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Morphology and molecular phylogeny constitute the structural elements of diatom taxonomy. These approaches do not, however, give information on the functioning of taxa. Additional methods to serve a more integrated and wide-ranging taxonomy have therefore been called for. Metabolic fingerprinting is one approach used within the field of metabolomics, often applied in classification of samples. Here we apply metabolic fingerprinting in a taxonomic study of a cryptic diatom species. Strains of the cosmopolitan diatom Chaetoceros socialis from two geographical areas; the north-east Atlantic and Arctic and the Gulf of Naples, were cultivated at three different temperatures; 2.5, 8 and 13 C. The strains from the two different geographical areas exhibited different growth rates as well as different photosynthetic efficiencies. Algal extracts, collected at the end of the growth experiments, were analysed by Ultra-Performance Liquid Chromatography High Resolution Mass Spectrometry. The two groups of strains were separated by principal component analysis of their metabolic fingerprints. Analysis of the data revealed both qualitative and quantitative differences in metabolite markers. These phenotypic differences reinforce differences also found for morphology, phylogenetic markers and growth rates, and point at different adaptive characteristics in organisms living under different temperature regimes.

In this study, we investigate how metabolic fingerprints are related to temperature. Six common northern temperate diatoms (Attheya longicornis, Chaetoceros socialis, Chaetoceros furcellatus, Porosira glacialis, Skeletonema marinoi, and Thalassiosira gravida) were cultivated at two different temperatures, 0.5 and 8.5 °C. To exclude metabolic variations due to differences in growth rates, the growth rates were kept similar by performing the experiments under light limited conditions but in exponential growth phase. Growth rates and maximum quantum yield of photosynthesis were measured and interpreted as physiological variables, and metabolic fingerprints were acquired by high-resolution mass spectrometry. The chemical diversity varied substantially between the two temperatures for the tested species, ranging from 31% similarity for C. furcellatus and P. glacialis to 81% similarity for A. longicornis. The chemical diversity was generally highest at the lowest temperature.

During normal sample preparation, storage in freezers and subsequent freeze/thaw cycles are commonly introduced. The effect of freeze/thaw cycles on the metabolic profiling of microalgal extracts using HR-MS was investigated. Methanolic extracts of monocultures of Arctic marine diatoms were analyzed immediately after extraction, after seven days of storage at −78 °C (one freeze/thaw cycle), and after additional seven days at −20 °C (two freeze/thaw cycles). Repeated direct infusion high-resolution mass spectrometry analysis of microalgae extracts of the same sample showed that reproducibility was ca. 90% when a fresh (unfrozen) sample was analyzed. The overall reproducibility decreased further by ca. 10% after the first freeze/thaw-cycle, and after one more freeze/thaw cycle the reproducibility decreased further by ca. 7%. The decrease in reproducibility after freeze-thaw cycles could be attributed to sample degradation and not to instrument variability.