Maximising environmental pressure-response relationship signals from diatom-based metabarcoding in rivers.

DNA metabarcoding has been performed on a large number of river phytobenthos samples collected from the UK, using rbcL primers optimised for diatoms. Within this dataset the composition of non-diatom sequence reads was studied and the effect of including these in models for evaluating the nutrient gradient was assessed. Whilst many non-diatom taxonomic groups were detected, few contained the full diversity expected in riverine environments. This may be due to the performance of the current primers in characterising the wider phytobenthic community and influenced by the sampling method employed, as both were developed specifically for diatoms. Nevertheless, the study identified considerable diversity in some groups, e.g. Eustigmatophyceae and a wider distribution than previously thought for freshwater Phaeophyceae. These results offer a strong case for the benefits of metabarcoding for expanding knowledge of aquatic biodiversity in the UK and elsewhere. Many of the ASVs associated with non-diatoms showed significant pressure responses; however, models that included non-diatoms had similar predictive strength to those based on diatoms alone. Whilst limitations of the primers for assessing non-diatoms may play a role in explaining these results, the diatoms provide a strong signal along the nutrient gradient and other algae, therefore, add little unique information. We recommend that future developments should use ASVs to calculate metrics, with links to reference databases made as a final step to generate lists of taxa to support interpretation. Any further exploration of the potential of non-diatoms would benefit from access to a well-curated reference database, similar to diat.barcode. Such a database does not yet exist, and we caution against the indiscriminate use of NCBI GenBank as a taxonomic resource as many rbcL sequences deposited have not been curated.

Plastisphere composition in a subtropical estuary: Influence of season, incubation time and polymer type on plastic biofouling.

Plastics are abundant artificial substrates in aquatic systems that host a wide variety of organisms (the plastisphere), including potential pathogens and invasive species. Plastisphere communities have many complex, but not well-understood ecological interactions. It is pivotal to investigate how these communities are influenced by the natural fluctuations in aquatic ecosystems, especially in transitional environments such as estuaries. Further study is needed in subtropical regions in the Southern Hemisphere, where plastic pollution is ever increasing. Here we applied DNA-metabarcoding (16S, 18S and ITS-2) as well Scanning Electron Microscopy (SEM) to assess the diversity of the plastisphere in the Patos Lagoon estuary (PLE), South Brazil. Through a one-year in situ colonization experiment, polyethylene (PE) and polypropylene (PP) plates were placed in shallow waters, and sampled after 30 and 90 days within each season. Over 50 taxa including bacteria, fungi and other eukaryotes were found through DNA analysis. Overall, the polymer type did not influence the plastisphere community composition. However, seasonality significantly affected community composition for bacteria, fungi and general eukaryotes. Among the microbiota, we found Acinetobacter sp., Bacillus sp., and Wallemia mellicola that are putative pathogens of aquatic organisms, such as algae, shrimp and fish, including commercial species. In addition, we identified organisms within genera that can potentially degrade hydrocarbons (e.g. Pseudomonas and Cladosporium spp). This study is the first to assess the full diversity and variation of the plastisphere on different polymers within a subtropical Southern Hemisphere estuary, significantly expanding knowledge on plastic pollution and the plastisphere in estuarine regions.

Monitoring of benthic eukaryotic communities in two tropical coastal lagoons through eDNA metabarcoding: a spatial and temporal approximation.

Tropical coastal lagoons are important ecosystems that support high levels of biodiversity and provide several goods and services. Monitoring of benthic biodiversity and detection of harmful or invasive species is crucial, particularly in relation to seasonal and spatial variation of environmental conditions. In this study, eDNA metabarcoding was used in two tropical coastal lagoons, Chacahua (CH) and Corralero (C) (Southern Mexican Pacific), to describe the benthic biodiversity and its spatial-temporal dynamics. The distribution of benthic diversity within the lagoons showed a very particular pattern evidencing a transition from freshwater to seawater. Although the two lagoon systems are similar in terms of the species composition of metazoans and microeukaryotes, our findings indicate that they are different in taxa richness and structure, resulting in regional partitioning of the diversity with salinity as the driving factor of community composition in CH. Harmful, invasive, non-indigenous species, bioindicators and species of commercial importance were detected, demonstrating the reach of this technique for biodiversity monitoring along with the continued efforts of building species reference libraries.

Floating plastics and their associated biota in the Western South Atlantic.

