Assessing patterns of metazoans in the global ocean using environmental DNA.

Documenting large-scale patterns of animals in the ocean and determining the drivers of these patterns is needed for conservation efforts given the unprecedented rates of change occurring within marine ecosystems. We used existing datasets from two global expeditions, Tara Oceans and Malaspina, that circumnavigated the oceans and sampled down to 4000 m to assess metazoans from environmental DNA (eDNA) extracted from seawater. We describe patterns of taxonomic richness within metazoan phyla and orders based on metabarcoding and infer the relative abundance of phyla using metagenome datasets, and relate these data to environmental variables. Arthropods had the greatest taxonomic richness of metazoan phyla at the surface, while cnidarians had the greatest richness in pelagic zones. Half of the marine metazoan eDNA from metagenome datasets was from arthropods, followed by cnidarians and nematodes. We found that mean surface temperature and primary productivity were positively related to metazoan taxonomic richness. Our findings concur with existing knowledge that temperature and primary productivity are important drivers of taxonomic richness for specific taxa at the ocean's surface, but these correlations are less evident in the deep ocean. Massive sequencing of eDNA can improve understanding of animal distributions, particularly for the deep ocean where sampling is challenging.

Seasonal and interannual variability of the free-living and particle-associated bacteria of a coastal microbiome.

Marine microbial communities differ genetically, metabolically, and ecologically according to their lifestyle, and they may respond differently to environmental changes. In this study, we investigated the seasonal dynamics of bacterial assemblies in the free-living (FL) and particle-associated (PA) fractions across a span of 6 years in the Blanes Bay Microbial Observatory in the Northwestern Mediterranean. Both lifestyles showed marked seasonality. The trends in alpha diversity were similar, with lower values in spring-summer than in autumn-winter. Samples from both fractions were grouped seasonally and the percentage of community variability explained by the measured environmental variables was comparable (32% in FL and 31% in PA). Canonical analyses showed that biotic interactions were determinants of bacterioplankton dynamics and that their relevance varies depending on lifestyles. Time-decay curves confirmed a high degree of predictability in both fractions. Yet, 'seasonal' Amplicon Sequence Variants (ASVs) (as defined by Lomb Scargle time series analysis) in the PA communities represented 46% of the total relative abundance while these accounted for 30% in the FL fraction. These results demonstrate that bacteria inhabiting both fractions exhibit marked seasonality, highlighting the importance of accounting for both lifestyles to fully comprehend the dynamics of marine prokaryotic communities.

A fungi hotspot deep in the ocean: explaining the presence of Gjaerumia minor in equatorial Pacific bathypelagic waters.

A plant parasite associated with the white haze disease in apples, the Basidiomycota Gjaerumia minor, has been found in most samples of the global bathypelagic ocean. An analysis of environmental 18S rDNA sequences on 12 vertical profiles of the Malaspina 2010 expedition shows that the relative abundance of this cultured species increases with depth while its distribution is remarkably different between the deep waters of the Pacific and Atlantic oceans, being present in higher concentrations in the former. This is evident from sequence analysis and a microscopic survey with a species-specific newly designed TSA-FISH probe. Several hints point to the hypothesis that G. minor is transported to the deep ocean attached to particles, and the absence of G. minor in bathypelagic Atlantic waters could then be explained by the absence of this organism in surface waters of the equatorial Atlantic. The good correlation of G. minor biomass with Apparent Oxygen Utilization, recalcitrant carbon and free-living prokaryotic biomass in South Pacific waters, together with the identification of the observed cells as yeasts and not as resting spores (teliospores), point to the possibility that once arrived at deep layers this species keeps on growing and thriving.

The nitroplast: A nitrogen-fixing organelle.

A bacterial endosymbiont of marine algae evolved to an organelle.

High amino acid osmotrophic incorporation by marine eukaryotic phytoplankton revealed by click chemistry.

