Discussion
We assessed the linearity between two common measurements for the size of yeast populations in laboratory cultures, cell density (OD, obtained from spectrophotometry) and cell count (obtained from flow cytometry), across all eight species of Saccharomyces (Figure 1). The steepness of the slope of this relationship is informative for researchers conducting studies across species and strain backgrounds, comparing the competitive fitness of stains, or specific aspects of their growth kinetics. We found that the average slope of the relationship between cell density and cell count significantly differs between species. Especially S. cerevisiae has a significantly steeper slope than most other species, i.e. they produce higher cell counts at lower OD values. Interestingly, the patterns in slope variation that we observed across the genus, closely follow the phylogenetic relationship of the species, with more closely related pairs (S. cerevisiae /S paradoxus , S. mikatae /S. jurei , and S. eubayanus /S. uvarum ) having more similar slopes (Figure 2). This suggests that the species- and strain- specificity of this relationship should be taken into account when setting up and interpreting results of competitive fitness assays, or when fine-tuning cell titers and inocula of non-commercial strains for industrial applications. It also shows the limitations of using only a single laboratory S. cerevisiae strain as a reference point when exploring the population biology of wild strains.
We then speculated that differences in cell size may explain variation in the steepness of the slopes between species. Variation in cell size is expected, given the large diversity of ecological niches these species inhabit (Table S1), and has been described in S. cerevisiae as a result of temperature and nutrient availability (Kellogg & Levin, 2022), affecting basic physiological functions such as protein synthesis and cell division rate . The intensity and the radius of light scattering in the spectrophotometer depends on cell size, thus affecting the absorbance of the microbial culture . Indeed, we found that species significantly differ in average cell size, withS. eubayanus having the smallest (1.16 ± 0.12 log µm2) and S. cerevisiae having the largest cells (1.4 ± 0.11 log µm2, Figure 3). But against our expectation, that strains with larger average cells would give lower cell counts at similar OD readings, increasing cell size did not predict a decrease in slopes from cell count – cell density regressions. Instead, we found the opposite to be true. Slopes increased slightly with increasing cell size (Figure 4), suggesting that at a given OD-value, strains with larger cells also produce higher cell counts.
Besides cell size, other species-specific cellular features may affect the cell count – cell density relationship. The species investigated here are ecologically and genetically vastly divergent and likely differ in the composition of their cell wall, determining its strength and rigidity . For instance, chitin, chitosan, and cell wall proteins have been shown to have different refractive indices that can affect OD readings . Wall-resident proteins have diversified rapidly over evolutionary time in Saccharomyces as a result of gene silencing through epigenetic mechanisms and environmentally induced expression regulation, providing adaptability to different habitats and lifestyles . Chitin and chitosan are also known to vary in structure between fungal species . Besides cell wall composition, the number and structure of bud scars may also affect the cell’s refractive index . Mother cells accumulate chitinous scar tissue from cytokinesis over their lifetime and different growth conditions (poor vs . rich media) can lead to variation in bud scar number . If species vary in the average number or structure of bud scars a mother cell carries (e.g. due to heritable differences in cell longevity or species-specific responses to nutrient availability), this may change their refractive indices and affect the translation of optical density readings into cell counts.