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Advances in high-throughput DNA sequencing methods reveal the vast diversity of marine protists. Amplicon sequencing of “barcode” genes, such as the 18S small subunit ribosomal RNA gene (henceforth, 18S gene), is a cost-effective and widely used genetic method for assessing the composition of marine protist communities. This method is now being applied from local to global scales to interrogate the causes and consequences of protist community variations. Significant efforts have been made to validate amplicon methods targeting prokaryotes, but the precision, accuracy, and quantitative potential of 18S gene amplicon sequencing methods for marine protists remain unclear. Here, we use artificial (mock) communities and environmental samples collected from the Santa Barbara Channel, CA to evaluate the precision and accuracy in an amplicon workflow targeting the V9 hypervariable region of the 18S gene for marine protists. Overall, we find that this amplicon workflow has high precision and reasonable accuracy, but the magnitude of analytical uncertainty can increase significantly unless certain procedural issues are avoided. Finally, we demonstrate the value of positive and negative controls in, and the quantitative potential of, amplicon sequencing assessments of marine protist communities.

This study examined diel shifts in metabolic functions of Microcystis spp. during a 48-h Lagrangian survey of a toxin-producing cyanobacterial bloom in western Lake Erie in the aftermath of the 2014 Toledo Water Crisis. Transcripts mapped to the genomes of recently sequenced lower Great Lakes Microcystis isolates showed distinct patterns of gene expression between samples collected across day (10:00 h, 16:00 h) and night (22:00 h, 04:00 h). Daytime transcripts were enriched in functions related to Photosystem II (e.g., psbA), nitrogen and phosphate acquisition, cell division (ftsHZ), heat shock response (dnaK, groEL), and uptake of inorganic carbon (rbc, bicA). Genes transcribed during nighttime included those involved in phycobilisome protein synthesis and Photosystem I core subunits. Hierarchical clustering and principal component analysis (PCA) showed a tightly clustered group of nighttime expressed genes, whereas daytime transcripts were separated from each other over the 48-h duration. Lack of uniform clustering within the daytime transcripts suggested that the partitioning of gene expression in Microcystis is dependent on both circadian regulation and physicochemical changes within the environment.

Reservoirs are a significant source of carbon (C) to the atmosphere, but their emission rates vary in space and time. We compared C emissions via diffusive and ebullitive pathways at several stations in six large hydropower reservoirs in the southeastern US that were previously sampled in summer 2012. We found that carbon dioxide (CO2) diffusion was the dominant flux pathway during 2012 and 2022, with only three exceptions where methane (CH4) diffusion or CH4 ebullition dominated. CH4 diffusion rates were positively associated with water temperature. However, we found no clear predictors of CH4 ebullition, which had extremely high variability, with rates ranging from 0 to 739 mg C m−2 day−1. For CO2 diffusion, the direction of the flux shifted between 2012 and 2022, where all but three stations across all reservoirs emitted CO2 in summer 2012, but every station sequestered CO2 in summer 2022. Here, indicators of greater algal production were associated with CO2 sequestration, including surface chlorophyll‐a concentration, surface dissolved oxygen saturation, and pH. Additional sampling campaigns outside the summer season highlighted the importance of seasonal phenology in primary production on the direction of CO2 diffusive fluxes, which shifted to positive CO2 fluxes by the end of August as productivity decreased. Our results demonstrate the importance of capturing CO2 sequestration in field and modeling measurements and understanding the seasonal drivers of these estimates. Measuring C emissions from multiple pathways in reservoirs and understanding their spatiotemporal responses and variability are vital to reducing uncertainties in global upscaling efforts. Plain Language Summary: Inland waters, including reservoirs used for many different purposes, contribute greenhouse gases like carbon dioxide and methane to the atmosphere. With 34 times more global warming potential than carbon dioxide, methane is of particular concern since reservoirs emit disproportionally large amounts of it. However, our ability to quantify reservoir emissions is limited by high variability in these emissions over space and time. In this study, we measured emissions from several stations across six reservoirs and compared these emissions to a previous study from 2012 to understand potential longer‐term variation in emissions and assess driver variables. Overall, we found that algal productivity was an important driver of fluxes among the reservoirs and could in fact lead to carbon dioxide sequestration. However, the seasonal phenology in algal productivity and related variables were also important to understand when considering emissions variability within a reservoir and over time. As we continue to collect emissions measurements from reservoirs, studies focused on multiple spatial and temporal scales (i.e., day and night) can improve upscaled estimates of greenhouse gas emissions to better quantify the role of reservoirs in the global carbon cycle. Key Points: Reservoirs are a major source of carbon emissions, but spatial and temporal variability leads to uncertain modeled or upscaled estimatesAcross six reservoirs, summer CO2 diffusion was the dominant flux pathway and showed sequestration, linked to increased algal productivityChanges in algal productivity influence the direction of CO2 diffusion with key implications for assessing net reservoir emissions [ABSTRACT FROM AUTHOR]