The osmotrophic uptake of dissolved organic compounds in the ocean is considered to be dominated by heterotrophic prokaryotes, whereas the role of planktonic eukaryotes is still unclear. We explored the capacity of natural eukaryotic plankton communities to incorporate the synthetic amino acid L-homopropargylglycine (HPG, analogue of methionine) using biorthogonal noncanonical amino acid tagging (BONCAT), and we compared it with prokaryotic HPG use throughout a 9-day survey in the NW Mediterranean. BONCAT allows to fluorescently identify translationally active cells, but it has never been applied to natural eukaryotic communities. We found a large diversity of photosynthetic and heterotrophic eukaryotes incorporating HPG into proteins, with dinoflagellates and diatoms showing the highest percentages of BONCAT-labelled cells (49 ± 25% and 52 ± 15%, respectively). Among them, pennate diatoms exhibited higher HPG incorporation in the afternoon than in the morning, whereas small (≤5 µm) photosynthetic eukaryotes and heterotrophic nanoeukaryotes showed the opposite pattern. Centric diatoms (e.g. Chaetoceros, Thalassiosira, and Lauderia spp.) dominated the eukaryotic HPG incorporation due to their high abundances and large sizes, accounting for up to 86% of the eukaryotic BONCAT signal and strongly correlating with bulk 3H-leucine uptake rates. When including prokaryotes, eukaryotes were estimated to account for 19-31% of the bulk BONCAT signal. Our results evidence a large complexity in the osmotrophic uptake of HPG, which varies over time within and across eukaryotic groups and highlights the potential of BONCAT to quantify osmotrophy and protein synthesis in complex eukaryotic communities.

The active free-living bathypelagic microbiome is largely dominated by rare surface taxa.

A persistent microbial seed bank is postulated to sustain the marine biosphere, and recent findings show that prokaryotic taxa present in the ocean's surface dominate prokaryotic communities throughout the water column. Yet, environmental conditions exert a tight control on the activity of prokaryotes, and drastic changes in these conditions are known to occur from the surface to deep waters. The simultaneous characterization of the total (DNA) and active (i.e. with potential for protein synthesis, RNA) free-living communities in 13 stations distributed across the tropical and subtropical global ocean allowed us to assess their change in structure and diversity along the water column. We observed that active communities were surprisingly more similar along the vertical gradient than total communities. Looking at the vertical connectivity of the active vs. the total communities, we found that taxa detected in the surface sometimes accounted for more than 75% of the active microbiome of bathypelagic waters (50% on average). These active taxa were generally rare in the surface, representing a small fraction of all the surface taxa. Our findings show that the drastic vertical change in environmental conditions leads to the inactivation and disappearance of a large proportion of surface taxa, but some surface-rare taxa remain active (or with potential for protein synthesis) and dominate the bathypelagic active microbiome.

Marine picoplankton metagenomes and MAGs from eleven vertical profiles obtained by the Malaspina Expedition.

The Ocean microbiome has a crucial role in Earth's biogeochemical cycles. During the last decade, global cruises such as Tara Oceans and the Malaspina Expedition have expanded our understanding of the diversity and genetic repertoire of marine microbes. Nevertheless, there are still knowledge gaps regarding their diversity patterns throughout depth gradients ranging from the surface to the deep ocean. Here we present a dataset of 76 microbial metagenomes (MProfile) of the picoplankton size fraction (0.2-3.0 µm) collected in 11 vertical profiles covering contrasting ocean regions sampled during the Malaspina Expedition circumnavigation (7 depths, from surface to 4,000 m deep). The MProfile dataset produced 1.66 Tbp of raw DNA sequences from which we derived: 17.4 million genes clustered at 95% sequence similarity (M-GeneDB-VP), 2,672 metagenome-assembled genomes (MAGs) of Archaea and Bacteria (Malaspina-VP-MAGs), and over 100,000 viral genomic sequences. This dataset will be a valuable resource for exploring the functional and taxonomic connectivity between the photic and bathypelagic tropical and sub-tropical ocean, while increasing our general knowledge of the Ocean microbiome.

Disentangling microbial networks across pelagic zones in the tropical and subtropical global ocean.

