Alkenone-producing species have been recently found in diverse lacustrine environments, albeit with taxonomic information derived indirectly from environmental genomic techniques. In this study, we isolated alkenone-producing algal species from Canadian saline lakes and established unialgal cultures of individual strains to identify their taxonomical and molecular biological characteristics. Water and sediments collected from the lakes were first enriched in artificial seawater medium over a range of salinities (5-40 g/L) to cultivate taxa in vitro. Unialgal cultures of seven haptophyte strains were isolated and categorized in the Isochrysis Glade using SSU and LSU rRNA gene analysis. The alkenone distributions within isolated strains were determined to be novel compared with other previously reported alkenone-producing haptophytes. While all strains produced the typical C-37 and C-38 range of isomers, one strain isolated from Canadian salt lakes also produced novel C-41 and C-42 alkenones that are temperature sensitive. In addition, we showed that all alkenone unsaturation indices (e.g., U-37(k) and U-37(k)') are temperature-dependent in culture experiments, and that alkenoate indices (e.g., U-37(A), U-38(A), RIA(38) and A(37)/A(38)) provide alternative options for temperature calibration based on these new lacustrine algal strains. Importantly, these indices show temperature dependence in culture experiments at temperatures below 10 degrees C, where traditional alkenone proxies were not as sensitive. We hypothesize that this suite of calibrations may be used for reconstructions of past water temperature in a broad range of lakes in the Canadian prairies. (C) 2018 Elsevier Ltd. All rights reserved.