The lack of information about plastic pollution in many marine regions hinders firm actions to manage human activities and mitigate their impacts. This study conducted for the first time a quali-quantitative evaluation of floating plastics and their associated biota from coastal and oceanic waters in South Brazil. Plastics were collected using a manta net, and were categorized according to their shape, size, malleability and polymer composition. Multi-marker DNA metabarcoding (16S, and 18S V4 and V9 rRNA regions) was performed to identify prokaryotes and eukaryotes associated to plastics. We found 371 likely plastic particles of several sizes, shapes and polymers, and the average concentration of plastics at the region was 4461 items.km-2 (SD ± 3914). Microplastics (0.5 - 5 mm) were dominant in most sampling stations, with fragments and lines representing the most common shapes. Diverse groups of prokaryotes (20 bacteria phyla) and eukaryotes (41 groups) were associated with plastics. Both the community composition and richness of epiplastic organisms were highly variable between individual plastics but, in general, were not influenced by plastic categories. Organisms with potential pathogenicity (e.g. Vibrio species. and Alexandrium tamarense), as well as potential plastic degraders (e.g. Ralstonia, Pseudomonas, and Alcanivorax species), were found. The information generated here is pivotal to support strategies to prevent the input and mitigate the impacts of plastics and their associated organisms on marine environments.

Parasites, pathogens, and other symbionts of copepods.

There is a large diversity of eukaryotic symbionts of copepods, dominated by epizootic protists such as ciliates, and metazoan parasites. Eukaryotic endoparasites, copepod-associated bacteria, and viruses are less well known, partly due to technical limitations. However, new molecular techniques, combined with a range of other approaches, provide a complementary toolkit for understanding the complete symbiome of copepods and how the symbiome relates to their ecological roles, relationships with other biota, and responses to environmental change. In this review we provide the most complete overview of the copepod symbiome to date, including microeukaryotes, metazoan parasites, bacteria, and viruses, and provide extensive literature databases to inform future studies.

Mechanistic strategies of microbial communities regulating lignocellulose deconstruction in a UK salt marsh.

BACKGROUND: Salt marshes are major natural repositories of sequestered organic carbon with high burial rates of organic matter, produced by highly productive native flora. Accumulated carbon predominantly exists as lignocellulose which is metabolised by communities of functionally diverse microbes. However, the organisms that orchestrate this process and the enzymatic mechanisms employed that regulate the accumulation, composition and permanence of this carbon stock are not yet known. We applied meta-exo-proteome proteomics and 16S rRNA gene profiling to study lignocellulose decomposition in situ within the surface level sediments of a natural established UK salt marsh. RESULTS: Our studies revealed a community dominated by Gammaproteobacteria, Bacteroidetes and Deltaproteobacteria that drive lignocellulose degradation in the salt marsh. We identify 42 families of lignocellulolytic bacteria of which the most active secretors of carbohydrate-active enzymes were observed to be Prolixibacteracea, Flavobacteriaceae, Cellvibrionaceae, Saccharospirillaceae, Alteromonadaceae, Vibrionaceae and Cytophagaceae. These families secreted lignocellulose-active glycoside hydrolase (GH) family enzymes GH3, GH5, GH6, GH9, GH10, GH11, GH13 and GH43 that were associated with degrading Spartina biomass. While fungi were present, we did not detect a lignocellulolytic contribution from fungi which are major contributors to terrestrial lignocellulose deconstruction. Oxidative enzymes such as laccases, peroxidases and lytic polysaccharide monooxygenases that are important for lignocellulose degradation in the terrestrial environment were present but not abundant, while a notable abundance of putative esterases (such as carbohydrate esterase family 1) associated with decoupling lignin from polysaccharides in lignocellulose was observed. CONCLUSIONS: Here, we identify a diverse cohort of previously undefined bacteria that drive lignocellulose degradation in the surface sediments of the salt marsh environment and describe the enzymatic mechanisms they employ to facilitate this process. Our results increase the understanding of the microbial and molecular mechanisms that underpin carbon sequestration from lignocellulose within salt marsh surface sediments in situ and provide insights into the potential enzymatic mechanisms regulating the enrichment of polyphenolics in salt marsh sediments. Video Abstract.

Soil fungal networks maintain local dominance of ectomycorrhizal trees.

