Rediscovering pelagic biodiversity: Progress, promise, and challenges of metabarcoding of microbes to mammals

Convenors:   Ann Bucklin (University of Connecticut, USA) and
Bengt Karlson (Swedish Meteorological and Hydrological Institute, Sweden)

Sponsor:   Scientific Committee for Ocean Research (SCOR), MetaZooGene Working Group (SCOR WG157)

Symposium Venue:   Svenska Mässan, Swedish Exhibition & Congress Centre (Gothenburg, Sweden)

Symposium Date:   September 13, 2019


Registered Participants ("*" indicates talk or poster presenters):

Adil Yousif Al-Handal   [adil.yousif@bioenv.gu.se] :   Goteborgs Universitet

Anders Andersson *   [doubleanders@gmail.com] :   KTH

Paola G Batta-Lona *   [paola.batta_lona@uconn.edu] :   University of Connecticut

Leocadio Blanco-Bercial *   [leocadio@bios.edu] :   Bermuda Institute of Ocean Sciences

Eileen Bresnan *   [Eileen.Bresnan@gov.scot] :   Marine Scotland Science

Ann Bucklin *   [ann.bucklin@uconn.edu] :   University of Connecticut

Li Chaolun *   [lcl@qdio.ac.cn] :   Institute of Oceanology, Chinese Academy of Sciences

Dave Clarke   [ruth.canning@marine.ie] :   Marine Institute

Astrid Cornils   [astrid.cornils@awi.de] :   Alfred Wegener Institute Helmholtz Centre for Polar and Marine Research

Thomas Dahlgren   [thda@norceresearch.no] :   NORCE Norwegian Research Centre, BERGEN, NORWAY

Lionel Eisenhauer   [lionel.eisenhauer@sintef.no] :   SINTEF Ocean AS

Elizaveta Ershova *   [elizaveta.ershova@uit.no] :   UiT The Arctic University of Norway

Tone Falkenhaug *   [tonef@hi.no] :   Institute of Marine Research

Alexandra Frenzel *   [alexandra.frenzel@uconn.edu] :   University of Connecticut

Carolina E. Gonzalez *   [carolgonzalez@udec.cl] :   Universidad de Concepcion

Junya Hirai *   [hirai@aori.u-tokyo.ac.jp] :   Atmosphere and Ocean Research Institute, The University of Tokyo

Jenny Huggett   [jenny.huggett@gmail.com] :   Department of Environmental Affairs: Oceans and Coasts

R.P.P.K. Jayasinghe *   [prabath_jayasinghe@yahoo.com] :   National Aquatic Resources Research and Development Agency (NARA)

Marie Johansen   [marie.johansen@smhi.se] :   SMHI

Bjorn Kallstrom *   [bjorn.kallstrom@gmbl.se] :   Gothenburg Marine Biological Laboratory

Bengt Karlson *   [bengt.karlson@smhi.se] :   Swedish Meteorological and Hydrological Institute, Oceanographic Research

Dr.H.M.P. Kithsiri   [palihikkaduwa@gmail.com] :   NATIONAL AQUATIC RESOURCES RESEARCH AND DEVELOPMENT AGENCY (NARA)

Silke Laakmann   [silke.laakmann@hifmb.de] :   Helmholtz Institute for Functional Marine Biodiversity at the University of Oldenburg (HIFMB)

Meike Latz   [meike.latz@gmx.de] :   KTH Royal Institute of Technology

Markus Lindh   [markus.lindh@smhi.se] :   SMHI

Ryuji Machida *   [ryujimachida@gmail.com] :   Biodiversity Research Centre, Academia Sinica

Maria Grazia Mazzocchi   [grazia.mazzocchi@szn.it] :   Stazione Zoologica Anton Dohrn

Christine Migwi   [tinamigwi@gmail.com] :   Kenya Marine and Fisheries Research Institute (KMFRI)

Masaki Miya *   [miya@chiba-muse.or.jp] :   Natural History Museum & Institute, Chiba

Tatiana Neretina *   [nertata@wsbs-msu.ru] :   N.A. Pertzov White Sea Biological Station Lomonosov Moscow State University

Mary Mar Noblezada-Payne *   [mmpnoblezada@gmail.com] :   Institute of Marine Fisheries and Oceanology, College of Fisheries and Ocean Sciences, University of the Philippines Visayas

