Genus-Wide Transcriptional Landscapes Reveal Correlated Gene Networks Underlying Microevolutionary Divergence in Diatoms.

Marine microbes like diatoms make up the base of marine food webs and drive global nutrient cycles. Despite their key roles in ecology, biogeochemistry, and biotechnology, we have limited empirical data on how forces other than adaptation may drive diatom diversification, especially in the absence of environmental change. One key feature of diatom populations is frequent extreme reductions in population size, which can occur both in situ and ex situ as part of bloom-and-bust growth dynamics. This can drive divergence between closely related lineages, even in the absence of environmental differences. Here, we combine experimental evolution and transcriptome landscapes (t-scapes) to reveal repeated evolutionary divergence within several species of diatoms in a constant environment. We show that most of the transcriptional divergence can be captured on a reduced set of axes, and that repeatable evolution can occur along a single major axis of variation defined by core ortholog expression comprising common metabolic pathways. Previous work has associated specific transcriptional changes in gene networks with environmental factors. Here, we find that these same gene networks diverge in the absence of environmental change, suggesting these pathways may be central in generating phenotypic diversity as a result of both selective and random evolutionary forces. If this is the case, these genes and the functions they encode may represent universal axes of variation. Such axes that capture suites of interacting transcriptional changes during diversification improve our understanding of both global patterns in local adaptation and microdiversity, as well as evolutionary forces shaping algal cultivation.

Multi-trait diversification in marine diatoms in constant and warmed environments.

Phytoplankton are photosynthetic marine microbes that affect food webs, nutrient cycles and climate regulation. Their roles are determined by correlated phytoplankton functional traits including cell size, chlorophyll content and cellular composition. Here, we explore patterns of evolution in interrelated trait values and correlations. Because both chance events and natural selection contribute to phytoplankton trait evolution, we used population bottlenecks to diversify six genotypes of Thalassiosirid diatoms. We then evolved them as large populations in two environments. Interspecific variation and within-species evolution were visualized for nine traits and their correlations using reduced axes (a trait-scape). Our main findings are that shifts in trait values resulted in movement of evolving populations within the trait-scape in both environments, but were more frequent when large populations evolved in a novel environment. Which trait relationships evolved was population-specific, but greater departures from ancestral trait correlations were associated with lower population growth rates. There was no single master trait that could be used to understand multi-trait evolution. Instead, repeatable multi-trait evolution occurred along a major axis of variation defined by several diatom traits and trait relationships. Because trait-scapes capture changes in trait relationships and values together, they offer an insightful way to study multi-trait variation.

Metabolic response to a heterologous poly-3-hydroxybutyrate (PHB) pathway in Phaeodactylum tricornutum.

The marine diatom Phaeodactylum tricornutum is an emerging host for metabolic engineering, but little is known about how introduced pathways are integrated into the existing metabolic framework of the host or influence transgene expression. In this study, we expressed the heterologous poly-3-hydroxybutyrate (PHB) pathway using episomal expression, which draws on the precursor acetyl coenzyme-A (AcCoA). By experimentally perturbing cultivation conditions, we gained insight into the regulation of the endogenous metabolism in transgenic lines under various environmental scenarios, as well as on alterations in AcCoA flux within the host cell. Biosynthesis of PHB led to distinct shifts in the metabolome of the host, and further analysis revealed a condition-dependent relationship between endogenous and transgenic metabolic pathways. Under N limitation, which induced a significant increase in neutral lipid content, both metabolic and transcriptomic data suggest that AcCoA was preferably shunted into the endogenous pathway for lipid biosynthesis over the transgenic PHB pathway. In contrast, supply of organic carbon in the form of glycerol supported both fatty acid and PHB biosynthesis, suggesting cross-talk between cytosolic and plastidial AcCoA precursors. This is the first study to investigate the transcriptomic and metabolomic response of diatom cell lines expressing a heterologous multi-gene pathway under different environmental conditions, providing useful insights for future engineering attempts for pathways based on the precursor AcCoA. KEY POINTS: • PHB expression had minimal effects on transcription of adjacent pathways. • N limitation favoured native lipid rather than transgenic PHB synthesis. • Glycerol addition allowed simultaneous lipid and PHB accumulation.

