Difficulties in distinguishing between morphologically similar species, such as Phoxinus lagowskii steindachneri and Phoxinus oxycephalus jouyi, whose distributions overlap in the Kinki region, are significant for assessments of biodiversity and conservation, in addition to taxonomic status. Morphological analyses were undertaken on Phoxinus specimens (n = 75) collected in the Ishi and Chihaya rivers (Yamato River basin, Osaka Prefecture), and compared with type specimens (syntypes of Phoxinus steindachneri, Sauvage, 1883 and paratypes of Leusiscus jouyi, Jordan and Snyder, 1991) and non-type museum specimens (Phoxinus oxycephalus jouyi). Mitochondrial DNA (mtDNA) analyses of 16 individuals were also undertaken. Two distinct types of Phoxinus were recognized, differing morphologically in the presence or absence of a black horizontal band on the side of the body and scale numbers above the lateral line, and separated by a principal component analysis of 9 characters. In addition, 12 haplotypes (identified in the present and previous studies) were variously associated within two genetically different groups, corresponding to two ecological types that inhabit the upper and middle parts of the Yamato River, respectively. Because reproductive isolation is already clearly established between the two types, their recognition as separate species (Phoxinus lagowskii steindachneri and P. oxycephalus jouyi) in the Ishi and Chihaya rivers is upheld.
