Simple nonlethal identification criteria for two endangered freshwater pearl mussels, Margaritifera laevis and Margaritifera togakushiensis , in Hokkaido, northern Japan

Species identification is a fundamental process for ecological studies and conservation practices, and simple nonlethal identification criteria are important for endangered species. This study developed species identification criteria for two endangered freshwater pearl mussels ( Margaritifera laevis and Margaritifera togakushiensis ) based on linear discriminant functions (LDFs) that were established considering intraspecific regional morphological differences from sites at Hokkaido, northern Japan. We collected a total of 1,110 mussels from 52 rivers across 32 watersheds in two geographical regions (east and west). Shell morphologies (length, height, and width) of the collected mussels were measured, followed by species identification with gel electrophoresis banding patterns of 16S rRNA polymerase chain reaction products. LDFs were constructed for two size classes (≥40 mm and < 40 mm) using forward stepwise procedures to determine key morphological differences between the two species and consideration of whether regional morphological differences improved identification accuracies. The LDFs revealed that the morphologies of the two species were clearly distinguished by the relationship between height and length for both size classes. Region‐specific LDFs produced better identification accuracies. Regardless of region, the maximum length of M. laevis exceeded 100 mm, whereas all M. togakushiensis were < 100 mm in length. Identification accuracies of the established LDFs were high for each of the five length classes (0–20 mm, 20–40 mm, 40–60 mm, 60–80 mm, and 80–100 mm) with 85–96% (mean: 92%) accuracy in the east and 67–96% (mean: 80%) in the west. These criteria for species identification will progress future ecological studies and conservation practices for freshwater mussels.