Quantification of marine benthic communities with metabarcoding.

DNA metabarcoding methods have been implemented in studies aimed at detecting and quantifying marine benthic biodiversity. In such surveys, universal barcodes are amplified and sequenced from environmental DNA. To quantify biodiversity with DNA metabarcoding, a relation between the number of DNA sequences of a species and its biomass and/or the abundance is required. However, this relationship is complicated by many factors, and it is often unknown. In this study, we validate estimates of biomass and abundance from molecular approaches with those from the traditional morphological approach. Abundance and biomass were quantified from 126 samples of benthic intertidal mudflat using traditional morphological approaches and compared with frequency of occurrence and relative read abundance estimates from a molecular approach. A relationship between biomass and relative read abundance was found for two widely dispersed annelid taxa (Pygospio and Scoloplos). None of the other taxons, however, showed such a relationship. We discuss how quantification of abundance and biomass using molecular approaches are hampered by the ecology of DNA i.e. all the processes that determine the amount of DNA in the environment, including the ecology of the benthic species as well as the compositional nature of sequencing data.

False-positive enterococci counts in seawater with the IDEXX Enterolert-E most probable number technique caused by Bacillus licheniformis.

Enterolert-E is an easy-to-use method for the enumeration of enterococci in water samples as an indicator of fecal pollution. This most probable number technique replaced the laborious and more time-consuming MEA-BEA plating method, and it is used extensively in ballast water testing and monitoring. In spring 2018, the Control Union Water ballast water test facility measured high enterococci concentrations in Wadden Sea water without any correlation with polluted freshwater input. By isolating bacteria from samples incubated in Enterolert-E culture medium, followed by analyses of colony morphology and DNA, it is shown that these erroneously high concentrations were caused by Bacillus licheniformis, a gram-positive rod-shaped chlorine-resistant bacterium. It is concluded that control analyses or the MEA-BEA method or dilution to reduce salinity must be performed when high enterococci concentrations are measured in water samples that are not suspected to be polluted.

Cascabel: A Scalable and Versatile Amplicon Sequence Data Analysis Pipeline Delivering Reproducible and Documented Results.

Marker gene sequencing of the rRNA operon (16S, 18S, ITS) or cytochrome c oxidase I (CO1) is a popular means to assess microbial communities of the environment, microbiomes associated with plants and animals, as well as communities of multicellular organisms via environmental DNA sequencing. Since this technique is based on sequencing a single gene, or even only parts of a single gene rather than the entire genome, the number of reads needed per sample to assess the microbial community structure is lower than that required for metagenome sequencing. This makes marker gene sequencing affordable to nearly any laboratory. Despite the relative ease and cost-efficiency of data generation, analyzing the resulting sequence data requires computational skills that may go beyond the standard repertoire of a current molecular biologist/ecologist. We have developed Cascabel, a scalable, flexible, and easy-to-use amplicon sequence data analysis pipeline, which uses Snakemake and a combination of existing and newly developed solutions for its computational steps. Cascabel takes the raw data as input and delivers a table of operational taxonomic units (OTUs) or Amplicon Sequence Variants (ASVs) in BIOM and text format and representative sequences. Cascabel is a highly versatile software that allows users to customize several steps of the pipeline, such as selecting from a set of OTU clustering methods or performing ASV analysis. In addition, we designed Cascabel to run in any linux/unix computing environment from desktop computers to computing servers making use of parallel processing if possible. The analyses and results are fully reproducible and documented in an HTML and optional pdf report. Cascabel is freely available at Github: https://github.com/AlejandroAb/CASCABEL.

Here are the polyps: in situ observations of jellyfish polyps and podocysts on bivalve shells.

