High‐throughput sequencing has the potential to describe biological communities with high efficiency yet comprehensive assessment of diversity with species‐level resolution remains one of the most challenging aspects of metabarcoding studies. We investigated the utility of curated ribosomal and mitochondrial nematode reference sequence databases for determining phylum‐specific species‐level clustering thresholds. We compiled 438 ribosomal and 290 mitochondrial sequences which identified 99% and 94% as the species delineation clustering threshold, respectively. These thresholds were evaluated in HTS data from mock communities containing 39 nematode species as well as environmental samples from Vietnam. We compared the taxonomic description of the mocks generated by two read‐merging and two clustering algorithms and the cluster‐free Dada2 pipeline. Taxonomic assignment with the RDP classifier was assessed under different training sets. Our results showed that 36/39 mock nematode species were identified across the molecular markers (18S: 32, JB2: 19, JB3: 21) in UClust_ref OTUs at their respective clustering thresholds, outperforming UParse_denovo and the commonly used 97% similarity. Dada2 generated the most realistic number of ASVs (18S: 83, JB2: 75, JB3: 82), collectively identifying 30/39 mock species. The ribosomal marker outperformed the mitochondrial markers in terms of species and genus‐level detections for both OTUs and ASVs. The number of taxonomic assignments of OTUs/ASVs was highest when the smallest reference database containing only nematode sequences was used and when sequences were truncated to the respective amplicon length. Overall, OTUs generated more species‐level detections, which were, however, associated with higher error rates compared to ASVs. Genus‐level assignments using ASVs exhibited higher accuracy and lower error rates compared to species‐level assignments, suggesting that this is the most reliable pipeline for rapid assessment of alpha diversity from environmental samples.

Scleractinian corals are bathed in a sea of planktonic and particle‐associated microorganisms. The metabolic products of corals influence the growth and composition of microorganisms, but interactions between corals and seawater microorganisms are underexplored. We conducted a field‐based survey to compare the biomass, diversity, composition, and functional capacity of microorganisms in small‐volume seawater samples collected adjacent to five coral species with seawater collected > 1 m away from the reef substrate on the same reefs. Seawater collected close to corals generally harbored copiotrophic‐type bacteria and its bacterial and archaeal composition was influenced by coral species as well as the local reef environment. Trends in picoplankton abundances were variable and either increased or decreased away from coral colonies based on coral species and picoplankton functional group. Genes characteristic of surface‐attached and potentially virulent microbial lifestyles were enriched in near‐coral seawater compared to reef seawater. There was a prominent association between the coral Porites astreoides and the coral symbiont Endozoicomonas, suggesting recruitment and/or shedding of these cells into the surrounding seawater. This evidence extends our understanding of potential species‐specific and reef site‐influenced microbial interactions that occur between corals and microorganisms within this near‐coral seawater environment that we propose to call the “coral ecosphere.” Microbial interactions that occur within the coral ecosphere could influence recruitment of coral‐associated microorganisms and facilitate the transfer of coral metabolites into the microbial food web, thus fostering reef biogeochemical cycling and a linkage between corals and the water column.

Summary: There are a few baseline reef‐systems available for understanding the microbiology of healthy coral reefs and their surrounding seawater. Here, we examined the seawater microbial ecology of 25 Northern Caribbean reefs varying in human impact and protection in Cuba and the Florida Keys, USA, by measuring nutrient concentrations, microbial abundances, and respiration rates as well as sequencing bacterial and archaeal amplicons and community functional genes. Overall, seawater microbial composition and biogeochemistry were influenced by reef location and hydrogeography. Seawater from the highly protected ‘crown jewel’ offshore reefs in Jardines de la Reina, Cuba had low concentrations of nutrients and organic carbon, abundant Prochlorococcus , and high microbial community alpha diversity. Seawater from the less protected system of Los Canarreos, Cuba had elevated microbial community beta‐diversity whereas waters from the most impacted nearshore reefs in the Florida Keys contained high organic carbon and nitrogen concentrations and potential microbial functions characteristic of microbialized reefs. Each reef system had distinct microbial signatures and within this context, we propose that the protection and offshore nature of Jardines de la Reina may preserve the oligotrophic paradigm and the metabolic dependence of the community on primary production by picocyanobacteria.

Coral reef health depends on an intricate relationship among the coral animal, photosynthetic algae, and a complex microbial community. The holobiont can impact the nutrient balance of their hosts amid an otherwise oligotrophic environment, including by cycling physiologically important nitrogen compounds. Here we use ¹⁵N-tracer experiments to produce the first simultaneous measurements of ammonium oxidation, nitrate reduction, and nitrous oxide (N₂O) production among five iconic species of reef-building corals (Acropora palmata, Diploria labyrinthiformis, Orbicella faveolata, Porites astreoides, and Porites porites) in the highly protected Jardines de la Reina reefs of Cuba. Nitrate reduction is present in most species, but ammonium oxidation is low potentially due to photoinhibition and assimilatory competition. Coral-associated rates of N₂O production indicate a widespread potential for denitrification, especially among D. labyrinthiformis, at rates of ~1 nmol cm⁻² d⁻¹. In contrast, A. palmata displays minimal active nitrogen metabolism. Enhanced rates of nitrate reduction and N₂O production are observed coincident with dark net respiration periods. Genomes of bacterial cultures isolated from multiple coral species confirm that microorganisms with the ability to respire nitrate anaerobically to either dinitrogen gas or ammonium exist within the holobiont. This confirmation of anaerobic nitrogen metabolisms by coral-associated microorganisms sheds new light on coral and reef productivity.