The effect of Ocean Acidification (OA) on marine biota is quasi-predictable at best. While perturbation studies, in the form of incubations under elevated pCO(2), reveal sensitivities and responses of individual species, one missing link in the OA story results from a chronic lack of pH data specific to a given species' natural habitat. Here, we present a compilation of continuous, high-resolution time series of upper ocean pH, collected using autonomous sensors, over a variety of ecosystems ranging from polar to tropical, open-ocean to coastal, kelp forest to coral reef. These observations reveal a continuum of month-long pH variability with standard deviations from 0.004 to 0.277 and ranges spanning 0.024 to 1.430 pH units. The nature of the observed variability was also highly site-dependent, with characteristic diel, semi-diurnal, and stochastic patterns of varying amplitudes. These biome-specific pH signatures disclose current levels of exposure to both high and low dissolved CO(2), often demonstrating that resident organisms are already experiencing pH regimes that are not predicted until 2100. Our data provide a first step toward crystallizing the biophysical link between environmental history of pH exposure and physiological resilience of marine organisms to fluctuations in seawater CO(2). Knowledge of this spatial and temporal variation in seawater chemistry allows us to improve the design of OA experiments: we can test organisms with a priori expectations of their tolerance guardrails, based on their natural range of exposure. Such hypothesis-testing will provide a deeper understanding of the effects of OA. Both intuitively simple to understand and powerfully informative, these and similar comparative time series can help guide management efforts to identify areas of marine habitat that can serve as refugia to acidification as well as areas that are particularly vulnerable to future ocean change.

Quantifying phytoplankton composition is critical to predicting marine ecosystem structure and function. DNA meta‐barcoding and high‐performance liquid chromatography (HPLC) pigment analysis are two widely used methods for assessing phytoplankton composition; however, comparing their performance has been done only rarely. Here, we integrate DNA meta‐barcoding and HPLC pigment observations to determine eukaryotic phytoplankton composition in the Santa Barbara Channel, California. We find that both methods identify the same four dominant eukaryotic phytoplankton taxa (diatoms, dinoflagellates, chlorophytes, and prymnesiophytes), but inter‐ and intra‐lineage variability in biomarker pigmentation (associated with both a lack of taxonomic specificity of biomarker pigments and intrinsic differences in accessory pigmentation) drives substantial disagreement between the methods. Covariation network analysis circumvents this disagreement and reveals that diverse assemblages of phytoplankton and other protists covary with distinct suites of biomarker pigments. Our results highlight the strengths and weaknesses of each method in characterizing phytoplankton composition and reveal novel insights into phytoplankton physiology that could only be gained by integrating the two methods. Finally, we suggest a path to monitor eukaryotic plankton communities on unprecedented spatiotemporal scales based on the covariation of unique phytoplankton and protistan assemblages with remotely sensible phytoplankton pigment concentrations. [ABSTRACT FROM AUTHOR]

