Copper and light shape coastal picophytoplankton communities via their combined effects on growth limitation and toxicity

Copper (Cu) and light are two resources that can limit phytoplankton growth at very low (deficiency) or very high (toxicity) levels. In this study, Cu and light interactive effects on picophytoplankton growth and community composition southern California were assessed during four bottle incubation experiments using a 7x7 matrix of overlapping Cu and light gradients. Consistent with prior knowledge, sensitivity to Cu in the two September experiments was greatest in Prochlorococcus, followed by Synechococcus, and then picoeukaryotes. Prochlorococcus abundance declined gradually with Cu additions >6 nM, whereas Synechococcus showed a sharp toxicity threshold at >10 nM added Cu. Synergistic effects between Cu and light exacerbated toxicity in both taxa, suggesting that shared stress response pathways become saturated and less effective when Cu and light are both high. An unexpected increase in ambient seawater Cu concentration prior to the two October experiments brought Prochlorococcus and Synechococcus close to their toxicity thresholds, potentiating their apparent sensitivity to light and leading to steeper population declines in the experiments. Addition of 10 mM nitrate at the start of one of the experiments did not rescue the populations from toxicity, suggesting that relief of N limitation did not allow for greater acclimation via de novo stress response enzyme synthesis. Across all experiments, picoeukaryotes were more resilient to high light and Cu, allowing them to persist or increase under conditions that limited Prochlorococcus and Synechococcus. This robustness combined with relief from competition for other resources (possibly phosphate) upon decline of the other two taxa, ultimately led picoeukaryotes to dominate the communities despite having very low baseline relative abundances. Selection for different Synechococcus clades and picoeukaryote species likely permitted each of these populations to thrive over a broader range of Cu and light combinations than would be possible for less biodiverse populations.

Methods
Trace metal concentrations (including Al, Cd, Co, Cu, Fe, Mn, Ni, Pb, V, and Zn) were determined on 0.22 micrometer filtered samples by inductively coupled plasma mass spectrometer (ICP-MS). The method’s LOQs were the following: dAl = 0.30 nM, dCd= 0.03 nM, dCo= 0.12 nM, dCu= 0.82 nM, dFe= 3.35 nM, dMn= 0.08 nM, dNi=0.23 nM, dPb= 0.03 nM, dV= 0.34 nM, dZn=1.39 nM.

Picophytoplankton were enumerated by flow cytometry at the Center for Aquatic Cytometry, Bigelow Laboratory for Ocean Sciences on a Bio-Rad ZE5 with 405 nm, 488 nm, and 640 nm lasers activated following standard methods (Poulton and Martin. 2010. Imaging flow cytometry for quantitative phytoplankton analysis — FlowCAM. In: Intergovernmental Oceanographic Commission of ©UNESCO. Karlson, Cusack, and Bresnan (editors). Microscopic and molecular methods for quantitative phytoplankton analysis. UNESCO. (IOC Manuals and Guides, no. 55.), 110 pages.)

Chlorophyll a was determined from cells filtered from 100mL of sample water and extracted in 5mL 90% acetone in the dark for 24h at -20oC. Extracted chlorophyll was measured fluorometrically using the Turner Trilogy non-acidified chlorophyll filter module (excitation 436/10nm, emission 685/10nm).