Defining marine bacterioplankton community assembly rules by contrasting the importance of environmental determinants and biotic interactions.

Bacterioplankton communities govern marine productivity and biogeochemical cycling, yet drivers of bacterioplankton assembly remain unclear. Here, we contrast the relative contribution of deterministic processes (environmental factors and biotic interactions) in driving temporal dynamics of bacterioplankton diversity at three different oceanographic time series locations, spanning 15° of latitude, which are each characterized by different environmental conditions and varying degrees of seasonality. Monthly surface samples (5.5 years) were analysed using 16S rRNA amplicon sequencing. The high- and mid-latitude sites of Maria Island and Port Hacking were characterized by high and intermediate levels of environmental heterogeneity, respectively, with both alpha diversity (72%; 24% of total variation) and beta diversity (32%; 30%) patterns within bacterioplankton assemblages explained by day length, ammonium, and mixed layer depth. In contrast, North Stradbroke Island, a sub-tropical location where environmental conditions are less variable, interspecific interactions were of increased importance in structuring bacterioplankton diversity (alpha: 33%; beta: 26%) with environment only contributing 11% and 13% to predicting diversity, respectively. Our results demonstrate that bacterioplankton diversity is the result of both deterministic environmental and biotic processes and that the importance of these different deterministic processes varies, potential in response to environmental heterogeneity.

Predictability of thermal fluctuations influences functional traits of a cosmopolitan marine diatom.

Evolutionary theory predicts that organismal plasticity should evolve in environments that fluctuate regularly. However, in environments that fluctuate less predictably, plasticity may be constrained because environmental cues become less reliable for expressing the optimum phenotype. Here, we examine how the predictability of +5°C temperature fluctuations impacts the phenotype of the marine diatom Thalassiosira pseudonana. Thermal regimes were informed by temperatures experienced by microbes in an ocean simulation and featured regular or irregular temporal sequences of fluctuations that induced mild physiological stress. Physiological traits (growth, cell size, complexity and pigmentation) were quantified at the individual cell level using flow cytometry. Changes in cellular complexity emerged as the first impact of predictability after only 8-11 days, followed by deleterious impacts on growth on days 13-16. Specifically, cells with a history of irregular fluctuation exposure exhibited a 50% reduction in growth compared with the stable reference environment, while growth was 3-18 times higher when fluctuations were regular. We observed no evidence of heat hardening (increasingly positive growth) with recurrent fluctuations. This study demonstrates that unpredictable temperature fluctuations impact this cosmopolitan diatom under ecologically relevant time frames, suggesting shifts in environmental stochasticity under a changing climate could have widespread consequences among ocean primary producers.

Temperature variability interacts with mean temperature to influence the predictability of microbial phenotypes.

Despite their relatively high thermal optima (Topt ), tropical taxa may be particularly vulnerable to a rising baseline and increased temperature variation because they live in relatively stable temperatures closer to their Topt . We examined how microbial eukaryotes with differing thermal histories responded to temperature fluctuations of different amplitudes (0 control, ±2, ±4°C) around mean temperatures below or above their Topt . Cosmopolitan dinoflagellates were selected based on their distinct thermal traits and included two species of the same genus (tropical and temperate Coolia spp.), and two strains of the same species maintained at different temperatures for >500 generations (tropical Amphidinium massartii control temperature and high temperature, CT and HT, respectively). There was a universal decline in population growth rate under temperature fluctuations, but strains with narrower thermal niche breadth (temperate Coolia and HT) showed ~10% greater reduction in growth. At suboptimal mean temperatures, cells in the cool phase of the fluctuation stopped dividing, fixed less carbon (C) and had enlarged cell volumes that scaled positively with elemental C, N, and P and C:Chlorophyll-a. However, at a supra-optimal mean temperature, fixed C was directed away from cell division and novel trait combinations developed, leading to greater phenotypic diversity. At the molecular level, heat-shock proteins, and chaperones, in addition to transcripts involving genome rearrangements, were upregulated in CT and HT during the warm phase of the supra-optimal fluctuation (30 ± 4°C), a stress response indicating protection. In contrast, the tropical Coolia species upregulated major energy pathways in the warm phase of its supra-optimal fluctuation (25 ± 4°C), indicating a broadscale shift in metabolism. Our results demonstrate divergent effects between taxa and that temporal variability in environmental conditions interacts with changes in the thermal mean to mediate microbial responses to global change, with implications for biogeochemical cycling.

