Coexisting picoplankton experience different relative grazing pressures across an ocean productivity gradient

Significance Mortality interactions are less understood than growth processes in marine microbial food webs but equally important for determining population increases or decreases with changing environmental conditions. Experimental studies in the California Current reveal new insights about complex microbial trophic interactions across ocean productivity gradients. High production is shown to drive intensified grazing on heterotrophic bacteria, but shared predation does not transfer up-shifted mortality to co-occurring picophytoplankton of similar size. We found order-of-magnitude variability in mortality ratios indicative of highly selective predators or environmental selection for microbes with tightly coupled tradeoffs in growth advantages and grazing vulnerabilities. Study results challenge simplistic representations of mortality processes in marine ecosystem models and clear distinctions between virus and grazer roles in diversity maintenance.

Table S1.Regression statistics for plotted variables in Figs.1-4.A = intercept; B = slope; R = correlation coefficient; p = significance of regression slope B. PP = primary production; P:H = phototroph:heterotroph ratio; Mort = grazing mortality; FALS = forward angle light scatter (cell size proxy).Font colors (black, red, blue) correspond to figure regression lines.Formats for exponential (Expon), logarithmic (Log) and Power functions are Y=A*10 B*X , Y=A+B*logX and Y=A*X B , respectively.Regression models are Ordinary Least Squares (OLS, Model 1) and Reduced Major Axis (RMA, Model 2).Values of p ≤ 0.05 are considered significant.Population designations and their associated fluorescence and scatter parameters were generated from the listmode files using FlowJo software (Tree Star, Inc.) and exported to spreadsheet format (e.g., Excel files).Several successive steps were used to define populations (Fig. S3).
Heterotrophic bacteria (HBAC) were distinguished from phytoplankton by absence of pigment fluorescence (Fig. S3b) and low DNA fluorescence (Fig. S3d).Prochlorococcus (PRO) and photosynthetic eukaryotes (PEUK) were separated from each other by their characteristic DNA, chlorophyll and light scatter signatures (Fig S3b & c).PEUK (mostly <2 µm pico-and nano-sized cells due to the small volume analyzed) generally had higher light scatter per cell than prokaryotes, although some PEUK had similar light scatter but higher DNA fluorescence.Note that light scatter signatures are only proportional to cell size, since scattered light is generated based on an object's size, shape, and internal structure or refractive index (Robertson and Button 1989;Olson et al. 1989;Olson et al. 1993).
In addition, a mixture of fluorescent polystyrene bead standards was analyzed on each run day to obtain mean estimates of scatter and fluorescence for normalization of each signal and to check instrument alignment."Normalization" refers to dividing the population mean scatter or fluorescence parameter by the corresponding mean parameter of the beads, which provides a stable basis for comparison of population cell characteristics within and between runs.The beads used were 0.5 µm yellow-green beads, 0.5 µm UV beads, and 1 µm yellow-green beads (Polysciences, Inc).Yellow-green beads are optimally excited by the 488 nm laser and emit in all visible wavelengths, whereas UV beads are excited mainly by the UV laser and emit in the blue (450 nm) range.Since most cells analyzed were ≤2-µm diameter (i.e., picoplankton), the 0.5 µm yellow-green bead signals were used to normalize all light scatter, orange and red fluorescence signals for all populations.The 0.5 µm UV beads were used to normalize blue fluorescence from Hoechst-bound DNA in all populations.
We assessed potential variability in relative cell sizes of phototrophic and heterotrophic bacteria across the CCE production gradient using the ratios of bead-normalized values of forward angle light scatter (FALS) of SYN or PEUK compared to HBAC (i.e., FALSSYN:FALSHBAC) for populations analyzed in the same samples (Landry et al. 2003(Landry et al. , 2022)).This derives from the near-linear relationship between FALS and Mie scattering cross section for cells in the submicron-micron size range (DuRand and Olson 1996).

Figure S1 .
Figure S1.Relationships of picoplankton growth rates to upper-euphotic-zone temperatures during CCE Process cruises.HBAC = heterotrophic bacteria; PRO = Prochlorococcus; SYN = Synechococcus; PEUK = photosynthetic picoeukaryotes.Regression relationships (Ordinary Least Squares because the X-axis variable temperature is precisely measured) are color coded to the cruise abbreviation legend and given in the format: regression intercept, regression slope, multiple R correlation coefficient, and significance (p) of regression slope.Regression statistics for all cruises combined are given in bold font, but those regression lines are not plotted.

Figure
Figure S2.Relationships between net growth rate of PRO and population abundances of HBAC and PRO for experiments conducted at intermediate values of primary production (10<PP<100 mg C m -3 d -1 ).
DuRand MD, Olson RJ. (1996) Contributions of phytoplankton light scattering and cell concentration changes to diel variations in beam attenuation in the equatorial Pacific from flow cytometric measurements of pico-, ultra and nanoplankton.Deep-Sea Res II 43:891-906.

Figure S3 .
Figure S3.Listmode (FCS 2.0) data were analyzed in FlowJo software to define HBAC and picophytoplankton populations PRO, SYN and PEUK.a) First, phycoerythrin fluorescence was plotted as a function of DNA fluorescence for all events, to separate SYN from the rest of the cells (PHYTO+HBAC).b) Next, the PHYTO+HBAC cells were separated into HBAC, PRO and PEUK by plotting chlorophyll fluorescence as a function of DNA fluorescence.c) All phytoplankton populations (PRO, SYN, and PEUK) are shown as a function of chlorophyll vs. 90° light scatter.d) HBAC are shown as a function of DNA fluorescence vs. forward angle light scatter.

Table S2 .
Environmental variables (temperature, nitrate, chlorophyll a), rates of primary production rates (PP) and cell abundances of heterotrophic bacteria (HBAC), Prochlorococcus (PRO), Synechococcus (SYN) and photosynthetic picoeukaryotes (PEUK) in the California Current Ecosystem.Data are averaged for experiments in the upper euphotic zone.Uncertainties are standard errors of mean (SEM) values.

Table S3 .
Measured rates of population growth (µ, d -1 ) and grazing mortality (m, d -1 ) for heterotrophic bacteria (HBAC), Prochlorococcus (PRO), Synechococcus (SYN) and photosynthetic picoeukaryotes (PEUK) in the California Current Ecosystem.Data are averaged for experiments in the upper euphotic zone.Uncertainties are standard errors of mean (SEM) values.