The mechanisms regulating community composition and local dominance of trees in species-rich forests are poorly resolved, but the importance of interactions with soil microbes is increasingly acknowledged. Here, we show that tree seedlings that interact via root-associated fungal hyphae with soils beneath neighbouring adult trees grow faster and have greater survival than seedlings that are isolated from external fungal mycelia, but these effects are observed for species possessing ectomycorrhizas (ECM) and not arbuscular mycorrhizal (AM) fungi. Moreover, survival of naturally-regenerating AM seedlings over ten years is negatively related to the density of surrounding conspecific plants, while survival of ECM tree seedlings displays positive density dependence over this interval, and AM seedling roots contain greater abundance of pathogenic fungi than roots of ECM seedlings. Our findings show that neighbourhood interactions mediated by beneficial and pathogenic soil fungi regulate plant demography and community structure in hyperdiverse forests.

Diverse groups of fungi are associated with plastics in the surface waters of the Western South Atlantic and the Antarctic Peninsula.

Marine plastic pollution has a range of negative impacts for biota and the colonization of plastics in the marine environment by microorganisms may have significant ecological impacts. However, data on epiplastic organisms, particularly fungi, is still lacking for many ocean regions. To evaluate plastic associated fungi and their geographic distribution, we characterised plastics sampled from surface waters of the western South Atlantic (WSA) and Antarctic Peninsula (AP), using DNA metabarcoding of three molecular markers (ITS2, 18S rRNA V4 and V9 regions). Numerous taxa from eight fungal phyla and a total of 64 orders were detected, including groups that had not yet been described associated with plastics. There was a varied phylogenetic assemblage of predominantly known saprotrophic taxa within the Ascomycota and Basidiomycota. We found a range of marine cosmopolitan genera present on plastics in both locations, i.e., Aspergillus, Cladosporium, Wallemia and a number of taxa unique to each region, as well as a high variation of taxa such as Chytridiomycota and Aphelidomycota between locations. Within these basal fungal groups we identified a number of phylogenetically novel taxa. This is the first description of fungi from the Plastisphere within the Southern Hemisphere, and highlights the need to further investigate the potential impacts of plastic associated fungi on other organisms and marine ecosystems.

Diet in the Driving Seat: Natural Diet-Immunity-Microbiome Interactions in Wild Fish.

Natural interactions between the diet, microbiome, and immunity are largely unstudied. Here we employ wild three-spined sticklebacks as a model, combining field observations with complementary experimental manipulations of diet designed to mimic seasonal variation in the wild. We clearly demonstrate that season-specific diets are a powerful causal driver of major systemic immunophenotypic variation. This effect occurred largely independently of the bulk composition of the bacterial microbiome (which was also driven by season and diet) and of host condition, demonstrating neither of these, per se, constrain immune allocation in healthy individuals. Nonetheless, through observations in multiple anatomical compartments, differentially exposed to the direct effects of food and immunity, we found evidence of immune-driven control of bacterial community composition in mucus layers. This points to the interactive nature of the host-microbiome relationship, and is the first time, to our knowledge, that this causal chain (diet → immunity → microbiome) has been demonstrated in wild vertebrates. Microbiome effects on immunity were not excluded and, importantly, we identified outgrowth of potentially pathogenic bacteria (especially mycolic-acid producing corynebacteria) as a consequence of the more animal-protein-rich summertime diet. This may provide part of the ultimate explanation (and possibly a proximal cue) for the dramatic immune re-adjustments that we saw in response to diet change.

Polychaete mucopolysaccharide alters sediment microbial diversity and stimulates ammonia-oxidising functional groups.

Sediment nitrogen cycling is a network of microbially mediated biogeochemical processes that are vital in regulating ecosystem functioning. Mucopolysaccharides (mucus) are produced by many invertebrates and have the potential to be an important source of organic carbon and nitrogen to sediment microorganisms. At present, we have limited understanding of how mucopolysaccharide moderates total sediment microbial communities and specific microbial functional groups that drive nitrogen cycling processes. To start addressing this knowledge gap, sediment slurries were incubated with and without Hediste diversicolor mucus. Changes in dissolved inorganic nitrogen (ammonia, nitrite and nitrate) concentrations and bacterial and archaeal community diversity were assessed. Our results showed that mucopolysaccharide addition supported a more abundant and distinct microbial community. Moreover, mucus stimulated the growth of bacterial and archaeal ammonia oxidisers, with a concomitant increase in nitrite and nitrate. Hediste diversicolor mucopolysaccharide appears to enhance sediment nitrification rates by stimulating and fuelling nitrifying microbial groups. We propose that invertebrate mucopolysaccharide secretion should be considered as a distinct functional trait when assessing invertebrate contributions to sediment ecosystem function. By including this additional trait, we can improve our mechanistic understanding of invertebrate-microbe interactions in nitrogen transformation processes and provide opportunity to generate more accurate models of global nitrogen cycling.

