The major objectives of the BioMarKs project were organized into 4 work-packages:
- WP-1: Establishing a baseline of unicellular eukaryote (protist) biodiversity in European coastal waters using massive parallel sequencing correlated to contextual physical, chemical, and biological metadata.
- WP-2: Applying the genetic and meta-data to environmental and evolutionary questions in marine protist diversity: how many, who, where, when and why?
- WP-3: Correlating change in marine protist diversity with global (ocean acidification) and local (anthropogenic pollution, ballast water) pressures.
- WP-4: Developing practical methods for monitoring eukaryotic microbial diversity change and for evaluating its economic implications.
Project activities and achievements
All activities and achievements of the BioMarKs project have been synthesized and organized in searchable databases implemented in the final BioMarKs web-browser (today @: http://biomarks.scrol.fr, later @: http://www.biomarks.eu). In particular the project led to the current completion of 50 papers (46 published and 4 submitted), with 24 in Journals with impact factors >5 (see below). Major achievements are given below by work-packages. BioMarKs partnership: P1: Roscoff; P2: Barcelona; P3: Exeter; P4: Marseille; P5: Naples; P6: Nice; P7: Oslo; P8: Kaiserslautern; P9: Geneva.
WP1. Baseline of unicellular eukaryote biodiversity in EU coastal waters.
The BioMarKs collection of eco-morpho-genetic samples from all around the EU coastline results from a great sampling effort that involved all BioMarKs partners. This consisted in:
- Design of original protocols to sample the entire size-range of planktonic (P1) and benthic (P9) marine protistan communities (genetic, morphological, and contextual material). These protocols were further used to sample the global oceans during the Tara-Oceans expeditions (2009-2014), and are currently part of an international effort to standardize marine microbial sampling.
- Application of our eco-morpho-genetic sampling protocols to build up a collection of 1,439 genetic and 1,717 morphological samples from entire protistan communities covering all organismic size-fractions, 3 depths, and 10 EU sites from Spitzbergen to the Black Sea (see Annex 1). All together 10 sampling campaigns were organized by at least 3 partner teams between 2009 and 2011, involving the use of local boats and standardized sampling protocols for microscopy and genetic analyses of holistic protist communities from the water column and sediments. Cruise reports and protocols are available on the BioMarKs website.
- Design of various metabarcoding systems and their application to the BioMarKs samples using Next Generation Sequencing (NGS; 25x 454 runs and 5x Illumina MiSeq runs). P1 developed the molecular biology protocols for successful extraction, amplification, and semi-quantitative sequencing of barcodes from total DNA and RNA from BioMarKs samples. Multiplex primers were designed to target all eukaryotes, only photosynthetic eukaryotes, or various groups of interest for each BioMarKs partners (see Annex 1). All together a collection of >220 million clean and complete environmental barcodes was produced to assess the biocomplexity of marine protists in EU marine coastal systems. In addition, hundreds of light, scanning electron, and transmission electron microscopy images from the same samples were acquired by P5, P7, P2, together with physico-chemical contextual parameters. Fluorescent In Situ Hybridization (FISH) data were acquired for several groups (all eukaryotes, MAST4, Chrysophyceae, Prymnesiophyceae) by P2.
WP2. How many EU marine protists, who, where, when and why?
Assessment of the taxonomic and ecological significance of marine coastal protists consisted in:
- Development of novel bioinformatics strategies and pipelines to extract clean DNA barcodes from the NGS raw datasets and cluster them into relevant Operational Taxononic Units (OTUs) (Mahe et al. submitted). Application of original strategy involving phylogenetic placement (Dunthorn et al. 2014) or network/graph theory (Forster et al. submitted) to unveil the extent of novel diversity in BioMarKs NGS datasets.
- Construction of reference taxonomic databases for each metabarcoding system. Group and/or gene-specific databases of taxonomically-known barcodes were assembled by each partner to serve as reference for taxonomic assignment of e-DNA/RNA metabarcodes. In particular a unique database of taxonomically curated rDNA sequences from all known eukaryotes (PR2 - Protist Ribosomal Reference database) has been assembled by experts within the consortium and published in Nucleic Acid Research in 2013.