Microbial interactions are vital in maintaining ocean ecosystem function, yet their dynamic nature and complexity remain largely unexplored. Here, we use association networks to investigate possible ecological interactions in the marine microbiome among archaea, bacteria, and picoeukaryotes throughout different depths and geographical regions of the tropical and subtropical global ocean. Our findings reveal that potential microbial interactions change with depth and geographical scale, exhibiting highly heterogeneous distributions. A few potential interactions were global, meaning they occurred across regions at the same depth, while 11-36% were regional within specific depths. The bathypelagic zone had the lowest proportion of global associations, and regional associations increased with depth. Moreover, we observed that most surface water associations do not persist in deeper ocean layers despite microbial vertical dispersal. Our work contributes to a deeper understanding of the tropical and subtropical global ocean interactome, which is essential for addressing the challenges posed by global change.

Differential apicomplexan presence predicts thermal stress mortality in the Mediterranean coral Paramuricea clavata.

Paramuricea clavata is an ecosystem architect of the Mediterranean temperate reefs that is currently threatened by episodic mass mortality events related to global warming. The microbiome may play an active role in the thermal stress susceptibility of corals, potentially holding the answer as to why corals show differential sensitivity to heat stress. To investigate this, the prokaryotic and eukaryotic microbiome of P. clavata collected from around the Mediterranean was characterised before experimental heat stress to determine if its microbial composition influences the thermal response of the holobiont. We found that members of P. clavata's microeukaryotic community were significantly correlated with thermal stress sensitivity. Syndiniales from the Dino-Group I Clade 1 were significantly enriched in thermally resistant corals, while the apicomplexan corallicolids were significantly enriched in thermally susceptible corals. We hypothesise that P. clavata mortality following heat stress may be caused by a shift from apparent commensalism to parasitism in the corallicolid-coral host relationship driven by the added stress. Our results show the potential importance of corallicolids and the rest of the microeukaryotic community of corals to understanding thermal stress response in corals and provide a useful tool to guide conservation efforts and future research into coral-associated microeukaryotes.

Global biogeography of the smallest plankton across ocean depths.

Tiny ocean plankton (picoplankton) are fundamental for the functioning of the biosphere, but the ecological mechanisms shaping their biogeography were partially understood. Comprehending whether these microorganisms are structured by niche versus neutral processes is relevant in the context of global change. We investigate the ecological processes (selection, dispersal, and drift) structuring global-ocean picoplanktonic communities inhabiting the epipelagic (0 to 200 meters), mesopelagic (200 to 1000 meters), and bathypelagic (1000 to 4000 meters) zones. We found that selection decreased, while dispersal limitation increased with depth, possibly due to differences in habitat heterogeneity and dispersal barriers such as water masses and bottom topography. Picoplankton ß-diversity positively correlated with environmental heterogeneity and water mass variability, but this relationship tended to be weaker for eukaryotes than for prokaryotes. Community patterns were more pronounced in the Mediterranean Sea, probably because of its cross-basin environmental heterogeneity and deep-water isolation. We conclude that different combinations of ecological mechanisms shape the biogeography of the ocean microbiome across depths.

Global perspective of environmental distribution and diversity of Perkinsea (Alveolata) explored by a meta-analysis of eDNA surveys.

Perkinsea constitutes a lineage within the Alveolata eukaryotic superphylum, mainly composed of parasitic organisms. Some described species represent significant ecological and economic threats due to their invasive ability and pathogenicity, which can lead to mortality events. However, the genetic diversity of these described species is just the tip of the iceberg. Environmental surveys targeting this lineage are still scarce and mainly limited to the Northern Hemisphere. Here, we aim to conduct an in depth exploration of the Perkinsea group, uncovering the diversity across a variety of environments, including those beyond freshwater and marine ecosystems. We seek to identify and describe putative novel organisms based on their genetic signatures. In this study, we conducted an extensive analysis of a metabarcoding dataset, focusing on the V4 region of the 18S rRNA gene (the EukBank dataset), to investigate the diversity, distribution and environmental preferences of the Perkinsea. Our results reveal a remarkable diversity within the Perkinsea, with 1568 Amplicon Sequence Variants (ASVs) identified across thousands of environmental samples. Surprisingly, we showed a substantial diversity of Perkinsea within soil samples (269 ASVs), challenging the previous assumption that this group is confined to marine and freshwater environments. In addition, we revealed that a notable proportion of Perkinsea ASVs (428 ASVs) could correspond to putative new organisms, encompassing the well-established taxonomic group Perkinsidae. Finally, our study shed light on previously unveiled taxonomic groups, including the Xcellidae, and revealed their environmental distribution. These findings demonstrate that Perkinsea exhibits far greater diversity than previously detected and surprisingly extends beyond marine and freshwater environments. The meta-analysis conducted in this study has unveiled the existence of previously unknown clusters within the Perkinsea lineage, solely identified based on their genetic signatures. Considering the ecological and economic importance of described Perkinsea species, these results suggest that Perkinsea may play a significant, yet previously unrecognized, role across a wide range of environments, spanning from soil environments to the abyssal zone of the open ocean with important implications for ecosystem functioning.