The evolution of trait correlations constrains phenotypic adaptation to high CO2 in a eukaryotic alga.

Microbes form the base of food webs and drive biogeochemical cycling. Predicting the effects of microbial evolution on global elemental cycles remains a significant challenge due to the sheer number of interacting environmental and trait combinations. Here, we present an approach for integrating multivariate trait data into a predictive model of trait evolution. We investigated the outcome of thousands of possible adaptive walks parameterized using empirical evolution data from the alga Chlamydomonas exposed to high CO2. We found that the direction of historical bias (existing trait correlations) influenced both the rate of adaptation and the evolved phenotypes (trait combinations). Critically, we use fitness landscapes derived directly from empirical trait values to capture known evolutionary phenomena. This work demonstrates that ecological models need to represent both changes in traits and changes in the correlation between traits in order to accurately capture phytoplankton evolution and predict future shifts in elemental cycling.

Taxonomic Variability in the Electron Requirement for Carbon Fixation Across Marine Phytoplankton.

Fast Repetition Rate fluorometry (FRRf) has been increasingly used to measure marine primary productivity by oceanographers to understand how carbon (C) uptake patterns vary over space and time in the global ocean. As FRRf measures electron transport rates through photosystem II (ETRPSII ), a critical, but difficult to predict conversion factor termed the "electron requirement for carbon fixation" (Φe,C ) is needed to scale ETRPSII to C-fixation rates. Recent studies have generally focused on understanding environmental regulation of Φe,C , while taxonomic control has been explored by only a handful of laboratory studies encompassing a limited diversity of phytoplankton species. We therefore assessed Φe,C for a wide range of marine phytoplankton (n = 17 strains) spanning multiple taxonomic and size classes. Data mined from previous studies were further considered to determine whether Φe,C variability could be explained by taxonomy versus other phenotypic traits influencing growth and physiological performance (e.g., cell size). We found that Φe,C exhibited considerable variability (~4-10 mol e-  · [mol C]-1 ) and was negatively correlated with growth rate (R2  = 0.7, P < 0.01). Diatoms exhibited a lower Φe,C compared to chlorophytes during steady-state, nutrient-replete growth. Inclusion of meta-analysis data did not find significant relationships between Φe,C and class, or growth rate, although confounding factors inherent to methodological inconsistencies between studies likely contributed to this. Knowledge of empirical relationships between Φe,C and growth rate coupled with recent improvements in quantifying phytoplankton growth rates in situ, facilitate up-scaling of FRRf campaigns to routinely derive Φe,C needed to assess ocean C-cycling.

Microbial tropicalization driven by a strengthening western ocean boundary current.

