Ploidy Distribution of the Harmful Bloom Forming Macroalgae Ulva spp. in Narragansett Bay, Rhode Island, USA, Using Flow Cytometry Methods

Abstract

Macroalgal blooms occur worldwide and have the potential to cause severe ecological and economic damage. Narragansett Bay, RI is a eutrophic system that experiences summer macroalgal blooms composed mostly of Ulva compressa and Ulva rigida, which have biphasic life cycles with separate haploid and diploid phases. In this study, we used flow cytometry to assess ploidy levels of U. compressa and U. rigida populations from five sites in Narragansett Bay, RI, USA, to assess the relative contribution of both phases to bloom formation. Both haploid gametophytes and diploid sporophytes were present for both species. Sites ranged from a relative overabundance of gametophytes to a relative overabundance of sporophytes, compared to the null model prediction of √2 gametophytes: 1 sporophyte. We found significant differences in cell area between ploidy levels for each species, with sporophyte cells significantly larger than gametophyte cells in U. compressa and U. rigida. We found no differences in relative growth rate between ploidy levels for each species. Our results indicate the presence of both phases of each of the two dominant bloom forming species throughout the bloom season, and represent one of the first studies of in situ Ulva life cycle dynamics.

Best practices in the flow cytometry of microalgae

“…In studies on microalgae, commonly-used buffers are LB01 (with streptophytes, Chlorophyta, Ochrophyta, raphidophytes; [4,11,30]), a MOPS-based buffer (3-(N-morpholino) propanesulfonic acid (used with dinoflagellates; [21,24]), or Otto buffers (with chrysophytes; [16,29]). Triton X-100 (to a final concentration of 0.05%-1%) may be added to improve the sample staining, though its effect varies across different groups of algae [2,7,14,19,34]. Phenols, tannins, and other secondary metabolites are commonly present in microalgae and may act as staining inhibitors or lower the quality of the FCM analysis.…”

“…It is also possible to distinguish taxa and identify cryptic species on the basis of differences in genome size [22,47]. When estimating genome size in microalgae using PI, caution should be taken as the emission spectrum of PI fluorescence can overlap with the autofluorescence of photosynthetic pigments, PI can bind polysaccharides from the remaining cell walls and thus contribute to background noise and increase of CV [34], and its ability to fluoresce in the presence of double-stranded RNA must be considered and, if necessary, eliminated by including RNAse in the staining protocol. In case of two peaks being observed in a FCM uniparametric histogram of a microalgal sample, the first peak (1C) is usually considered to represent G 1 -phase cells and the second (2C) belonging to G 2 cells (Figure 1; [4, 11, 16]).…”

“…Due to the often challenging preparation of microalgal FCM samples and the frequently high contents of secondary metabolites interfering with the analysis, it is unusual to obtain measurements having CV values at or below 3%, although CVs are typically below 10% [9,21,34].…”

“…Although the number of available algal standards is now rising, it is still a negligible number in contrast to algal diversity. Examples of standards include the green microalga Chlamydomonas reinhardtii CC‐400 cw15 mt+, which, being a cell‐wall deficient mutant, is therefore easy to use (2C = 0.24 pg; available at Chlamydomonas Resource Center, University of Minnesota; [14,34]). The chrysophyte Synura sphagnicola CCAC 2959 B (2C = 0.4 pg; otherwise designated LO234K‐E, and available at The Central Collection of Algal Cultures [CCAC]) was recently introduced as an internal standard by Olefeld et al [16].…”

Meet the challenges of analyzing small genomes using flow cytometry

“…Talhinhas and colleagues [4] further discussed the lack of appropriate FCM standards, which is yet another important issue accompanying analysis of small genomes. In recent years, the number of newly introduced FCM standards is slowly rising up, with, for example, Saccharomyces cerevisiae, Aspergillus fumigatus or Chlamydomonas reinhardtii possessing very small genome sizes (1C values of 24.1, 29.2, and 0.12 pg, respectively; [8,9]). In the previous work, Talhinhas et al [10] introduced additional fungal FCM standards with various genome sizes.…”