Most Scyphozoan jellyfish species have a metagenic life cycle involving a benthic, asexually reproducing polyp stage and a sexually reproducing medusa stage. Medusae can be large and conspicuous and most can be identified using morphological characteristics. Polyps on the other hand are small, live a cryptic life attached to hard substrates and often are difficult or impossible to distinguish based on morphology alone. Consequently, for many species the polyp stage has not been identified in the natural environment. We inspected hard substrates in various habitats for the presence of Scyphozoan polyps. Three polyps were found on Dogger Bank, Central North Sea, attached to the inside of the umbo of empty valves of the bivalves Mactra stultorum and Spisula subtruncata. One polyp was accompanied by four podocysts. With this knowledge, the inside of bivalve shells washed ashore in Oostende (Belgium) was inspected and supposed podocysts on the inside of empty valves of Cerastoderma edule and Spisula elliptica were found. Polyps and podocysts were identified to species level by 18S rDNA and mitochondrial COI sequencing. The three polyps found on Dogger Bank all belonged to the compass jellyfish Chrysaora hysoscella. One podocyst from the Oostende beach also belonged to this species but another podocyst belonged to Cyanea lamarkii. These are the first in situ observations of C. hysoscella and C. lamarckii polyps and podocysts in the natural environment. Mactra, Cerastoderma and Spisula species are abundant in many North Sea regions and empty bivalve shells could provide an abundant settling substrate for jellyfish polyps in the North Sea and other areas. Several new strategies to increase the detection of polyps on bivalve shells are presented.

Strong population structure but no equilibrium yet: Genetic connectivity and phylogeography in the kelp Saccharina latissima (Laminariales, Phaeophyta).

Kelp aquaculture is globally developing steadily as human food source, along with other applications. One of the newer crop species is Saccharina latissima, a northern hemisphere kelp inhabiting temperate to arctic rocky shores. To protect and document its natural genetic variation at the onset of this novel aquaculture, as well as increase knowledge on its taxonomy and phylogeography, we collected new genetic data, both nuclear and mitochondrial, and combined it with previous knowledge to estimate genetic connectivity and infer colonization history. Isolation-with-migration coalescent analyses demonstrate that gene flow among the sampled locations is virtually nonexistent. An updated scenario for the origin and colonization history of S. latissima is developed as follows: We propose that the species (or species complex) originated in the northwest Pacific, crossed to the northeast Pacific in the Miocene, and then crossed the Bering Strait after its opening ~5.5 Ma into the Arctic and northeast Atlantic. It subsequently crossed the Atlantic from east to west. During the Pleistocene, it was compressed in the south with evidence for northern refugia in Europe. Postglacial recolonization led to secondary contact in the Canadian Arctic. Saccharina cichorioides is shown to probably belong to the S. latissima species complex and to derive from ancestral populations in the Asian North Pacific. Our novel approach of comparing inferred gene flow based on coalescent analysis versus Wright's island model suggests that equilibrium levels of differentiation have not yet been reached in Europe and, hence, that genetic differentiation is expected to increase further if populations are left undisturbed.

Characterization and Temperature Dependence of Arctic Micromonas polaris Viruses.

Global climate change-induced warming of the Artic seas is predicted to shift the phytoplankton community towards dominance of smaller-sized species due to global warming. Yet, little is known about their viral mortality agents despite the ecological importance of viruses regulating phytoplankton host dynamics and diversity. Here we report the isolation and basic characterization of four prasinoviruses infectious to the common Arctic picophytoplankter Micromonas. We furthermore assessed how temperature influenced viral infectivity and production. Phylogenetic analysis indicated that the putative double-stranded DNA (dsDNA) Micromonas polaris viruses (MpoVs) are prasinoviruses (Phycodnaviridae) of approximately 120 nm in particle size. One MpoV showed intrinsic differences to the other three viruses, i.e., larger genome size (205 ± 2 vs. 191 ± 3 Kb), broader host range, and longer latent period (39 vs. 18 h). Temperature increase shortened the latent periods (up to 50%), increased the burst size (up to 40%), and affected viral infectivity. However, the variability in response to temperature was high for the different viruses and host strains assessed, likely affecting the Arctic picoeukaryote community structure both in the short term (seasonal cycles) and long term (global warming).

Where are the polyps? Molecular identification, distribution and population differentiation of Aurelia aurita jellyfish polyps in the southern North Sea area.