Limited congruence exhibited across microbial, meiofaunal and macrofaunal benthic assemblages in a heterogeneous coastal environment.

One of the most common approaches for investigating the ecology of spatially complex environments is to examine a single biotic assemblage present, such as macroinvertebrates. Underlying this approach are assumptions that sampled and unsampled taxa respond similarly to environmental gradients and exhibit congruence across different sites. These assumptions were tested for five benthic groups of various sizes (archaea, bacteria, microbial eukaryotes/protists, meiofauna and macrofauna) in Plymouth Sound, a harbour with many different pollution sources. Sediments varied in granulometry, hydrocarbon and trace metal concentrations. Following variable reduction, canonical correspondence analysis did not identify any associations between sediment characteristics and assemblage composition of archaea or macrofauna. In contrast, variation in bacteria was associated with granulometry, trace metal variations and bioturbation (e.g. community bioturbation potential). Protists varied with granulometry, hydrocarbon and trace metal predictors. Meiofaunal variation was associated with hydrocarbon and bioturbation predictors. Taxon turnover between sites varied with only three out of 10 group pairs showing congruence (meiofauna-protists, meiofauna-macrofauna and protists-macrofauna). While our results support using eukaryotic taxa as proxies for others, the lack of congruence suggests caution should be applied to inferring wider indicator or functional interpretations from studies of a single biotic assemblage.

Active bacterioplankton community response to dissolved 'free' deoxyribonucleic acid (dDNA) in surface coastal marine waters.

Seawater contains dissolved 'free' DNA (dDNA) that is part of a larger <0.2 µm pool of DNA (D-DNA) including viruses and uncharacterised bound DNA. Previous studies have shown that bacterioplankton readily degrade dDNA, and culture-based approaches have identified several potential dDNA-utilising taxa. This study characterised the seasonal variation in D-DNA concentrations at Station L4, a coastal marine observatory in the Western English Channel, and linked changes in concentration to cognate physicochemical and biological factors. The impact of dDNA addition on active bacterioplankton communities at Station L4 was then determined using 16S rRNA high-throughput sequencing and RNA Stable Isotope Probing (RNA SIP) with 13C-labelled diatom-derived dDNA. Compared to other major bacterioplankton orders, the Rhodobacterales actively responded to dDNA additions in amended microcosms and RNA SIP identified two Rhodobacterales populations most closely associated with the genera Halocynthiibacter and Sulfitobacter that assimilated the 13C-labelled dDNA. Here we demonstrate that dDNA is a source of dissolved organic carbon for some members of the major bacterioplankton group the Marine Roseobacter Clade. This study enhances our understanding of roles of specific bacterioplankton taxa in dissolved organic matter cycling in coastal waters with potential implications for nitrogen and phosphorus regeneration processes.

Partitioning of soil phosphorus among arbuscular and ectomycorrhizal trees in tropical and subtropical forests.

Partitioning of soil phosphorus (P) pools has been proposed as a key mechanism maintaining plant diversity, but experimental support is lacking. Here, we provided different chemical forms of P to 15 tree species with contrasting root symbiotic relationships to investigate plant P acquisition in both tropical and subtropical forests. Both ectomycorrhizal (ECM) and arbuscular mycorrhizal (AM) trees responded positively to addition of inorganic P, but strikingly, ECM trees acquired more P from a complex organic form (phytic acid). Most ECM tree species and all AM tree species also showed some capacity to take up simple organic P (monophosphate). Mycorrhizal colonisation was negatively correlated with soil extractable P concentration, suggesting that mycorrhizal fungi may regulate organic P acquisition among tree species. Our results support the hypothesis that ECM and AM plants partition soil P sources, which may play an ecologically important role in promoting species coexistence in tropical and subtropical forests.

Community responses to seawater warming are conserved across diverse biological groupings and taxonomic resolutions.

