population diversity and geographical features. We believe that the microsatellite markers developed in this study would be useful in exploring their genetic diversity in other populations and contribute to the conservation of this species in the future.