Gene expression dynamics of natural assemblages of heterotrophic flagellates during bacterivory.

BACKGROUND: Marine heterotrophic flagellates (HF) are dominant bacterivores in the ocean, where they represent the trophic link between bacteria and higher trophic levels and participate in the recycling of inorganic nutrients for regenerated primary production. Studying their activity and function in the ecosystem is challenging since most of the HFs in the ocean are still uncultured. In the present work, we investigated gene expression of natural HF communities during bacterivory in four unamended seawater incubations. RESULTS: The most abundant species growing in our incubations belonged to the taxonomic groups MAST-4, MAST-7, Chrysophyceae, and Telonemia. Gene expression dynamics were similar between incubations and could be divided into three states based on microbial counts, each state displaying distinct expression patterns. The analysis of samples where HF growth was highest revealed some highly expressed genes that could be related to bacterivory. Using available genomic and transcriptomic references, we identified 25 species growing in our incubations and used those to compare the expression levels of these specific genes. Video Abstract CONCLUSIONS: Our results indicate that several peptidases, together with some glycoside hydrolases and glycosyltransferases, are more expressed in phagotrophic than in phototrophic species, and thus could be used to infer the process of bacterivory in natural assemblages.

Choanoflagellates alongside diverse uncultured predatory protists consume the abundant open-ocean cyanobacterium Prochlorococcus.

Prochlorococcus is a key member of open-ocean primary producer communities. Despite its importance, little is known about the predators that consume this cyanobacterium and make its biomass available to higher trophic levels. We identify potential predators along a gradient wherein Prochlorococcus abundance increased from near detection limits (coastal California) to >200,000 cells mL-1 (subtropical North Pacific Gyre). A replicated RNA-Stable Isotope Probing experiment involving the in situ community, and labeled Prochlorococcus as prey, revealed choanoflagellates as the most active predators of Prochlorococcus, alongside a radiolarian, chrysophytes, dictyochophytes, and specific MAST lineages. These predators were not appropriately highlighted in multiyear conventional 18S rRNA gene amplicon surveys where dinoflagellates and other taxa had highest relative amplicon abundances across the gradient. In identifying direct consumers of Prochlorococcus, we reveal food-web linkages of individual protistan taxa and resolve routes of carbon transfer from the base of marine food webs.

Disentangling temporal associations in marine microbial networks.

BACKGROUND: Microbial interactions are fundamental for Earth's ecosystem functioning and biogeochemical cycling. Nevertheless, they are challenging to identify and remain barely known. Omics-based censuses are helpful in predicting microbial interactions through the statistical inference of single (static) association networks. Yet, microbial interactions are dynamic and we have limited knowledge of how they change over time. Here, we investigate the dynamics of microbial associations in a 10-year marine time series in the Mediterranean Sea using an approach inferring a time-resolved (temporal) network from a single static network. RESULTS: A single static network including microbial eukaryotes and bacteria was built using metabarcoding data derived from 120 monthly samples. For the decade, we aimed to identify persistent, seasonal, and temporary microbial associations by determining a temporal network that captures the interactome of each individual sample. We found that the temporal network appears to follow an annual cycle, collapsing, and reassembling when transiting between colder and warmer waters. We observed higher association repeatability in colder than in warmer months. Only 16 associations could be validated using observations reported in literature, underlining our knowledge gap in marine microbial ecological interactions. CONCLUSIONS: Our results indicate that marine microbial associations follow recurrent temporal dynamics in temperate zones, which need to be accounted for to better understand the functioning of the ocean microbiome. The constructed marine temporal network may serve as a resource for testing season-specific microbial interaction hypotheses. The applied approach can be transferred to microbiome studies in other ecosystems. Video Abstract.

