Development of molecular identification methods for and , and their hybrids.

BACKGROUND

As hybridization can reduce biodiversity or cause extinction, it is important to identify both purebred parental species and their hybrids prior to conserving them. The Suwon tree frog, is an endangered wildlife species in Korea that shares its habitat and often hybridizes with the Japanese tree frog, . In particular, , , and their hybrids often have abnormal ovaries and gonads, which are known causes that could threaten their existence.

METHODS

We collected 57 individuals from six localities where is known to be present. High-resolution melting curve (HRM) analysis of the mitochondrial 12S ribosomal RNA gene was performed to determine the maternal species. Thereafter, the DNA sequences of five nuclear genes (, , , , and ) were analyzed to determine their parental species and hybrid status.

RESULTS

The HRM analysis showed that the melting temperature of was in the range of 79.0-79.3 °C, and that of was 77.7-78.0 °C, which clearly distinguished the two tree frog species. DNA sequencing of the five nuclear genes revealed 37 single-nucleotide polymorphism (SNP) sites, and STRUCTURE analysis showed a two-group structure as the most likely grouping solution. No heterozygous position in the purebred parental sequences with Q values ≥ 0.995 were found, which clearly distinguished the two treefrog species from their hybrids; 11 individuals were found to be , eight were found to be , and the remaining 38 individuals were found to be hybrids.

CONCLUSION

Thus, it was possible to unambiguously identify the parental species and their hybrids using HRM analysis and DNA sequencing methods. This study provided fundamental information for conservation and restoration research.