Evaluation of the rbcL marker for metabarcoding of marine diatoms and inference of population structure of selected genera

Diatoms are one of the most important phytoplankton groups in the world’s oceans. There are responsible for up to 40% of the photosynthetic activity in the Ocean, and they play an important role in the silicon and carbon cycles by decoupling carbon from atmospheric interactions through sinking and export. These processes are strongly influenced by the taxonomic composition of diatom assemblages. Traditionally, these have been assessed using microscopy, which in some cases is not reliable or reproducible. Next-generation sequencing enabled us to study diversity in a high-throughput manner and uncover new distribution patterns and diversity. However, phylogenetic markers used for this purpose, such as various 18S rDNA regions, are often insufficient because they cannot distinguish between some taxa. In this work, we demonstrate the performance of the chloroplast-encoded rbcL marker for metabarcoding marine diatoms compared to microscopy and 18S-V9 metabarcoding using a series of monthly samples from the Gulf of Trieste (GoT), northern Adriatic Sea. We demonstrate that rbcL is able to detect more taxa compared to 18S-V9 metabarcoding or microscopy, while the overall structure of the diatom assemblage was comparable to the other two methods with some variations, that were taxon dependent. In total, 6 new genera and 22 new diatom species for the study region were identified. We were able to spot misidentification of genera obtained with microscopy such as Pseudo-nitzschia galaxiae, which was mistaken for Cylindrotheca closterium, as well as genera that were completely overlooked, such as Minidiscus and several genera from the Cymatosiraceae family. Furthermore, on the example of two well-studied genera in the region, namely Chaetoceros and particularly Pseudo-nitzschia, we show how the rbcL method can be used to infer even deeper phylogenetic and ecologically significant differences at the species population level. Despite a very thorough community analysis obtained by rbcL the incompleteness of reference databases was still evident, and we shed light on possible improvements. Our work has further implications for studies dealing with taxa distribution and population structure, as well as carbon and silica flux models and networks

Evaluation of the rbcL marker for metabarcoding of marine diatoms and inference of population structure of selected genera

Diatoms are one of the most important phytoplankton groups in the world’s oceans. There are responsible for up to 40% of the photosynthetic activity in the Ocean, and they play an important role in the silicon and carbon cycles by decoupling carbon from atmospheric interactions through sinking and export. These processes are strongly influenced by the taxonomic composition of diatom assemblages. Traditionally, these have been assessed using microscopy, which in some cases is not reliable or reproducible. Next-generation sequencing enabled us to study diversity in a high-throughput manner and uncover new distribution patterns and diversity. However, phylogenetic markers used for this purpose, such as various 18S rDNA regions, are often insufficient because they cannot distinguish between some taxa. In this work, we demonstrate the performance of the chloroplast-encoded rbcL marker for metabarcoding marine diatoms compared to microscopy and 18S-V9 metabarcoding using a series of monthly samples from the Gulf of Trieste (GoT), northern Adriatic Sea. We demonstrate that rbcL is able to detect more taxa compared to 18S-V9 metabarcoding or microscopy, while the overall structure of the diatom assemblage was comparable to the other two methods with some variations, that were taxon dependent. In total, 6 new genera and 22 new diatom species for the study region were identified. We were able to spot misidentification of genera obtained with microscopy such as Pseudo-nitzschia galaxiae, which was mistaken for Cylindrotheca closterium, as well as genera that were completely overlooked, such as Minidiscus and several genera from the Cymatosiraceae family. Furthermore, on the example of two well-studied genera in the region, namely Chaetoceros and particularly Pseudo-nitzschia, we show how the rbcL method can be used to infer even deeper phylogenetic and ecologically significant differences at the species population level. Despite a very thorough community analysis obtained by rbcL the incompleteness of reference databases was still evident, and we shed light on possible improvements. Our work has further implications for studies dealing with taxa distribution and population structure, as well as carbon and silica flux models and networks

Toxicity of the diatom genus Pseudo-nitzschia (Bacillariophyceae)

Diatoms of the genus Pseudo-nitzschia H. Peragallo are known to produce domoic acid (DA), a toxin involved in amnesic shellfish poisoning (ASP). Strains of the same species are often classified as both toxic and nontoxic, and it is largely unknown whether this difference is also genetic. In the Northern Adriatic Sea, there are virtually no cases of ASP, but DA occasionally occurs in shellfish samples. So far, three species—P. delicatissima (Cleve) Heiden, P. multistriata (H. Takano) H. Takano, and P. calliantha Lundholm, Moestrup, & Hasle—have been identified as producers of DA in the Adriatic Sea. By means of enzme-linked immunosorbent assay (ELISA), high-performance liquid chromatography with UV and visible spectrum detection (HPLC-UV/VIS), and liquid chromatography with tandem mass spectrometry (LC-MS/MS), we reconfirmed the presence of DA in P. multistriata and P. delicatissima and detect for the first time in the Adriatic Sea DA in P. galaxiae Lundholm, & Moestrup. Furthermore, we attempted to answer the question of the distribution of DA production among Pseudo-nitzschia species and strains by sequencing the internal transcribed spacer (ITS) phylogenetic marker and the dabA DA biosynthesis gene and coupling this with toxicity data. Results show that all subclades of the Pseudo-nitzschia genus contain toxic species and that toxicity appears to be strain dependent, often with geographic partitioning. Amplification of dabA was successful only in toxic strains of P. multistriata and the presence of the genetic architecture for DA production in non-toxic strains was thus not confirmed