Mixotrophy in the bloom-forming genus Phaeocystis and other haptophytes.

Phaeocystis is a globally widespread marine phytoplankton genus, best known for its colony-forming species that can form large blooms and odorous foam during bloom decline. In the North Sea, Phaeocystis globosa typically becomes abundant towards the end of the spring bloom, when nutrients are depleted and the share of mixotrophic protists increases. Although mixotrophy is widespread across the eukaryotic tree of life and is also found amongst haptophytes, a mixotrophic nutrition has not yet been demonstrated in Phaeocystis. Here, we sampled two consecutive Phaeocystis globosa spring blooms in the coastal North Sea. In both years, bacterial cells were observed inside 0.6 - 2% of P. globosa cells using double CARD-FISH hybridizations in combination with laser scanning confocal microscopy. Incubation experiments manipulating light and nutrient availability showed a trend towards higher occurrence of intracellular bacteria under P-deplete conditions. Based on counts of bacteria inside P. globosa cells in combination with theoretical values of prey digestion times, maximum ingestion rates of up to 0.08 bacteria cell-1 h-1 were estimated. In addition, a gene-based predictive model was applied to the transcriptome assemblies of seven Phaeocystis strains and 24 other haptophytes to assess their trophic mode. This model predicted a phago-mixotrophic feeding strategy in several (but not all) strains of P. globosa, P. antarctica and other haptophytes that were previously assumed to be autotrophic. The observation of bacterial cells inside P. globosa and the gene-based model predictions strongly suggest that the phago-mixotrophic feeding strategy is widespread among members of the Phaeocystis genus and other haptophytes, and might contribute to their remarkable success to form nuisance blooms under nutrient-limiting conditions.

Global patterns and rates of habitat transitions across the eukaryotic tree of life.

The successful colonization of new habitats has played a fundamental role during the evolution of life. Salinity is one of the strongest barriers for organisms to cross, which has resulted in the evolution of distinct marine and non-marine (including both freshwater and soil) communities. Although microbes represent by far the vast majority of eukaryote diversity, the role of the salt barrier in shaping the diversity across the eukaryotic tree is poorly known. Traditional views suggest rare and ancient marine/non-marine transitions but this view is being challenged by the discovery of several recently transitioned lineages. Here, we investigate habitat evolution across the tree of eukaryotes using a unique set of taxon-rich phylogenies inferred from a combination of long-read and short-read environmental metabarcoding data spanning the ribosomal DNA operon. Our results show that, overall, marine and non-marine microbial communities are phylogenetically distinct but transitions have occurred in both directions in almost all major eukaryotic lineages, with hundreds of transition events detected. Some groups have experienced relatively high rates of transitions, most notably fungi for which crossing the salt barrier has probably been an important aspect of their successful diversification. At the deepest phylogenetic levels, ancestral habitat reconstruction analyses suggest that eukaryotes may have first evolved in non-marine habitats and that the two largest known eukaryotic assemblages (TSAR and Amorphea) arose in different habitats. Overall, our findings indicate that the salt barrier has played an important role during eukaryote evolution and provide a global perspective on habitat transitions in this domain of life.

Long-term patterns of an interconnected core marine microbiota.