Coccolithophores are an important group of calcifying marine phytoplankton. Although coccolithophores are not silicified, some species exhibit a requirement for Si in the calcification process. These species also possess a novel protein (SITL) that resembles the SIT family of Si transporters found in diatoms. However, the nature of Si transport in coccolithophores is not yet known, making it difficult to determine the wider role of Si in coccolithophore biology. Here, we show that coccolithophore SITLs act as Na+‐coupled Si transporters when expressed in heterologous systems and exhibit similar characteristics to diatom SITs. We find that CbSITL from Coccolithus braarudii is transcriptionally regulated by Si availability and is expressed in environmental coccolithophore populations. However, the Si requirement of C. braarudii and other coccolithophores is very low, with transport rates of exogenous Si below the level of detection in sensitive assays of Si transport. As coccoliths contain only low levels of Si, we propose that Si acts to support the calcification process, rather than forming a structural component of the coccolith itself. Si is therefore acting as a micronutrient in coccolithophores and natural populations are only likely to experience Si limitation in circumstances where dissolved silicon (DSi) is depleted to extreme levels. [ABSTRACT FROM AUTHOR]

This study examines an unprecedented bloom of Emiliania huxleyi along the California coast during the NE Pacific warm anomaly of 2014–2015. Observations of coccolithophore populations from microscopy and flow cytometry, surface current data derived from high‐frequency radar, and satellite ocean color imagery were used to track the population dynamics of the bloom in the Santa Barbara Channel. Results show a coastal bloom of mostly E. huxleyi that reached cell concentrations up to 5.7 × 106 cells per liter and a maximum spatial extent of 1,220 km2. We speculate that the rare cooccurrence of warm water, high water column stability, and an extensive preceding diatom bloom during the anomaly contributed to the development of this bloom. Flow cytometry measurements provided insight on the phases of bloom development (e.g., growth versus senescence) with calcified cells comprising up to 64% of particles containing chlorophyll a and detached‐coccolith:cell ratios ranging from 10 to >100. Lagrangian particle trajectories estimated during two nonoverlapping 48‐ and 72‐hr periods showed the changes in the surface structure of the bloom due to advection by surface currents and nonconservative biological and physical processes. Time rates of change of particulate inorganic carbon were estimated along particle trajectories, with rates ranging from −4 to 6 μmol·L−1·day−1. The approach presented here is likely to be useful for understanding the evolution of coastal phytoplankton bloom events in a general setting. Plain Language Summary: This study examines an unprecedented bloom of the coccolithophore Emiliania huxleyi, a single‐celled algae that is covered by calcium carbonate plates, along the California coast during a period of unusually warm water during 2014–2015. We used microscopy and flow cytometry, surface current data, and satellite ocean color imagery to track how the bloom changed through time in the Santa Barbara Channel. Results show a coastal bloom of mostly E. huxleyi that reached cell concentrations up to 5.7 × 106 cells per liter and covered up to 1,220 km2. We speculate that the rare cooccurrence of environmental conditions, including warm water, high water column stability, and an extensive preceding diatom bloom during the anomaly, contributed to the development of this bloom. Flow cytometry measurements provided insight on the phases of bloom development (e.g., growth versus senescence) and hourly surface current data to simulate how currents may have moved the coccolithophores during the course of the bloom. In addition, we combined this data with satellite imagery to estimate how the amount of calcium carbonate in this bloom changed through time. The approach presented here is likely to be useful for understanding the rise and fall of phytoplankton blooms along the coast. Key Points: Unprecedented coastal bloom of the coccolithophore Emiliania huxleyi during 2014/2015 warming anomaly occurring along the California coastLagrangian particle trajectories showed changes in bloom structure due to advection by surface currents and nonconservative processesBloom phases identified based on particle trajectories and satellite‐derived estimates of change in particulate inorganic carbon [ABSTRACT FROM AUTHOR]

