Influence of Ocean Acidification on a Natural Winter-to-Summer Plankton Succession : First Insights from a Long-Term Mesocosm Study Draw Attention to Periods of Low Nutrient Concentrations

Every year, the oceans absorb about 30% of anthropogenic carbon dioxide (CO2) leading to a re-equilibration of the marine carbonate system and decreasing seawater pH. Today, there is increasing awareness that these changes-summarized by the term ocean acidification (OA)-could differentially affect the competitive ability of marine organisms, thereby provoking a restructuring of marine ecosystems and biogeochemical element cycles. In winter 2013, we deployed ten pelagic mesocosms in the Gullmar Fjord at the Swedish west coast in order to study the effect of OA on plankton ecology and biogeochemistry under close to natural conditions. Five of the ten mesocosms were left unperturbed and served as controls (similar to 380 mu atm pCO(2)), whereas the others were enriched with CO2-saturated water to simulate realistic end-of-the-century carbonate chemistry conditions (mu 760 mu atm pCO(2)). We ran the experiment for 113 days which allowed us to study the influence of high CO2 on an entire winter-to-summer plankton succession and to investigate the potential of some plankton organisms for evolutionary adaptation to OA in their natural environment. This paper is the first in a PLOS collection and provides a detailed overview on the experimental design, important events, and the key complexities of such a "long-term mesocosm" approach. Furthermore, we analyzed whether simulated end-of-the-century carbonate chemistry conditions could lead to a significant restructuring of the plankton community in the course of the succession. At the level of detail analyzed in this overview paper we found that CO2-induced differences in plankton community composition were non-detectable during most of the succession except for a period where a phytoplankton bloom was fueled by remineralized nutrients. These results indicate: (1) Long-term studies with pelagic ecosystems are necessary to uncover OA-sensitive stages of succession. (2) Plankton communities fueled by regenerated nutrients may be more responsive to changing carbonate chemistry than those having access to high inorganic nutrient concentrations and may deserve particular attention in future studies.Peer reviewe

Significance of N2 fixing planktonic symbioses for open ocean ecosystems

Di-nitrogen (N2) fixers, also called diazotrophs, are able to reduce atmospheric N2 into bioavailable nitrogen, giving them an advantage in open ocean regions with low dissolved inorganic nitrogen concentrations. The focus of this thesis are three lineages of symbiotic heterocystous filamentous types (het-1, het-2 and het-3), that associate with several genera of microalgae called diatoms (collectively referred to as Diatom Diazotroph Associations, DDAs). Other major cyanobacterial diazotrophs in the ocean are the filamentous Trichodesmium spp., and the unicellular UCYN-A, UCYN-B and UCYN-C. Although widespread in the tropics and subtropics, and first described in the early 20th century, the DDAs are an understudied group of diazotrophs. Hence, our knowledge of their distribution, abundance, activity, and how these are constrained by the environment is limited. Initially we investigated the abundances and distributions of eight cyanobacterial diazotrophs, and two proposed micro-algal hosts of UCYN-A1 and A2, in the western tropical south Pacific (WTSP), using quantitative polymerase chain reaction (qPCR). Trichodesmium spp. was the most abundant diazotroph and het-1 was the most abundant DDA symbiont. Using correlation analysis a distinct vertical separation was observed between UCYN-A and the other diazotrophs (Trichodesmium spp., UCYN-B and DDA symbionts). The most influential environmental parameter on the diazotroph abundances in the WTSP was temperature, and in order to investigate this further we compiled qPCR data from 11 publicly available datasets from four ocean basins. Using a weighted meta-analysis we found that temperature was a robust factor governing the diazotroph abundances (except for UCYN-A) across ocean basins. Attempting to identify differences in environmental impacts on two of the DDA symbiont strains (het-1 and het-2), we applied a new statistical tool called piecewise structural equation model, on qPCR abundance data from the western tropical North Atlantic. We saw that the two strains had a direct positive interaction between each other, but two parameters (salinity and dissolved inorganic phosphorous) differed. Based on a direct positive effect of salinity on het-1, and an indirect negative effect of salinity on het-2, we concluded that het-2 prefers intermediate salinities (30-35 PSU), which is consistent with where observations of het-2 blooms have been made. Although DDA and UCYN-A symbionts both are major contributors of new N, and are symbiotic, they have several unique differences. The host partners differ in phylogeny (diatom vs prymnesiophyte), size (80-250 vs 7-10 µm) and the symbiotic life history (colonial vs solitary). The larger, colonial nature of DDAs make them difficult to collect, and hence they are often under-sampled and undetected. In fact, after reviewing 46 qPCR studies we found that &lt; 30% of the studies (13 out of 46) quantified all three DDA symbionts, compared to UCYN-A (96%, 44 out of 46). In order to study the DDA symbiont gene expressions we developed a highly specific DDA symbiont microarray (748 probes), which was applied on samples collected in the South China Sea. Although the gene expression levels were highly variable, we observed an upregulation of the nifH gene (for N2 fixation) in the night. Investigating environmental impact on overall gene expression levels, we found that fluorescence, temperature and salinity was most important. Temperature and salinity also constrained abundances, but fluorescence could be seen as a proxy for either other phytoplankton or light availability, suggesting that daylight and host influence DDA symbiont gene expression levels. The results of this thesis broaden our understanding of the DDAs and how their ambient environment influences them. It has also opened up new possibilities for in depth analysis of these complex environmental impacts. Lastly, it has provided new analysis tools for further development on how the symbionts and hosts potentially impact each other’s activities.At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p

