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Ecological assessment of freshwater ecosystems based on diatom metrics is an important issue for attaining environmental sustainability. The present study aimed to evaluate differences in the diatom–stressor relationship in relatively least disturbed streams in the Konya closed river basin using multivariate analyses and to bio-assess streams by the application of different ecoregional diatom indices. Cocconeis euglypta, Cymbella excisa, Cocconeis placentula, and Achnanthidium minutissimum are the most contributing species to the dissimilarity of sampling stations between rainy (spring) and dry (summer and fall) seasons and also between altitude (A2 800- < 1600 m and A3 ≥ 1600 m) groups. The first two axes of canonical correspondence analysis revealed a significant (82.8%) relationship between diatom species and stressors. Diatom species displayed distinct responses to environmental variables (electrical conductivity, Ni, Cu, B, and altitude) playing important roles on the distribution of species. Diatom indices indicate different ecological statuses of stations, from bad to high. European diatom indices except Duero Diatom Index (DDI) and Trophic Diatom Index (TDI) showed good responses to the eco-assessment of streams and indicated high ecological status for the least disturbed sampling stations symbolized as S16, S20, S24, S25, S27-29, S37, and S39. These results were also supported by abiotic evaluation. Although TIT was more competitive in the bio-assessment of streams among diatom indices, it is necessary to increase its species list by determining their trophic weights in future studies. Therefore, the use of ecoregion-specific diatom indices is suggested along with increasing the number of used species to correctly interpret the water quality.

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