High levels of intra-specific variation are commonly observed in natural microbial populations, yet the consequences of this variation for ecological and evolutionary processes remains poorly understood. Protists are excellent experimental models for investigating fundamental and applied questions in ecology and evolution, but studying intra-specific variation remains a challenge due to a lack of molecular resources to aid in quantifying and distinguishing strains during experiments. Here we present a molecular method, quantitative microsatellite genotyping, to accurately quantify strain specific frequencies from microcosm experiments of the marine flagellate Oxyrrhis marina, both between many pairs of strains and between strains in a multi-strain mixture. We find that for pairs of strains the method is effective for relative frequencies as low as 0.02 and with around 99% accuracy. The method is able to quantify four strains reasonably well, though less accurately than for pairs (range 92%-97% accuracy). This makes accessible a cheap and easy to implement method for quantifying strain (or allele) frequencies, and is suitable for use in a broad range of single celled eukaryotes (Protists) where copy number should correlate well with number of individuals (i.e. cells). This opens up the possibility of examining the role of intra-specific variation using experimental protist microcosms.

MS calibration data DNA PairsThis file contains data for extracted and mixed nDNA (i.e. black data in Fig.1), and shows microsatellite peak heights and peak areas for each of two strains. DNA is mixed in known ratios, and peak height and peak area ratios are calculated.MS calibration data CELL PairsThis data contains calibrations for mixtures of cells from pairs of strains (i.e. red data in Fig.1).MS calibration data CELL MultiThis data file contains calibration data for multistrain testing of the genotyping assay (i.e. data analysed in SI 1).Dynamics example dataExample dynamics from a microcosm experiment of two strains of Oxyrrhis over 13 days under two pCO2 levels, using the method for strain frequency determination described here.