A Short Comparison of Two Marine Planktonic Diazotrophic Symbioses Highlights an Un-quantified Disparity

Some N2-fixing cyanobacteria form symbiosis with diverse protists. In the plankton two groups of diazotrophic symbioses are described: (1) a collective group of diatoms which associate with heterocystous cyanobacteria (Diatom Diazotroph Associations, DDA), and (2) the microalgal prymnesiophyte Braarudosphaera bigelowii and its relatives which associate with the unicellular cyanobacterium Candidatus Atelocyanobacterium thalassa (hereafter as UCYN-A). Both symbiotic systems co-occur, and in both partnerships the symbionts function as a nitrogen (N) source. In this perspective, we provide a brief comparison between the DDAs and the prymnesiophyte-UCYN-A symbioses highlighting similarities and differences in both systems, and present a bias in the attention and current methodology that has led to an under-detection and under-estimation of the DDAs

Abundance of symbiotic and free-living diazotrophic cyanobacteria in the western tropical South Pacific during L'Atalante cruise OUTPACE in 2015

Eight different cyanobacterial diazotrophs (Trichodesmium spp., UCYN-A1, UCYN-A2, UCYN-B, UCYN-C, het-1, het-2 and het-3), and two phytoplankton hosts (UCYN-A1 and A2 hosts), were quantified by their nifH genes (18S rRNA for the hosts) in the western tropical South Pacific in spring 2015. The qPCR data was generated at 18 stations along a west to east transect from New Caledonia to Tahiti, covering oligotrophic to ultra-oligotrophic waters

Piecewise Structural Equation Model (SEM) Disentangles the Environmental Conditions Favoring Diatom Diazotroph Associations (DDAs) in the Western Tropical North Atlantic (WTNA)

Diatom diazotroph associations (DDAs) are important components in the world's oceans, especially in the western tropical north Atlantic (WTNA), where blooms have a significant impact on carbon and nitrogen cycling. However, drivers of their abundances and distribution patterns remain unknown. Here, we examined abundance and distribution patterns for two DDA populations in relation to the Amazon River (AR) plume in the WTNA. Quantitative PCR assays, targeting two DDAs (het-1 and het-2) by their symbiont's nifH gene, served as input in a piecewise structural equation model (SEM). Collections were made during high (spring 2010) and low (fall 2011) flow discharges of the AR. The distributions of dissolved nutrients, chlorophyll-a, and DDAs showed coherent patterns indicative of areas influenced by the AR. A symbiotic Hemiaulus hauckii-Richelia (het-2) bloom (> 10(6) cells L-1) occurred during higher discharge of the AR and was coincident with mesohaline to oceanic (30-35) sea surface salinities (SSS), and regions devoid of dissolved inorganic nitrogen (DIN), low concentrations of both DIP (> 0.1 mu mol L-1) and Si (> 1.0 mu mol L-1). The Richelia (het-1) associated with Rhizosolenia was only present in 2010 and at lower densities (10-1.76 x 10(5) nifH copies L-1) than het-2 and limited to regions of oceanic SSS (> 36). The het-2 symbiont detected in 2011 was associated with H. membranaceus (> 10(3) nifH copies L-1) and were restricted to regions with mesohaline SSS (31.8-34.3), immeasurable DIN, moderate DIP (0.1-0.60 mu mol L-1) and higher Si (4.19-22.1 mu mol L-1). The piecewise SEM identified a profound direct negative effect of turbidity on the het-2 abundance in spring 2010, while DIP and water turbidity had a more positive influence in fall 2011, corroborating our observations of DDAs at subsurface maximas. We also found a striking difference in the influence of salinity on DDA symbionts suggesting a niche differentiation and preferences in oceanic and mesohaline salinities by het-1 and het-2, respectively. The use of the piecewise SEM to disentangle the complex and concomitant hydrography of the WTNA acting on two biogeochemically relevant populations was novel and underscores its use to predict conditions favoring abundance and distributions of microbial populations