The Southern Ocean, a region that will be an ocean acidification hotspot in the near future, is home to a uniquely adapted fauna that includes a diversity of lightly-calcified invertebrates. We exposed the larvae of the echinoid Sterechinus neumayeri to environmental levels of CO2 in McMurdo Sound (control: 410 µatm, ω= 1.35) and mildly elevated pCO2 levels, both near the level of the aragonite saturation horizon (510 matm pCO2, V= 1.12), and to under-saturating conditions (730 µatm, ω= 0.82). Early embryological development was normal under these conditions with the exception of the hatching process, which was slightly delayed. Appearance of the initial calcium carbonate (CaCO3) spicule nuclei among the primary mesenchyme cells of the gastrulae was synchronous between control and elevated pCO2 treatments. However, by prism (7 days after the initial appearance of the spicule nucleus), elongating arm rod spicules were already significantly shorter in the highest CO2 treatment. Unfed larvae in the 730 matm pCO2 treatment remained significantly smaller than unfed control larvae at days 15-30, and larvae in the 510 matm treatment were significantly smaller at day 20. At day 30, the arm lengths were more differentiated between 730 matm and control CO2 treatments than were body lengths as components of total length. Arm length is the most plastic morphological aspect of the echinopluteus, and appears to exhibit the greatest response to high pCO2/low pH/low carbonate, even in the absence of food. Thus, while the effects of elevated pCO2 representative of near future climate scenarios are proportionally minor on these early developmental stages, the longer term effects on these long-lived invertebrates is still unknown. [ABSTRACT FROM AUTHOR]

Ocean acidification (OA) is expected to have a major impact on marine species, particularly during early life-history stages. These effects appear to be species-specific and may include reduced survival, altered morphology, and depressed metabolism. However, less information is available regarding the bioenergetics of development under elevated CO2 conditions. We examined the biochemical and morphological responses of Strongylocentrotus purpuratus during early development under ecologically relevant levels of pCO2 (365, 1030, and 1450 µatm) that may occur during intense upwelling events. The principal findings of this study were (1) lipid utilization rates and protein content in S. purpuratus did not vary with pCO2; (2) larval growth was reduced at elevated pCO2 despite similar rates of energy utilization; and (3) relationships between egg phospholipid content and larval length were found under control but not high pCO2 conditions. These results suggest that this species may either prioritize endogenous energy toward development and physiological function at the expense of growth, or that reduced larval length may be strictly due to higher costs of growth under OA conditions. This study highlights the need to further expand our knowledge of the physiological mechanisms involved in OA response in order to better understand how present populations may respond to global environmental change. [ABSTRACT FROM AUTHOR]

The Southern Ocean, a region that will be an ocean acidification hotspot in the near future, is home to a uniquely adapted fauna that includes a diversity of lightly-calcified invertebrates. We exposed the larvae of the echinoid Sterechinus neumayeri to environmental levels of CO2 in McMurdo Sound (control: 410 µatm, ω= 1.35) and mildly elevated pCO2 levels, both near the level of the aragonite saturation horizon (510 matm pCO2, V= 1.12), and to under-saturating conditions (730 µatm, ω= 0.82). Early embryological development was normal under these conditions with the exception of the hatching process, which was slightly delayed. Appearance of the initial calcium carbonate (CaCO3) spicule nuclei among the primary mesenchyme cells of the gastrulae was synchronous between control and elevated pCO2 treatments. However, by prism (7 days after the initial appearance of the spicule nucleus), elongating arm rod spicules were already significantly shorter in the highest CO2 treatment. Unfed larvae in the 730 matm pCO2 treatment remained significantly smaller than unfed control larvae at days 15-30, and larvae in the 510 matm treatment were significantly smaller at day 20. At day 30, the arm lengths were more differentiated between 730 matm and control CO2 treatments than were body lengths as components of total length. Arm length is the most plastic morphological aspect of the echinopluteus, and appears to exhibit the greatest response to high pCO2/low pH/low carbonate, even in the absence of food. Thus, while the effects of elevated pCO2 representative of near future climate scenarios are proportionally minor on these early developmental stages, the longer term effects on these long-lived invertebrates is still unknown. [ABSTRACT FROM AUTHOR]