For many species of metagenic jellyfish the location of the benthic polyps is unknown. To gain insight in the distribution, species composition and population structure of scyphozoan jellyfish polyps in the southern North Sea area, polyp samples were collected from natural and artificial substrates (settling plates, marina floats and wrecks) at ten inshore locations in the Netherlands, seven offshore locations in the North Sea and in the Gullmar Fjord in Sweden. Polyps were identified to species level by sequencing both a fragment of 18S rDNA and a fragment of mitochondrial COI, and comparing these sequences to reference sequences available in GenBank and to newly obtained sequences from medusae collected in the area. All polyps sequenced did belong to Aurelia aurita. For this species, molecular diversity in mitochondrial COI was high, with 50 haplotypes among 183 polyps. Population differentiation was detected between the Dogger Bank and other-more coastal-locations, indicating extremely low connectivity. No significant differences were found between coastal samples. The location of polyps of Cyanea capillata, Cyanea lamarckii, Chrysaora hysoscella and Rhizostoma octopus in the study area remains unresolved.

Virus production in phosphorus-limited Micromonas pusilla stimulated by a supply of naturally low concentrations of different phosphorus sources, far into the lytic cycle.

Earlier studies show that the proliferation of phytoplankton viruses can be inhibited by depletion of soluble reactive phosphorus (SRP; orthophosphate). In natural marine waters, phytoplankton phosphorus (P) availability is, however, largely determined by the supply rate of SRP (e.g. through remineralization) and potentially by the source of P as well (i.e. the utilization of soluble non-reactive P; SNP). Here we show how a steady low supply of P (mimicking natural P recycling) to virally infected P-limited Micromonas pusilla stimulates virus proliferation. Independent of the degree of P limitation prior to infection (0.32 and 0.97µmax chemostat cultures), SRP supply resulted in 2-fold higher viral burst sizes (viruses lysed per host cell) as compared with no addition (P starvation). Delaying these spikes during the infection cycle showed that the added SRP was utilized for extra M. pusilla virus (MpV) production far into the lytic cycle (18 h post-infection). Moreover, P-limited M. pusilla utilized several SNP compounds with high efficiency and with the same extent of burst size stimulation as for SRP. Finally, addition of virus-free MpV lysate (representing a complex SNP mixture) to newly infected cells enhanced MpV production, implicating host-associated alkaline phosphatase activity, and highlighting its important role in oligotrophic environments.

Population Genetic Structure, Abundance, and Health Status of Two Dominant Benthic Species in the Saba Bank National Park, Caribbean Netherlands: Montastraea cavernosa and Xestospongia muta.

Saba Bank, a submerged atoll in the Caribbean Sea with an area of 2,200 km2, has attained international conservation status due to the rich diversity of species that reside on the bank. In order to assess the role of Saba Bank as a potential reservoir of diversity for the surrounding reefs, we examined the population genetic structure, abundance and health status of two prominent benthic species, the coral Montastraea cavernosa and the sponge Xestospongia muta. Sequence data were collected from 34 colonies of M. cavernosa (nDNA ITS1-5.8S-ITS2; 892 bp) and 68 X. muta sponges (mtDNA I3-M11 partition of COI; 544 bp) on Saba Bank and around Saba Island, and compared with published data across the wider Caribbean. Our data indicate that there is genetic connectivity between populations on Saba Bank and the nearby Saba Island as well as multiple locations in the wider Caribbean, ranging in distance from 100s-1000s km. The genetic diversity of Saba Bank populations of M. cavernosa (π = 0.055) and X. muta (π = 0.0010) was comparable to those in other regions in the western Atlantic. Densities and health status were determined along 11 transects of 50 m2 along the south-eastern rim of Saba Bank. The densities of M. cavernosa (0.27 ind. m-2, 95% CI: 0.12-0.52) were average, while the densities of X. muta (0.09 ind. m-2, 95% CI: 0.02-0.32) were generally higher with respect to other Caribbean locations. No disease or bleaching was present in any of the specimens of the coral M. cavernosa, however, we did observe partial tissue loss (77.9% of samples) as well as overgrowth (48.1%), predominantly by cyanobacteria. In contrast, the majority of observed X. muta (83.5%) showed signs of presumed bleaching. The combined results of apparent gene flow among populations on Saba Bank and surrounding reefs, the high abundance and unique genetic diversity, indicate that Saba Bank could function as an important buffer for the region. Either as a natural source of larvae to replenish genetic diversity or as a storehouse of diversity that can be utilized if needed for restoration practices.

