Paleolimnological records from temperate South-Central Chilean lakes revealed a recent and marked increase in the relative abundances of the key-planktonic diatom species Asterionella formosa and Aulacoseira distans. To better understand these changes, we studied the ecological niches of planktonic diatoms in 42 temperate Chilean lakes (36-43 degrees S). We used morphological analysis of spring and early summer diatom communities, as well as sub-fossil sedimentary assemblages along gradients in mixing regime, light climate, and water chemistry. For Asterionella formosa and the species belonging to the genus Aulacoseira, morphology based enumerations were combined with molecular phylogenies using the 18S rDNA and rbcL genes to determine their taxonomic position and explore potential hidden diversity. Analysis of extant and sub-fossil communities revealed similar patterns, with small Discostella species (D. mascarenica and D. pseudostelligera) and Urosolenia eriensis being characteristic for large, well-mixed lakes, whereas Aulacoseira pusilla (previously erroneously identified as A. distans in Chile) is more dominant in smaller, warmer, and stratified water bodies. This suggests that recent increases in the latter taxon might be related to changes in the physical lake properties in the region, rather than a recent increase in nutrient concentrations. By contrast, Asterionella formosa occurred in lakes with a high Si concentration. Phylogenetic data of the most common species belonging to the genera Aulacoseira and Asterionella showed little evidence for hidden diversity in most of the taxa, except for the Aulacoseira granulata complex. This indicates that the Chilean strains are closely related to populations in the Northern Hemisphere. The results of this study will allow for more accurate diatom-based reconstructions of limnological changes in South-Central Chilean lakes. This is crucial to better understand climate dynamics in the region, as these lakes are known to be sensitive to alterations in windiness and precipitation in response to changes in the intensity and latitudinal position of the Southern Hemisphere Westerlies and the El Nino-Southern Oscillation.