Otolith Morphology Resolves Taxonomic Ambiguities in Four Freshwater Minnows (Cyprinidae)

Taxonomic ambiguities within the subfamily Smiliogastrinae, particularly involving the genera Puntius and Pethia, have led to ongoing debates about species classification and phylogenetic relationships. In this study, we used otolith shape and morphometric analysis to differentiate the four species of subfamily Smiliogastrinae (Puntius chola, Puntius sophore, Pethia conchonius, and Pethia ticto). We collected 134 fish samples from the local fish market of Aligarh. Otolith shape was analysed through wavelet coefficients, revealing significant differences among the species (p = 0.001) by PERMANOVA. The Kruskal−Wallis and the post-hoc test identified significant differences between the four species in several otolith morphometric parameters. Canonical analysis of principal coordinates indicated that the first two axes explained 61.3 and 25.3% of the variance for wavelet coefficients and 93.2 and 6.5% for otolith morphometric indices. Canonical analysis scores tested by ANOVA-like permutation test revealed significant separation (p = 0.001). The wavelet coefficients showed a higher classification rate (89.55%) than the otolith morphometric parameters (81.34%) through cross-validation classification. UPGMA cluster analysis revealed two major groups, indicating close relationships between Pethia conchonius and Pethia ticto, and between Puntius chola and Puntius sophore. These findings support the use of otolith morphology as a complementary tool for resolving taxonomic uncertainties in Smiliogastrinae.