Todd D. O'Brien *   [Todd.OBrien@noaa.gov] :   NOAA Fisheries

Kennedy Omondi Oduor   [oduorkenn@gmail.com] :   Kenya Marine and Fisheries Research Institute (KMFRI)

Katja Peijenburg *   [lottejuliabouwman@gmail.com] :   Naturalis Biodiversity Center

Katja Peijnenburg *   [K.T.C.A.Peijnenburg@uva.nl] :   Naturalis Biodiversity Center

Janna Peters   [janna.peters@senckenberg.de] :   DZMB Senckenberg

Mai Hoang Philipsen   [hmai@chalmers.se] :   Chalmers University of Technology

Maria Prager *   [maria.prager@scilifelab.se] :   SciLifeLab, Dept. Ecology, Environment and Plant Sciences (DEEP), Stockholm University; Dept. Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet; Gothenburg Global Biodiversity Centre (GGBC), University of Gothenburg

Jasmin Renz   [jrenz@senckenberg.de] :   DZMB Senckenberg

Henna Savela *   [henna.savela@ymparisto.fi] :   Finnish Environment Institute, Marine Research Centre

Erik Selander   [erik.selander@marine.gu.se] :   Dept. Marine Sciences, U of Gothenburg

Anne Sell   [anne.sell@thuenen.de] :   Thunen Institute of Sea Fisheries

David Stankovic *   [david.stankovic@nib.si] :   Marine biology station Piran, National Institute of Biology, Slovenia

Sergio Stefanni *   [sergio.stefanni@szn.it] :   Stazione Zoologica Anton Dohrn

Peter Thor   [peter.thor@smhi.se] :   Swedish Meteorological and Hydrological Institute

Joelle van der Sprong *   [joelle.vandersprong@student.uva.nl] :   University of Amsterdam

Deborah Wall-Palmer *   [dmwallpalmer@gmail.com] :   Naturalis Biodiversity Center

Agata Weydmann-Zwolicka *   [agata.weydmann@ug.edu.pl] :   University of Gdansk, Department of Marine Plankton Research

Melissa Wojcicki *   [melissa.wojcicki@uconn.edu] :   University of Connecticut



A comprehensive catalogue of Baltic Sea bacterioplankton genomes

Johannes Alneberg, Christin Bennke, Sara Beier, Carina Bunse, Christopher Quince, Karolina Ininbergs, Lasse Riemann, Martin Ekman, Klaus Jurgens, Matthias Labrenz, Jarone Pinhassi, Anders F. Andersson

Presented-by:   Anders Andersson   (doubleanders@gmail.com)


Each liter of seawater contains a billion microorganisms that play key roles in the marine ecosystem by driving the nutrient cycles and by forming the basis of the food web. Yet, our knowledge about aquatic microorganisms - e.g. how ecosystem functions are distributed across taxa, how their abundances and activities are regulated, how they evolve and adapt to changing conditions - is still limited. Here we have conducted large-scale genome reconstruction from metagenome samples spanning the environmental gradients of the Baltic Sea and assembled a catalogue of Baltic Sea prokaryotic genomes. From the 123 samples we reconstructed 1961 metagenome-assembled genomes (MAGs) that were further clustered into 352 prokaryotic species-level clusters (Baltic Sea clusters; BACL). The genomes were widely distributed over the prokaryotic tree of life, representing 20 different phyla. 320 out of the 352 BACLs could not be classified to the species-level, and the corresponding numbers for genus- and family-level were 180 and 56, respectively. Thus, the dataset contains substantial genomic novelty. The genomes recruit on average 1/3 of the metagenome reads of the prokaryotic size fraction of the samples and thus represent a significant proportion of the planktonic prokaryotes of the Baltic Sea. The catalogue of genomes will provide an important resource for future studies on brackish ecosystems and provides an unprecedented opportunity to investigate links between genomes and ecosystems.