Exposing the grey seal as a major predator of harbour porpoises.

Harbour porpoises (Phocoena phocoena) stranding in large numbers around the southern North Sea with fatal, sharp-edged mutilations have spurred controversy among scientists, the fishing industry and conservationists, whose views about the likely cause differ. The recent detection of grey seal (Halichoerus grypus) DNA in bite marks on three mutilated harbour porpoises, as well as direct observations of grey seal attacks on porpoises, have identified this seal species as a probable cause. Bite mark characteristics were assessed in a retrospective analysis of photographs of dead harbour porpoises that stranded between 2003 and 2013 (n = 1081) on the Dutch coastline. There were 271 animals that were sufficiently fresh to allow macroscopic assessment of grey seal-associated wounds with certainty. In 25% of these, bite and claw marks were identified that were consistent with the marks found on animals that had tested positive for grey seal DNA. Affected animals were mostly healthy juveniles that had a thick blubber layer and had recently fed. We conclude that the majority of the mutilated harbour porpoises were victims of grey seal attacks and that predation by this species is one of the main causes of death in harbour porpoises in The Netherlands. We provide a decision tree that will help in the identification of future cases of grey seal predation on porpoises.

Coral calcification under daily oxygen saturation and pH dynamics reveals the important role of oxygen.

Coral reefs are essential to many nations, and are currently in global decline. Although climate models predict decreases in seawater pH (∼0.3 units) and oxygen saturation (∼5 percentage points), these are exceeded by the current daily pH and oxygen fluctuations on many reefs (pH 7.8-8.7 and 27-241% O2 saturation). We investigated the effect of oxygen and pH fluctuations on coral calcification in the laboratory using the model species Acropora millepora. Light calcification rates were greatly enhanced (+178%) by increased seawater pH, but only at normoxia; hyperoxia completely negated this positive effect. Dark calcification rates were significantly inhibited (51-75%) at hypoxia, whereas pH had no effect. Our preliminary results suggest that within the current oxygen and pH range, oxygen has substantial control over coral growth, whereas the role of pH is limited. This has implications for reef formation in this era of rapid climate change, which is accompanied by a decrease in seawater oxygen saturation owing to higher water temperatures and coastal eutrophication.

Sharing the slope: depth partitioning of agariciid corals and associated Symbiodinium across shallow and mesophotic habitats (2-60 m) on a Caribbean reef.

BACKGROUND: Scleractinian corals and their algal endosymbionts (genus Symbiodinium) exhibit distinct bathymetric distributions on coral reefs. Yet, few studies have assessed the evolutionary context of these ecological distributions by exploring the genetic diversity of closely related coral species and their associated Symbiodinium over large depth ranges. Here we assess the distribution and genetic diversity of five agariciid coral species (Agaricia humilis, A. agaricites, A. lamarcki, A. grahamae, and Helioseris cucullata) and their algal endosymbionts (Symbiodinium) across a large depth gradient (2-60 m) covering shallow to mesophotic depths on a Caribbean reef. RESULTS: The five agariciid species exhibited distinct depth distributions, and dominant Symbiodinium associations were found to be species-specific, with each of the agariciid species harbouring a distinct ITS2-DGGE profile (except for a shared profile between A. lamarcki and A. grahamae). Only A. lamarcki harboured different Symbiodinium types across its depth distribution (i.e. exhibited symbiont zonation). Phylogenetic analysis (atp6) of the coral hosts demonstrated a division of the Agaricia genus into two major lineages that correspond to their bathymetric distribution ("shallow": A. humilis / A. agaricites and "deep": A. lamarcki / A. grahamae), highlighting the role of depth-related factors in the diversification of these congeneric agariciid species. The divergence between "shallow" and "deep" host species was reflected in the relatedness of the associated Symbiodinium (with A. lamarcki and A. grahamae sharing an identical Symbiodinium profile, and A. humilis and A. agaricites harbouring a related ITS2 sequence in their Symbiodinium profiles), corroborating the notion that brooding corals and their Symbiodinium are engaged in coevolutionary processes. CONCLUSIONS: Our findings support the hypothesis that the depth-related environmental gradient on reefs has played an important role in the diversification of the genus Agaricia and their associated Symbiodinium, resulting in a genetic segregation between coral host-symbiont communities at shallow and mesophotic depths.