Western boundary currents (WBCs) redistribute heat and oligotrophic seawater from the tropics to temperate latitudes, with several displaying substantial climate change-driven intensification over the last century. Strengthening WBCs have been implicated in the poleward range expansion of marine macroflora and fauna, however, the impacts on the structure and function of temperate microbial communities are largely unknown. Here we show that the major subtropical WBC of the South Pacific Ocean, the East Australian Current (EAC), transports microbial assemblages that maintain tropical and oligotrophic (k-strategist) signatures, to seasonally displace more copiotrophic (r-strategist) temperate microbial populations within temperate latitudes of the Tasman Sea. We identified specific characteristics of EAC microbial assemblages compared with non-EAC assemblages, including strain transitions within the SAR11 clade, enrichment of Prochlorococcus, predicted smaller genome sizes and shifts in the importance of several functional genes, including those associated with cyanobacterial photosynthesis, secondary metabolism and fatty acid and lipid transport. At a temperate time-series site in the Tasman Sea, we observed significant reductions in standing stocks of total carbon and chlorophyll a, and a shift towards smaller phytoplankton and carnivorous copepods, associated with the seasonal impact of the EAC microbial assemblage. In light of the substantial shifts in microbial assemblage structure and function associated with the EAC, we conclude that climate-driven expansions of WBCs will expand the range of tropical oligotrophic microbes, and potentially profoundly impact the trophic status of temperate waters.

First description of the environmental niche of the epibenthic dinoflagellate species Coolia palmyrensis, C. malayensis, and C. tropicalis (Dinophyceae) from Eastern Australia.

Environmental variables such as temperature, salinity, and irradiance are significant drivers of microalgal growth and distribution. Therefore, understanding how these variables influence fitness of potentially toxic microalgal species is particularly important. In this study, strains of the potentially harmful epibenthic dinoflagellate species Coolia palmyrensis, C. malayensis, and C. tropicalis were isolated from coastal shallow water habitats on the east coast of Australia and identified using the D1-D3 region of the large subunit (LSU) ribosomal DNA (rDNA). To determine the environmental niche of each taxon, growth was measured across a gradient of temperature (15-30°C), salinity (20-38), and irradiance (10-200 µmol photons · m-2  · s-1 ). Specific growth rates of Coolia tropicalis were highest under warm temperatures (27°C), low salinities (ca. 23), and intermediate irradiance levels (150 µmol photons · m-2  · s-1 ), while C. malayensis showed the highest growth at moderate temperatures (24°C) and irradiance levels (150 µmol photons · m-2  · s-1 ) and growth rates were consistent across the range of salinity levels tested (20-38). Coolia palmyrensis had the highest growth rate of all species tested and favored moderate temperatures (24°C), oceanic salinity (35), and high irradiance (>200 µmol photons · m-2  · s-1 ). This is the first study to characterize the environmental niche of species from the benthic harmful algal bloom genus Coolia and provides important information to help define species distributions and inform risk management.

Drift in ocean currents impacts intergenerational microbial exposure to temperature.

Microbes are the foundation of marine ecosystems [Falkowski PG, Fenchel T, Delong EF (2008) Science 320(5879):1034-1039]. Until now, the analytical framework for understanding the implications of ocean warming on microbes has not considered thermal exposure during transport in dynamic seascapes, implying that our current view of change for these critical organisms may be inaccurate. Here we show that upper-ocean microbes experience along-trajectory temperature variability up to 10 °C greater than seasonal fluctuations estimated in a static frame, and that this variability depends strongly on location. These findings demonstrate that drift in ocean currents can increase the thermal exposure of microbes and suggests that microbial populations with broad thermal tolerance will survive transport to distant regions of the ocean and invade new habitats. Our findings also suggest that advection has the capacity to influence microbial community assemblies, such that regions with strong currents and large thermal fluctuations select for communities with greatest plasticity and evolvability, and communities with narrow thermal performance are found where ocean currents are weak or along-trajectory temperature variation is low. Given that fluctuating environments select for individual plasticity in microbial lineages, and that physiological plasticity of ancestors can predict the magnitude of evolutionary responses of subsequent generations to environmental change [Schaum CE, Collins S (2014) Proc Biol Soc 281(1793):20141486], our findings suggest that microbial populations in the sub-Antarctic (∼40°S), North Pacific, and North Atlantic will have the most capacity to adapt to contemporary ocean warming.

Roadmaps and Detours: Active Chlorophyll- a Assessments of Primary Productivity Across Marine and Freshwater Systems.

