The benthic diatom species Achnanthidium minutissimum belongs to a species complex with a challenging taxonomy. Achnanthidium minutissimum has been reported to be a widespread and abundant species occurring in a broad range of freshwater habitats. However, differentiating and delimiting it from other Achnanthidium species is challenging due to the small size and great similarity of the different species, often with overlaps in morphological features. Therefore, reports of the occurrence of these taxa probably come with a large uncertainty due to potential misidentification. To gain a better understanding of the boundaries between species within the A. minutissimum species complex, we applied an integrative taxonomic approach and investigated the congruence between morphological, molecular, and ecophysiological variability among 13 monoclonal strains isolated from Germany, Sweden, and Spitsbergen. In addition to the characterization of valve morphology, we assessed their growth under different temperatures and salt concentrations and compared sequences of the rbcL marker gene as well as of a broad set of homologous loci sampled by genome skimming. Molecular and ecophysiological variability was mostly congruent with scanning electron microscopy-based morphological identification; the main exception was that two pairs of strains identified as A. cf. microcephalum and A. jackii could be distinguished neither in their ecophysiological profiles nor in their DNA sequences. Extending this integrated taxonomic approach to more strains will be beneficial for a better understanding of the morphological, molecular, and niche differentiation among different Achnanthidium species. The added value of the combined morphological–molecular–ecophysiological approach is an improved delineation of morphological features applicable for species differentiation and a better understanding of ecological differentiation.