Abundance and distribution of archaeal acetyl-CoA/propionyl-CoA carboxylase genes indicative for putatively chemoautotrophic Archaea in the tropical Atlantic's interior.

Recently, evidence suggests that dark CO2 fixation in the pelagic realm of the ocean does not only occur in the suboxic and anoxic water bodies but also in the oxygenated meso- and bathypelagic waters of the North Atlantic. To elucidate the significance and phylogeny of the key organisms mediating dark CO2 fixation in the tropical Atlantic, we quantified functional genes indicative for CO2 fixation. We used a Q-PCR-based assay targeting the bifunctional acetyl-CoA/propionyl-CoA carboxylase (accA subunit), a key enzyme powering inter alia the 3-hydroxypropionate/4-hydroxybutyrate cycle (HP/HB) and the archaeal ammonia monooxygenase (amoA). Quantification of accA-like genes revealed a consistent depth profile in the upper mesopelagial with increasing gene abundances from subsurface layers towards the oxygen minimum zone (OMZ), coinciding with an increase in archaeal amoA gene abundance. Gene abundance profiles of metabolic marker genes (accA, amoA) were correlated with thaumarchaeal 16S rRNA gene abundances as well as CO2 fixation rates to link the genetic potential to actual rate measurements. AccA gene abundances correlated with archaeal amoA gene abundance throughout the water column (r(2)  = 0.309, P < 0.0001). Overall, a substantial genetic predisposition of CO2 fixation was present in the dark realm of the tropical Atlantic in both Archaea and Bacteria. Hence, dark ocean CO2 fixation might be more widespread among prokaryotes inhabiting the oxygenated water column of the ocean's interior than hitherto assumed.

Putative ammonia-oxidizing Crenarchaeota in suboxic waters of the Black Sea: a basin-wide ecological study using 16S ribosomal and functional genes and membrane lipids.

Within the upper 400 m at western, central and eastern stations in the world's largest stratified basin, the Black Sea, we studied the qualitative and quantitative distribution of putative nitrifying Archaea based on their genetic markers (16S rDNA, amoA encoding for the alpha-subunit of archaeal ammonia monooxygenase), and crenarchaeol, the specific glycerol diphytanyl glycerol tetraether of pelagic Crenarchaeota within the Group I.1a. Marine Crenarchaeota were the most abundant Archaea (up to 98% of the total archaeal 16S rDNA copies) in the suboxic layers with oxygen levels as low as 1 microM including layers where previously anammox bacteria were described. Different marine crenarchaeotal phylotypes (both 16S rDNA and amoA) were found at the upper part of the suboxic zone as compared with the base of the suboxic zone and the upper 15-30 m of the anoxic waters with prevailing sulfide concentrations of up to 30 microM. Crenarchaeol concentrations were higher in the sulfidic chemocline as compared with the suboxic zone. These results indicate an abundance of putative nitrifying Archaea at very low oxygen levels within the Black Sea and might form an important source of nitrite for the anammox reaction.

Archaeal nitrification in the ocean.

