Problems and pitfalls in using HPLC pigment analysis to distinguish Lake Michigan phytoplankton taxa

High pressure liquid chromatography (HPLC) has become a standard for analysis of phytoplankton pigment in marine and freshwaters. In marine systems, such data have been combined with optimization algorithms (e.g., CHEMTAX) to quantitatively predict the taxa present in a given sample; such studies are less common in freshwaters, particularly in oligo- and mesotrophic lakes. HPLC/CHEMTAX methods were compared with taxonomic identification and quantification using traditional microscopy and volume to chlorophyll a conversions in Lake Michigan phytoplankton communities collected on six cruises during summer (June to August) 2008. Chlorophyll a reached maxima (approximately 1.5 μg L− 1 nearshore and 0.5 μg L− 1 offshore) in late June/early July, with the exception of the offshore metalimnion where chlorophyll a peaked (3.0 μg L− 1) in early July. Taxonomic groups were consistently misidentified by HPLC/CHEMTAX, relative to microscope methods; of 18 points of comparisons, only 5 were significantly related (judged by regression of relative proportion of chlorophyll a estimated by HPLC/CHEMTAX versus cell counts/volume). Confusion between diatoms and chrysophytes was particularly serious. For example, in late July when counts indicated that biovolume at epilimnetic stations was dominated by diatoms, HPLC/CHEMTAX indicated dominance by chrysophytes. Grouping diatoms and chrysophytes as a single taxon improved comparisons, so that there was significant agreement in 7 out of 18 cases. While some specific improvement to the HPLC method (e.g., selecting a smaller subset of pigments relevant to the specific phytoplankton assemblages found) might help, our work emphasizes the point made repeatedly in the literature, that HPLC methods cannot replace microscopy.