Assessing phytoplankton productivity over space and time remains a core goal for oceanographers and limnologists. Fast Repetition Rate fluorometry (FRRf) provides a potential means to realize this goal with unprecedented resolution and scale yet has not become the "go-to" method despite high expectations. A major obstacle is difficulty converting electron transfer rates to equivalent rates of C-fixation most relevant for studies of biogeochemical C-fluxes. Such difficulty stems from methodological inconsistencies and our limited understanding of how the electron requirement for C-fixation (Φe,C) is influenced by the environment and by differences in the composition and physiology of phytoplankton assemblages. We outline a "roadmap" for limiting methodological bias and to develop a more mechanistic understanding of the ecophysiology underlying Φe,C. We 1) re-evaluate core physiological processes governing how microalgae invest photosynthetic electron transport-derived energy and reductant into stored carbon versus alternative sinks. Then, we 2) outline steps to facilitate broader uptake and exploitation of FRRf, which could transform our knowledge of aquatic primary productivity. We argue it is time to 3) revise our historic methodological focus on carbon as the currency of choice, to 4) better appreciate that electron transport fundamentally drives ecosystem biogeochemistry, modulates cell-to-cell interactions, and ultimately modifies community biomass and structure.

Thermal niche evolution of functional traits in a tropical marine phototroph.

Land-based plants and ocean-dwelling microbial phototrophs known as phytoplankton, are together responsible for almost all global primary production. Habitat warming associated with anthropogenic climate change has detrimentally impacted marine primary production, with the effects observed on regional and global scales. In contrast to slower-growing higher plants, there is considerable potential for phytoplankton to evolve rapidly with changing environmental conditions. The energetic constraints associated with adaptation in phytoplankton are not yet understood, but are central to forecasting how global biogeochemical cycles respond to contemporary ocean change. Here, we demonstrate a number of potential trade-offs associated with high-temperature adaptation in a tropical microbial eukaryote, Amphidinium massartii (dinoflagellate). Most notably, the population became high-temperature specialized (higher fitness within a narrower thermal envelope and higher thermal optimum), and had a greater nutrient requirement for carbon, nitrogen and phosphorus. Evidently, the energetic constraints associated with living at elevated temperature alter competiveness along other environmental gradients. While high-temperature adaptation led to an irreversible change in biochemical composition (i.e., an increase in fatty acid saturation), the mechanisms underpinning thermal evolution in phytoplankton remain unclear, and will be crucial to understanding whether the trade-offs observed here are species-specific or are representative of the evolutionary constraints in all phytoplankton.

Toxicology of Gambierdiscus spp. (Dinophyceae) from Tropical and Temperate Australian Waters.

Ciguatera Fish Poisoning (CFP) is a human illness caused by the consumption of marine fish contaminated with ciguatoxins (CTX) and possibly maitotoxins (MTX), produced by species from the benthic dinoflagellate genus Gambierdiscus. Here, we describe the identity and toxicology of Gambierdiscus spp. isolated from the tropical and temperate waters of eastern Australia. Based on newly cultured strains, we found that four Gambierdiscus species were present at the tropical location, including G. carpenteri, G. lapillus and two others which were not genetically identical to other currently described species within the genus, and may represent new species. Only G. carpenteri was identified from the temperate location. Using LC-MS/MS analysis we did not find any characterized microalgal CTXs (P-CTX-3B, P-CTX-3C, P-CTX-4A and P-CTX-4B) or MTX-1; however, putative maitotoxin-3 (MTX-3) was detected in all species except for the temperate population of G. carpenteri. Using the Ca2+ influx SH-SY5Y cell Fluorescent Imaging Plate Reader (FLIPR) bioassay we found CTX-like activity in extracts of the unidentified Gambierdiscus strains and trace level activity in strains of G. lapillus. While no detectable CTX-like activity was observed in tropical or temperate strains of G. carpenteri, all species showed strong maitotoxin-like activity. This study, which represents the most comprehensive analyses of the toxicology of Gambierdiscus strains isolated from Australia to date, suggests that CFP in this region may be caused by currently undescribed ciguatoxins and maitotoxins.