Marine Crenarchaeota are the most abundant single group of prokaryotes in the ocean, but their physiology and role in marine biogeochemical cycles are unknown. Recently, a member of this clade was isolated from a sea aquarium and shown to be capable of nitrification, tentatively suggesting that Crenarchaeota may play a role in the oceanic nitrogen cycle. We enriched a crenarchaeote from North Sea water and showed that its abundance, and not that of bacteria, correlates with ammonium oxidation to nitrite. A time series study in the North Sea revealed that the abundance of the gene encoding for the archaeal ammonia monooxygenase alfa subunit (amoA) is correlated with a decline in ammonium concentrations and with the abundance of Crenarchaeota. Remarkably, the archaeal amoA abundance was 1-2 orders of magnitude higher than those of bacterial nitrifiers, which are commonly thought to mediate the oxidation of ammonium to nitrite in marine environments. Analysis of Atlantic waters of the upper 1,000 m, where most of the ammonium regeneration and oxidation takes place, showed that crenarchaeotal amoA copy numbers are also 1-3 orders of magnitude higher than those of bacterial amoA. Our data thus suggest a major role for Archaea in oceanic nitrification.

Diversity of anoxygenic phototrophic sulfur bacteria in the microbial mats of the Ebro Delta: a combined morphological and molecular approach.

The diversity of purple and green sulfur bacteria in the multilayered sediments of the Ebro Delta was investigated. Specific oligonucleotide primers for these groups were used for the selective amplification of 16S rRNA gene sequences. Subsequently, amplification products were separated by denaturing gradient gel electrophoresis and sequenced, which yielded a total of 32 sequences. Six of the sequences were related to different cultivated members of the green sulfur bacteria assemblage, whereas seven fell into the cluster of marine or halophilic Chromatiaceae. Six sequences were clustered with the family Ectothiorhodospiraceae, three of the six being closely related to chemotrophic bacteria grouped together with Halorhodospira genus, and the other three forming a group related to the genus Ectothiorhodospira. The last thirteen sequences constituted a cluster where no molecular isolate from microbial mats has so far been reported. Our results indicate that the natural diversity in the ecosystem studied has been significantly underestimated in the past and point out the presence of novel species not related to all known purple sulfur bacteria. Furthermore, the detection of green sulfur bacteria, after only an initial step of enrichment, suggests that -- with the appropriate methodology -- several genera, such as Prosthecochloris, could be established as regular members of marine microbial mats.

Characterization of functional bacterial groups in a hypersaline microbial mat community (Salins-de-Giraud, Camargue, France).

A photosynthetic microbial mat was investigated in a large pond of a Mediterranean saltern (Salins-de-Giraud, Camargue, France) having water salinity from 70 per thousand to 150 per thousand (w/v). Analysis of characteristic biomarkers (e.g., major microbial fatty acids, hydrocarbons, alcohols and alkenones) revealed that cyanobacteria were the major component of the pond, in addition to diatoms and other algae. Functional bacterial groups involved in the sulfur cycle could be correlated to these biomarkers, i.e. sulfate-reducing, sulfur-oxidizing and anoxygenic phototrophic bacteria. In the first 0.5 mm of the mat, a high rate of photosynthesis showed the activity of oxygenic phototrophs in the surface layer. Ten different cyanobacterial populations were detected with confocal laser scanning microscopy: six filamentous species, with Microcoleus chthonoplastes and Halomicronema excentricum as dominant (73% of total counts); and four unicellular types affiliated to Microcystis, Chroococcus, Gloeocapsa, and Synechocystis (27% of total counts). Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments confirmed the presence of Microcoleus, Oscillatoria, and Leptolyngbya strains (Halomicronema was not detected here) and revealed additional presence of Phormidium, Pleurocapsa and Calotrix types. Spectral scalar irradiance measurements did not reveal a particular zonation of cyanobacteria, purple or green bacteria in the first millimeter of the mat. Terminal-restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA gene fragments of bacteria depicted the community composition and a fine-scale depth-distribution of at least five different populations of anoxygenic phototrophs and at least three types of sulfate-reducing bacteria along the microgradients of oxygen and light inside the microbial mat.