- Organization of 9 BioMarKs meetings between 2009 and 2013 to discuss the eco/taxonomic novelties in BioMarkS datasets at various systemic levels and organize collaborative work. All meetings’ abstracts and presentations are available on the BioMarKs website.
The main scientific outcomes of each partner are listed in a section below. Overall, to our surprise the exploration of marine protistan biodiversity based on 454-NGS was far from reaching saturation but still unveiled a wealth of novel biodiversity, especially in marine sediments (Chambouvet et al. 2014; Berney et al. 2013; Richard et al. 2012; etc). A highlight of this work was the discovery of a diverse and primitive clade of aquatic fungi that do not produce chitin-rich cell wall and thus challenges current concepts of the fungal tree of life. The cryptomycota were published in Nature (Jones et al. 2011). In 2012, we shifted to Illumina sequencing of V9 and later V4 rDNA metabarcodes and could finally approach saturation of eukaryotic diversity in BioMarKs samples. This allowed a unique exploration of the phylogenetic and geographical patterning of EU marine protist biodiversity published in Current Biology (Logares et al. 2014). We found that abundant and rare assemblages of marine protists present contrasting structuring patterns and phylogenetic characteristics, despite a remarkable consistency in their relative proportions across individual samples.
WP3. Marine protist biodiversity changes.
In order to test for marine protistan biodiversity change over time and facing anthropogenic pressures, we (i) built up global-scale datasets of morpho-genetic protistan biodiversities across gradients of oceans carbonate chemistry (Beaufort et al. 2011); (ii) applied the BioMarKs morpho-genetic sampling protocol to the large-scale acidification mesocosm experiment realized in an Arctic fjord (Svalbard, 2010) in frame of the EU-FP7 project EPOCA; (iii) sampled ballast water from several ships in the port of Rotterdam. Unfortunately material from this last activity was lost during a freezer breakup. The genetic datasets associated to the Arctic ocean acidification experiment was delivered to both EPOCA and BioMarKs consortium in 2012 and will be analyzed in 2014. The highlight of WP3 was a global-scale morpho-genetic study of coccolithophores demonstrating a general pattern of cells’ de-calcification when oceanic waters get more acidic, but with a striking exception in the most acidic water mass of modern oceans where specific genotypes are displaying hyper-calcified phenotypes. This first study of the adaptation of coccolithophore calcification across global scales in both modern and past oceans, linking morphological and genetic information, was published in Nature in 2011 (Beaufort, .., de Vargas, 2011). Furthermore, during the last months of the project, we generated massive metabarcoding datasets from multi-years time-series sampling in both Roscoff and Blanes (Spain), at sites that are regularly sampled by local marine institute (e.g. the SOMLIT station for Roscoff). This added a new and important time dimension to the original goal of the project centered on a spatial assessment of marine protistan biodiversity and this effort will be extended to time-series samples at P7 (Oslo) and P5 (Naples).
WP4. Biomonitoring marine eukaryotic microbial diversity.
Over the course of the projects, we developed a series of state-of-the-art sampling and molecular ecology protocols, databases of reference barcodes, NGS-sequencing strategies based on both total DNA and RNA extracts, and bioinformatics algorithms, providing a complete toolbox for modern, cheap and accurate biomonitoring of marine eukaryotic biodiversity. All protocols are freely available on the BioMarKs website. Logically, several BioMarKs partners were contacted by governmental agencies or private companies requesting help to develop simple NGS-based assays to answer biodiversity related environmental questions -see sections 7 and 10 below. Nevertheless, the withdrawal of our Italian partner after the project was approved seriously impacted achievements within WP4, which was critically relying on Dr. Kooistra and Zingone, respectively the EMBRC program manager and a world expert in marine toxic algae and plankton-related environmental issues.