Phytoplankton Functional Groups Variation and Influencing Factors in a Shallow Temperate Lake.

The present study was carried out in Luoma Lake, a shallow lake in temperate eastern China. Based on a two-year study, the dynamics of phytoplankton functional groups and influencing factors were analyzed. A total of 178 taxa were identified and sorted into 20 codons, according to the phytoplankton functional group classification. In order to find the environmental factors driving phytoplankton variations, fifteen groups were analyzed in detail using redundancy analysis. Groups P (Fragilaria crotonensis), X2 (Chlamydomonas globosa, C. microsphaera and Chroomonas acuta), and MP (Navicula rotaeana) were dominant during low temperature periods, whereas groups X2, S1 (Pseudanabaena limnetica), and W1 (Euglena sp.) were dominant during high temperature periods. Water temperature, total phosphorus, and ammonium were the significant driving factors explaining phytoplankton succession. Furthermore, total phosphorus and ammonium could be broadly used in risk management for potential algal blooms in Luoma Lake.

Effect of copper on multiple successional stages of a marine fouling assemblage.

Copper based paints are used to prevent fouling on the hulls of ships. The widely documented effect of copper on hull assemblages may be primarily due to direct effects on the invertebrates themselves or indirect effects from copper absorbed into the microbial biofilm before settlement has commenced. Artificial units of habitat were exposed to varied regimes of copper to examine (1) the photosynthetic efficiency and pigments of early-colonising biofilms, and (2) subsequent macroinvertebrate assemblage change in response to the different regimes of copper. Macroinvertebrate assemblages were found to be less sensitive to the direct effects of copper than indirect effects as delivered through biofilms that have been historically exposed to copper, with some species more tolerant than others. This raises further concern for the efficacy of copper as a universal antifoulant on the hulls of ships, which may continue to assist the invasion of copper-tolerant invertebrate species.

Active water transport in unicellular algae: where, why, and how.

The occurrence of active water transport (net transport against a free energy gradient) in photosynthetic organisms has been debated for several decades. Here, active water transport is considered in terms of its roles, where it is found, and the mechanisms by which it could occur. First there is a brief consideration of the possibility of active water transport into plant xylem in the generation of root pressure and the refilling of embolized xylem elements, and from an unsaturated atmosphere into terrestrial organisms living in habitats with limited availability of liquid water. There is then a more detailed consideration of volume and osmotic regulation in wall-less freshwater unicells, and the possibility of generation of buoyancy in marine phytoplankton such as large-celled diatoms. Calculations show that active water transport is a plausible mechanism to assist cells in upwards vertical movements, requires less energy than synthesis of low-density organic solutes, and potentially on a par with excluding certain ions from the vacuole.

Fine particle pollution during megafires contains potentially toxic elements.

Wildfires that raged across Australia during the 2019-2020 'Black Summer' produced an enormous quantity of particulate matter (PM) pollution, with plumes that cloaked many urban centres and ecosystems along the eastern seaboard. This has motivated a need to understand the magnitude and nature of PM exposure, so that its impact on both built and natural environments can be more accurately assessed. Here we present the potentially toxic fingerprint of PM captured by building heating, ventilation, and air conditioning filters in Sydney, Australia during the peak of the Wildfires, and from ambient urban emissions one year later (Reference period). Atmospheric PM and meteorological monitoring data were also assessed to determine the magnitude and source of high PM exposure. The wildfires were a major source of PM pollution in Sydney, exceeding the national standards on 19 % of days between November-February. Wildfire particles were finer and more spherical compared to Reference PM, with count median diameters of 892.1 ± 23.1 versus 1484.8 ± 96.7 nm (mean ± standard error). On an equal-mass basis, differences in potentially toxic elements were predominantly due to higher SO42--S (median 20.4 vs 4.7 mg g-1) and NO3--N (2.4 vs 1.2 mg g-1) in Wildfire PM, and higher PO43--P (10.4 vs 1.4 mg g-1) in Reference PM. Concentrations of remaining elements were similar or lower than Reference PM, except for enrichments to F-, Cl-, dissolved Mn, and particulate Mn, Co and Sb. Fractional solubilities of trace elements were similar or lower than Reference PM, except for enhanced Hg (12.1 vs 1.0 %) and greater variability in Cd, Hg and Mn solubility, which displayed upper quartiles exceeding that of Reference PM. These findings contribute to our understanding of human and ecosystem exposures to the toxic components of mixed smoke plumes, especially in regions downwind of the source.

