Development of novel high-resolution melting markers for the assessment of hybridization between Mauremys sinensis and Mauremys reevesii in South Korea

The Chinese striped-necked turtle Mauremys sinensis was first reported in the wild in South Korea in 2012. Owing to its potential to hybridize with the native and endangered Mauremys reevesii in South Korea, leading to ecosystem disturbance, M. sinensis was designated as an ecosystem-disturbing species (EDS; hereafter) in 2020. Such hybridization between M. sinensis and M. reevesii has previously been reported in Taiwan. To protect and conserve the native South Korean M. reevesii population, the extent of its hybridization needed to be examined. Therefore, we developed novel high-resolution melting markers to elucidate the status of hybridization between Mauremys sinensis and Mauremys reevesii in South Korea. We identified the presence of M. sinensis, M. reevesii, and their hybrid across 47 sites in South Korea. Among these sites, five locations were confirmed as areas of sympatry for both M. sinensis and M. reevesii, with one hybrid individual identified at the Jeonju-si site. In total, hybrids were identified at four different sites. For more definitive species identification, blood samples were collected from the specimens and subjected to mitochondrial DNA (mtDNA) cytochrome c oxidase subunit I (COI) gene and nuclear DNA (nDNA) R35 gene analysis, leading to the identification of two more hybrids from two other sites. To date, no hybrids of M. sinensis have been confirmed to reproduce in the wild, yet this requires further research for validation. The high-resolution melting (HRM) marker in the R35 gene developed in this study yielded three distinct curves that were designated as M. sinensis, M. reevesii, and M. sinensis x M. reevesii, aligning with those of the R35 gene sequencing. The novel markers developed in this study serve as a valuable tool that can be used in future research on rapid detection of hybridization.