Genetic introgression among charr species in the Shiretoko Peninsula, Japan

Introgressive hybridization is an important source of genetic variation and a driver of evolutionary diversification in fishes. On the Shiretoko Peninsula, a UNESCO World Natural Heritage site in northern Japan, the Southern Asian Dolly Varden Salvelinus curilus (SADV) is the only charr species present in most rivers. However, previous studies have suggested that some SADV individuals possess mitochondrial DNA (mtDNA) from the White-spotted charr Salvelinus leucomaenis, implying historical introgression. To quantify and map this introgression, we estimated the proportion of non-SADV mtDNA (%noSADV) across 40 rivers using environmental DNA (eDNA) metabarcoding. Because White-spotted charr were absent from the study rivers, %noSADV could be interpreted as reflecting mitochondrial introgression within SADV populations. Indeed, %noSADV estimates from eDNA concentrations were strongly correlated with those from conventional catch surveys in 10 rivers (r = 0.96), supporting the reliability of the eDNA-based estimates. In addition, a minor proportion of Salvelinus eDNA was assigned to Salvelinus malma, a charr species that does not occur in Japan. Tissue-based analysis confirmed that some SADV individuals indeed carried S. malma mtDNA, suggesting an additional, independent introgression event. Across the 40 rivers, %noSADV ranged 0%-97.2%, including 0%-0.47% assigned to S. malma mtDNA. Generalized linear models indicated significant positive associations between %noSADV assigned to S. leucomaenis and both latitude and forest coverage. These findings demonstrate that (1) results from mtDNA-based analyses of taxa in which genetic introgression occurs frequently should be interpreted with caution and (2) in cases where one of the donor species is absent, eDNA metabarcoding can be a useful tool for estimating the population-level proportion of introgression over broad spatial scales and uncovering hidden evolutionary legacies in freshwater fishes. This approach provides valuable insights for evolutionary ecology and conservation genetics, especially for rare fish species in regions where conventional, tissue-based surveys are logistically constrained.