Response of planktonic microbial assemblages to disturbance in an urban sub-tropical estuary.

Microbes are sensitive indicators of estuarine processes because they respond rapidly to dynamic disturbance events. As most of the world's population lives in urban areas and climate change-related disturbance events are becoming more frequent, estuaries bounded by cities are experiencing increasing stressors, at the same time that their ecosystem services are required more than ever. Here, using a multidisciplinary approach, we determined the response of planktonic microbial assemblages in response to seasonality and a rainfall disturbance in an urban estuary bounded by Australia's largest city, Sydney. We used molecular barcoding (16S, 18S V4 rRNA) and microscopy-based identification to compare microbial assemblages at locations with differing characteristics and urbanisation histories. Across 142 samples, we identified 8,496 unique free-living bacterial zOTUs, 8,175 unique particle associated bacterial zOTUs, and 1,920 unique microbial eukaryotic zOTUs. Using microscopy, we identified only the top <10% abundant, larger eukaryotic taxa (>10 µm), however quantification was possible. The site with the greater history of anthropogenic impact showed a more even community of associated bacteria and eukaryotes, and a significant increase in dissolved inorganic nitrogen following rainfall, when compared to the more buffered site. This coincided with a reduced proportional abundance of Actinomarina and Synechococcus spp., a change in SAR 11 clades, and an increase in the eukaryotic microbial groups Dinophyceae, Mediophyceae and Bathyoccocaceae, including a temporary dominance of the harmful algal bloom dinoflagellate Prorocentrum cordatum (syn. P. minimum). Finally, a validated hydrodynamic model of the estuary supported these results, showing that the more highly urbanised and upstream location consistently experienced a higher magnitude of salinity reduction in response to rainfall events during the study period. The best abiotic variables to explain community dissimilarities between locations were TDP, PN, modelled temperature and salinity (r = 0.73) for the free living bacteria, TP for the associated bacteria (r = 0.43), and modelled temperature (r = 0.28) for the microbial eukaryotic communities. Overall, these results show that a minor disturbance such as a brief rainfall event can significantly shift the microbial assemblage of an anthropogenically impacted area within an urban estuary to a greater degree than a seasonal change, but may result in a lesser response to the same disturbance at a buffered, more oceanic influenced location. Fine scale research into the factors driving the response of microbial communities in urban estuaries to climate related disturbances will be necessary to understand and implement changes to maintain future estuarine ecosystem services.

Mucospheres produced by a mixotrophic protist impact ocean carbon cycling.

Mixotrophic protists (unicellular eukaryotes) that engage in both phototrophy (photosynthesis) and phago-heterotrophy (engulfment of particles)-are predicted to contribute substantially to energy fluxes and marine biogeochemical cycles. However, their impact remains largely unquantified. Here we describe the sophisticated foraging strategy of a widespread mixotrophic dinoflagellate, involving the production of carbon-rich 'mucospheres' that attract, capture, and immobilise microbial prey facilitating their consumption. We provide a detailed characterisation of this previously undescribed behaviour and reveal that it represents an overlooked, yet quantitatively significant mechanism for oceanic carbon fluxes. Following feeding, the mucospheres laden with surplus prey are discarded and sink, contributing an estimated 0.17-1.24 mg m-2 d-1 of particulate organic carbon, or 0.02-0.15 Gt to the biological pump annually, which represents 0.1-0.7% of the estimated total export from the euphotic zone. These findings demonstrate how the complex foraging behaviour of a single species of mixotrophic protist can disproportionally contribute to the vertical flux of carbon in the ocean.