Temperature variability is a major driver of ecological pattern, with recent changes in average and extreme temperatures having significant impacts on populations, communities and ecosystems. In the marine realm, very few experiments have manipulated temperature in situ, and current understanding of temperature effects on community dynamics is limited. We developed new technology for precise seawater temperature control to examine warming effects on communities of bacteria, microbial eukaryotes (protists) and metazoans. Despite highly contrasting phylogenies, size spectra and diversity levels, the three community types responded similarly to seawater warming treatments of +3°C and +5°C, highlighting the critical and overarching importance of temperature in structuring communities. Temperature effects were detectable at coarse taxonomic resolutions and many taxa responded positively to warming, leading to increased abundances at the community-level. Novel field-based experimental approaches are essential to improve mechanistic understanding of how ocean warming will alter the structure and functioning of diverse marine communities.

Coastal bacterioplankton community response to diatom-derived polysaccharide microgels.

Phytoplankton-derived polysaccharide microgels, including transparent exopolymer particles (TEP), are a major component of the marine organic carbon pool. Previous studies have made correlative links between phytoplankton material and bacterioplankton, and performed experiments that assess general responses to phytoplankton, yet there is a lack of direct empirical evidence of specific bacterioplankton responses to natural phytoplankton polysaccharide microgels. In this study, we used diatom produced TEP in controlled incubation experiments to determine the impact of polysaccharide microgels on a coastal bacterioplankton community. Quantification of bacterial 16S rRNA gene transcripts showed that the addition of TEP caused an increase in bacterioplankton activity. Similarly, high-throughput sequencing of RT-PCR amplified bacterial 16S rRNA gene transcripts showed that active bacterioplankton community structure and diversity also changed in response to microgels. Alteromonadales and Rhodobacterales increased in abundance in response to TEP, suggesting that both bacterioplankton taxa utilize diatom-derived microgels. However, through assessing 13 C-labelled TEP uptake via RNA Stable Isotope Probing, we show that only the Alteromonadales (genus Alteromonas) assimilated the TEP carbon. This study adds utilization of diatom-derived TEP to the metabolic repertoire of the archetypal copiotrophic bacterioplankton Alteromonas, and indicates that the Rhodobacterales may utilize TEP for other purposes (e.g. attachment sites).

Multi-year assessment of coastal planktonic fungi reveals environmental drivers of diversity and abundance.

Mycoplankton have so far been a neglected component of pelagic marine ecosystems, having been poorly studied relative to other plankton groups. Currently, there is a lack of understanding of how mycoplankton diversity changes through time, and the identity of controlling environmental drivers. Using Fungi-specific high-throughput sequencing and quantitative PCR analysis of plankton DNA samples collected over 6 years from the coastal biodiversity time series site Station L4 situated off Plymouth (UK), we have assessed changes in the temporal variability of mycoplankton diversity and abundance in relation to co-occurring environmental variables. Mycoplankton diversity at Station L4 was dominated by Ascomycota, Basidiomycota and Chytridiomycota, with several orders within these phyla frequently abundant and dominant in multiple years. Repeating interannual mycoplankton blooms were linked to potential controlling environmental drivers, including nitrogen availability and temperature. Specific relationships between mycoplankton and other plankton groups were also identified, with seasonal chytrid blooms matching diatom blooms in consecutive years. Mycoplankton α-diversity was greatest during periods of reduced salinity at Station L4, indicative of riverine input to the ecosystem. Mycoplankton abundance also increased during periods of reduced salinity, and when potential substrate availability was increased, including particulate organic matter. This study has identified possible controlling environmental drivers of mycoplankton diversity and abundance in a coastal sea ecosystem, and therefore sheds new light on the biology and ecology of an enigmatic marine plankton group. Mycoplankton have several potential functional roles, including saprotrophs and parasites, that should now be considered within the consensus view of pelagic ecosystem functioning and services.

Polychaete burrows harbour distinct microbial communities in oil-contaminated coastal sediments.