BACKGROUND: Ocean microbes constitute ~ 70% of the marine biomass, are responsible for ~ 50% of the Earth's primary production and are crucial for global biogeochemical cycles. Marine microbiotas include core taxa that are usually key for ecosystem function. Despite their importance, core marine microbes are relatively unknown, which reflects the lack of consensus on how to identify them. So far, most core microbiotas have been defined based on species occurrence and abundance. Yet, species interactions are also important to identify core microbes, as communities include interacting species. Here, we investigate interconnected bacteria and small protists of the core pelagic microbiota populating a long-term marine-coastal observatory in the Mediterranean Sea over a decade. RESULTS: Core microbes were defined as those present in > 30% of the monthly samples over 10 years, with the strongest associations. The core microbiota included 259 Operational Taxonomic Units (OTUs) including 182 bacteria, 77 protists, and 1411 strong and mostly positive (~ 95%) associations. Core bacteria tended to be associated with other bacteria, while core protists tended to be associated with bacteria. The richness and abundance of core OTUs varied annually, decreasing in stratified warmers waters and increasing in colder mixed waters. Most core OTUs had a preference for one season, mostly winter, which featured subnetworks with the highest connectivity. Groups of highly associated taxa tended to include protists and bacteria with predominance in the same season, particularly winter. A group of 13 highly-connected hub-OTUs, with potentially important ecological roles dominated in winter and spring. Similarly, 18 connector OTUs with a low degree but high centrality were mostly associated with summer or autumn and may represent transitions between seasonal communities. CONCLUSIONS: We found a relatively small and dynamic interconnected core microbiota in a model temperate marine-coastal site, with potential interactions being more deterministic in winter than in other seasons. These core microbes would be essential for the functioning of this ecosystem over the year. Other non-core taxa may also carry out important functions but would be redundant and non-essential. Our work contributes to the understanding of the dynamics and potential interactions of core microbes possibly sustaining ocean ecosystem function.

Patterns of eukaryotic diversity from the surface to the deep-ocean sediment.

Remote deep-ocean sediment (DOS) ecosystems are among the least explored biomes on Earth. Genomic assessments of their biodiversity have failed to separate indigenous benthic organisms from sinking plankton. Here, we compare global-scale eukaryotic DNA metabarcoding datasets (18S-V9) from abyssal and lower bathyal surficial sediments and euphotic and aphotic ocean pelagic layers to distinguish plankton from benthic diversity in sediment material. Based on 1685 samples collected throughout the world ocean, we show that DOS diversity is at least threefold that in pelagic realms, with nearly two-thirds represented by abundant yet unknown eukaryotes. These benthic communities are spatially structured by ocean basins and particulate organic carbon (POC) flux from the upper ocean. Plankton DNA reaching the DOS originates from abundant species, with maximal deposition at high latitudes. Its seafloor DNA signature predicts variations in POC export from the surface and reveals previously overlooked taxa that may drive the biological carbon pump.

Dominant marine heterotrophic flagellates are adapted to natural planktonic bacterial abundances.

Grazing controls bacterial abundances and composition in many ecosystems. In marine systems, heterotrophic flagellates (HFs) are important predators. Assemblages of HFs are primarily formed by species still uncultured; therefore, many aspects of their trophic behaviour are poorly known. Here, we assessed the functional response of the whole assemblage and of four taxa grown in an unamended seawater incubation. We used fluorescently labelled bacteria to create a prey gradient of two orders of magnitude in abundance and estimated ingestion rates. Natural HFs had a half-saturation constant of 6.7 × 105 prey ml-1 , a value lower than that of cultured flagellates and within the range of marine planktonic bacterial abundances. Minorisa minuta was well adapted to low prey abundances and very efficient in ingesting bacteria. MAST-4 and MAST-7 were also well adapted to the typical marine abundances but less voracious. In contrast, Paraphysomonas imperforata, a typical cultured species, did not achieve ingestion rate saturation even at the highest prey concentration assayed. Our study, beside to set the basis for the fundamental differences between cultured and uncultured bacterial grazers, indicate that the examined predator taxa have different functional responses, suggesting that they occupy distinct ecological niches according to their grazing strategies and prey preferences.

Metagenome assembled genomes are for eukaryotes too.

Environmental community sequencing is suitable for producing metagenome assembled genomes (MAGs) of prokaryotes, but there is the perception that it cannot work for eukaryotes. In this issue of Cell Genomics, Delmont et al1 process a massive sequencing dataset from marine plankton to produce 683 eukaryotic MAGs; the study also includes useful functional information.