In-depth characterization of diazotroph activity across the western tropical South Pacific hotspot of N2 fixation (OUTPACE cruise)

Here we report N2 fixation rates from a  ∼ 4000km transect in the western and central tropical South Pacific, a particularly undersampled region in the world ocean. Water samples were collected in the euphotic layer along a west to east transect from 160°E to 160°W that covered contrasting trophic regimes, from oligotrophy in the Melanesian archipelago (MA) waters to ultra-oligotrophy in the South Pacific Gyre (GY) waters. N2 fixation was detected at all 17 sampled stations with an average depth-integrated rate of 631±286µmol N m−2 d−1 (range 196–1153µmol N m−2 d−1) in MA waters and of 85±79µmol N m−2 d−1 (range 18–172µmol N m−2 d−1) in GY waters. Two cyanobacteria, the larger colonial filamentous Trichodesmium and the smaller UCYN-B, dominated the enumerated diazotroph community ( > 80%) and gene expression of the nifH gene (cDNA > 105 nifH copies L−1) in MA waters. Single-cell isotopic analyses performed by nanoscale secondary ion mass spectrometry (nanoSIMS) at selected stations revealed that Trichodesmium was always the major contributor to N2 fixation in MA waters, accounting for 47.1–83.8% of bulk N2 fixation. The most plausible environmental factors explaining such exceptionally high rates of N2 fixation in MA waters are discussed in detail, emphasizing the role of macro- and micro-nutrient (e.g., iron) availability, seawater temperature and currents

Piecewise Structural Equation Model (SEM) Disentangles the Environmental Conditions Favoring Diatom Diazotroph Associations (DDAs) in the Western Tropical North Atlantic (WTNA)

Diatom diazotroph associations (DDAs) are important components in the world’s oceans, especially in the western tropical north Atlantic (WTNA), where blooms have a significant impact on carbon and nitrogen cycling. However, drivers of their abundances and distribution patterns remain unknown. Here, we examined abundance and distribution patterns for two DDA populations in relation to the Amazon River (AR) plume in the WTNA. Quantitative PCR assays, targeting two DDAs (het-1 and het-2) by their symbiont’s nifH gene, served as input in a piecewise structural equation model (SEM). Collections were made during high (spring 2010) and low (fall 2011) flow discharges of the AR. The distributions of dissolved nutrients, chlorophyll-a, and DDAs showed coherent patterns indicative of areas influenced by the AR. A symbiotic Hemiaulus hauckii-Richelia (het-2) bloom (&gt;106 cells L-1) occurred during higher discharge of the AR and was coincident with mesohaline to oceanic (30–35) sea surface salinities (SSS), and regions devoid of dissolved inorganic nitrogen (DIN), low concentrations of both DIP (&gt;0.1 μmol L-1) and Si (&gt;1.0 μmol L-1). The Richelia (het-1) associated with Rhizosolenia was only present in 2010 and at lower densities (10-1.76 × 105nifH copies L-1) than het-2 and limited to regions of oceanic SSS (&gt;36). The het-2 symbiont detected in 2011 was associated with H. membranaceus (&gt;103nifH copies L-1) and were restricted to regions with mesohaline SSS (31.8–34.3), immeasurable DIN, moderate DIP (0.1–0.60 μmol L-1) and higher Si (4.19–22.1 μmol L-1). The piecewise SEM identified a profound direct negative effect of turbidity on the het-2 abundance in spring 2010, while DIP and water turbidity had a more positive influence in fall 2011, corroborating our observations of DDAs at subsurface maximas. We also found a striking difference in the influence of salinity on DDA symbionts suggesting a niche differentiation and preferences in oceanic and mesohaline salinities by het-1 and het-2, respectively. The use of the piecewise SEM to disentangle the complex and concomitant hydrography of the WTNA acting on two biogeochemically relevant populations was novel and underscores its use to predict conditions favoring abundance and distributions of microbial populations