Previous studies have shown that the bioturbating polychaete Hediste (Nereis) diversicolor can affect the composition of bacterial communities in oil-contaminated sediments, but have not considered diversity specifically within bioturbator burrows or the impact on microbial eukaryotes. We tested the hypothesis that H. diversicolor burrows harbour different eukaryotic and bacterial communities compared with un-bioturbated sediment, and that bioturbation stimulates oil degradation. Oil-contaminated sediment was incubated with or without H. diversicolor for 30 days, after which sediment un-affected by H. diversicolor and burrow DNA/RNA samples were analysed using quantitative reverse transcription PCR (Q-RT-PCR) and high-throughput sequencing. Fungi dominated both burrow and un-bioturbated sediment sequence libraries; however, there was significant enrichment of bacterivorous protists and nematodes in the burrows. There were also significant differences between the bacterial communities in burrows compared with un-bioturbated sediment. Increased activity and relative abundance of aerobic hydrocarbon-degrading bacteria in the burrows coincided with the significant reduction in hydrocarbon concentration in the bioturbated sediment. This study represents the first detailed assessment of the effect of bioturbation on total microbial communities in oil-contaminated sediments. In addition, it further shows that bioturbation is a significant factor in determining microbial diversity within polluted sediments and plays an important role in stimulating bioremediation.

Intertidal epilithic bacteria diversity changes along a naturally occurring carbon dioxide and pH gradient.

Intertidal epilithic bacteria communities are important components of coastal ecosystems, yet few studies have assessed their diversity and how it may be affected by changing environmental parameters. Submarine CO2 seeps produce localised areas of CO2-enriched seawater with reduced pH levels. We utilised the seawater pH/CO2 gradient at Levante Bay (Italy) to test the hypothesis that epilithic bacteria communities are modified by exposure to seawater with the varying chemical parameters. Biofilms were sampled from three sites exposed to seawater with different pH/CO2 levels and diversity determined using high-throughput sequencing of 16S rRNA genes. Seawater pCO2 concentrations were increased from ambient at site 1 to 621 µatm at site 2 and 1654 µatm site 3, similar to the predicated future oceans beyond 2050 and 2150, respectively. Alpha diversity of total bacteria communities and Cyanobacteria communities was significantly different between sites (anova P < 0.05). Comparison between sites showed that bacteria communities and Cyanobacteria communities were significantly different (anosim P < 0.01; permanova P < 0.01). Proteobacteria, Bacteroidetes and Cyanobacteria dominated all communities; however, there were differences between sites in the relative abundance of specific orders. This study provides the most detailed assessment of intertidal epilithic bacteria diversity and shows that diversity is significantly different along a seawater pH/CO2 gradient. This information supports the evaluation of the impacts of future ocean acidification on coastal marine ecosystems.

Seasonal microbial community dynamics correlate with phytoplankton-derived polysaccharides in surface coastal waters.

Phytoplankton produce large amounts of polysaccharide gel material known as transparent exopolymer particles (TEP). We investigated the potential links between phytoplankton-derived TEP and microbial community structure in the sea surface microlayer and underlying water at the English Channel time-series station L4 during a spring diatom bloom, and in two adjacent estuaries. Major changes in bacterioneuston and bacterioplankton community structure occurred after the peak of the spring bloom at L4, and coincided with the significant decline of microlayer and water column TEP. Increased abundance of Flavobacteriales and Rhodobacterales in bacterioneuston and bacterioplankton communities at L4 was significantly related to the TEP decline, indicating that both taxa could be responsible. The results suggest that TEP is an important factor in determining microbial diversity in coastal waters, and that TEP utilisation could be a niche occupied by Flavobacteriales and Rhodobacterales.

High-throughput sequencing reveals neustonic and planktonic microbial eukaryote diversity in coastal waters.

Neustonic organisms inhabit the sea surface microlayer (SML) and have important roles in marine ecosystem functioning. Here, we use high-throughput 18S rRNA gene sequencing to characterize protist and fungal diversity in the SML at a coastal time-series station and compare with underlying plankton assemblages. Protist diversity was higher in February (pre-bloom) compared to April (spring bloom), and was lower in the neuston than in the plankton. Major protist groups, including Stramenopiles and Alveolata, dominated both neuston and plankton assemblages. Chrysophytes and diatoms were enriched in the neuston in April, with diatoms showing distinct changes in community composition between the sampling periods. Pezizomycetes dominated planktonic fungi assemblages, whereas fungal diversity in the neuston was more varied. This is the first study to utilize a molecular-based approach to characterize neustonic protist and fungal assemblages, and provides the most comprehensive diversity assessment to date of this ecosystem. Variability in the SML microeukaryote assemblage structure has potential implications for biogeochemical and food web processes at the air-sea interface.