Inter-comparison of marine microbiome sampling protocols

Research on marine microbial communities is growing, but studies are hard to compare because of variation in seawater sampling protocols. To help researchers in the inter-comparison of studies that use different seawater sampling methodologies, as well as to help them design future sampling campaigns, we developed the EuroMarine Open Science Exploration initiative (EMOSE). Within the EMOSE framework, we sampled thousands of liters of seawater from a single station in the NW Mediterranean Sea (Service d\u27Observation du Laboratoire Arago [SOLA], Banyuls-sur-Mer), during one single day. The resulting dataset includes multiple seawater processing approaches, encompassing different material-type kinds of filters (cartridge membrane and flat membrane), three different size fractionations (>0.22 µm, 0.22–3 µm, 3–20 µm and >20 µm), and a number of different seawater volumes ranging from 1 L up to 1000 L. We show that the volume of seawater that is filtered does not have a significant effect on prokaryotic and protist diversity, independently of the sequencing strategy. However, there was a clear difference in alpha and beta diversity between size fractions and between these and “whole water” (with no pre-fractionation). Overall, we recommend care when merging data from datasets that use filters of different pore size, but we consider that the type of filter and volume should not act as confounding variables for the tested sequencing strategies. To the best of our knowledge, this is the first time a publicly available dataset effectively allows for the clarification of the impact of marine microbiome methodological options across a wide range of protocols, including large-scale variations in sampled volume

Productivity, pressure, and new perspectives: impacts of the COVID-19 pandemic on marine early-career researchers

The worldwide disruption caused by the beginning of the COVID-19 pandemic has dramatically impacted the activities of marine scientists working towards the goals of the UN Ocean Decade. As in other disciplines, marine early-career researchers (ECRs) are essential contributors to the development of novel and innovative science. Based on a survey of 322 of our peers, we show that the pandemic negatively impacted marine ECRs in ways that further exacerbate existing structural challenges such as social isolation, job insecurity, and short-term contracts, competitive funding, and work pressure. Furthermore, we find that the success and wellbeing of marine ECRs depends heavily on networking opportunities, gaining practical experience, collecting data, and producing publications, all of which were disrupted by the pandemic. Our analysis shows that those in the earliest stages of their careers feel most vulnerable to long-term career disadvantage as a result of the pandemic. This paper contributes to the empirical body of work about the impacts of the pandemic on marine science and offers recommendations on how marine ECRs should be supported to achieve the UN Ocean Decade’s goal of producing “the science we need for the ocean we want”

Inter-comparison of marine microbiome sampling protocols

International audienceAbstract Research on marine microbial communities is growing, but studies are hard to compare because of variation in seawater sampling protocols. To help researchers in the inter-comparison of studies that use different seawater sampling methodologies, as well as to help them design future sampling campaigns, we developed the EuroMarine Open Science Exploration initiative (EMOSE). Within the EMOSE framework, we sampled thousands of liters of seawater from a single station in the NW Mediterranean Sea (Service d'Observation du Laboratoire Arago [SOLA], Banyuls-sur-Mer), during one single day. The resulting dataset includes multiple seawater processing approaches, encompassing different material-type kinds of filters (cartridge membrane and flat membrane), three different size fractionations (>0.22 µm, 0.22–3 µm, 3–20 µm and >20 µm), and a number of different seawater volumes ranging from 1 L up to 1000 L. We show that the volume of seawater that is filtered does not have a significant effect on prokaryotic and protist diversity, independently of the sequencing strategy. However, there was a clear difference in alpha and beta diversity between size fractions and between these and “whole water” (with no pre-fractionation). Overall, we recommend care when merging data from datasets that use filters of different pore size, but we consider that the type of filter and volume should not act as confounding variables for the tested sequencing strategies. To the best of our knowledge, this is the first time a publicly available dataset effectively allows for the clarification of the impact of marine microbiome methodological options across a wide range of protocols, including large-scale variations in sampled volume

EMOSE (2017) Inter-Comparison of Marine Plankton Metagenomic Analysis Methods

The scientific objective of the first Edition (2017) of EMOSE is to review methods in marine plankton metagenomic analysis, essentially comparing the different filtration, sequencing and analysis pipelines used by recent initiatives, i.e. Tara Oceans, Malaspina and the Ocean Sampling Day. Already published data from these projects will be complemented by a new set of data obtained from a comparative experiment of protocols used in the three initiatives. The bulk of the analyses will be done during a 5‐day hands‐on session in Porto, and a virtual research environment will be used to plan the analysis and to work‐up a review paper