Metabarcoding analysis of salp diets and trophic relationships in mesopelagic food webs

Paola G. Batta-Lona, Joel K. Llopiz, Annette Govindarajan, Ann Bucklin

Presented-by:   Paola G Batta-Lona   (paola.batta_lona@uconn.edu)


Salps are among the most important and efficient grazers and diurnal migrators within the mesopelagic zone (200m-1,000m). In the NW Atlantic Ocean, the dynamics of these deep sea ecosystems are impacted by seasonal and annual environmental variation, driving changes in biodiversity and biomass of phytoplankton and zooplankton, and altering dynamics of the mesopelagic food web. DNA-based analysis of gut contents allows highly precise and broad coverage information about the diet of salps, and is yielding new insights into the energy flow through pelagic food webs. Salps were collected using mid water-trawls from the NW Atlantic Slope Water during the summers of 2016, 2017 and 2018. Selected specimens were removed from samples immediately on collection and flash-frozen in liquid nitrogen. Salps were partially thawed for dissection of gut contents and tissue for molecular species identification. Salpa aspera and Iasis zonaria were paired by species and stage for comparison of gut contents by metabarcoding analysis. Extracted DNA was sequenced for the V4 hypervariable region of the 18s rRNA gene using an Illumina MiSeq. Sequences and Operational Taxonomic Units (OTU's) were resolved and classified using custom scripts and reference databases. Multivariate statistical approaches were used to compare prey composition among different salp species over the 3 years. DNA metabarcoding analysis of consumed prey is yielding new insights into the trophic relationships of salps and their importance in particle and energy transfer in mesopelagic food webs.




Annual Cycle Of The Mesozooplankton In The Sargasso Sea

Leocadio Blanco-Bercial

Presented-by:   Leocadio Blanco-Bercial   (leocadio@bios.edu)


The diversity and specific composition of zooplankton communities have a direct effect on the ecosystem services (e.g. carbon export) and structure of ocean food webs. In areas where diversity is high, a detailed characterization of the whole community can represent a hurdle that prevents a complete understanding of those processes and interactions. One such region is the Sargasso Sea, where zooplankton diversity is among the highest in the world oceans. As a consequence, at the Bermuda Atlantic Time-series Study location, most research has focused on zooplankton biomass or on particular groups. To provide new insight into the total community composition at the BATS site, in this study the zooplankton community is investigated by means of metabarcoding of the 18S V9 hypervariable region. Day and night samples from a full year (2015) are analyzed. Results are discussed in terms of diel and seasonal variability and the identity of the taxonomic units driving these changes. The signature of diel vertical migration is driven by those groups with a higher representation in the metabarcoding reads, meanwhile the seasonality signal emerged after logarithmic transformation. Similar to temperate regions, four seasons can be differentiated based on community composition (post-spring bloom, summer stratification, fall mixing event and winter mixing), and do not correspond with, for example, the associated biomass patterns.




Linking juvenile and adult pteropods from the Atlantic Ocean through DNA barcoding, with special attention to the elusive Pseudothecosomata

Lotte J. Bouwman1, Alice Burridge1,2, Jaap de Boer1, Catharina M.H. de Weerd1, Erica Goetze3, Katja T.C.A. Peijnenburg1,2

1 Marine Biodiversity Group, Naturalis Biodiversity Center, Darwinweg 2, 2333 CR Leiden, The Netherlands.
2 Institute for Biodiversity and Ecosystem Dynamics (IBED), University of Amsterdam, P. O. Box 942480 1090 GE Amsterdam, The Netherlands.
3 Department of Oceanography, University of Hawai'i at Manoa, Honolulu, HI 96822, USA

Presented-by:   Katja Peijenburg   (lottejuliabouwman@gmail.com)


Pteropods are a group of holoplanktonic gastropods, commonly known as 'sea butterflies', which are widely regarded as biodindicators of the effects of ocean acidification because of their delicate aragonite shells. However, identification of pteropod shells remains problematic, particularly of juvenile shells and those in the suborder Pseudothecosomata, which can look very different from their adult morphologies. Consistent identification of all pteropods is important to better characterize their diversity, abundance and fluxes. Here, we used a combination of state-of-the-art imaging of juvenile shells sampled during three Atlantic cruises and DNA barcoding techniques (CO1, 28S) to link unknown juvenile shell morphologies to their more easily identifiable adult forms. Furthermore, an integrative taxonomic approach identified hidden diversity within the Peracle genus, with at least 10 species occurring in the Atlantic Ocean. To encourage and aid in the future identification of shelled pteropods, especially juvenile shells that are commonly found in plankton tows and sediment trap samples, as well as in metabarcoding studies, we prepared an open access online identification portal sharing all our images and DNA barcodes.




The use of metabarcoding to investigate the microbial and fungal communities at the Scottish Coastal Observatory monitoring site at Stonehaven in the north east of Scotland

Eileen Bresnan, Catherine Collins, Rowena Stern and Joe Taylor

Presented-by:   Eileen Bresnan   (Eileen.Bresnan@gov.scot)


The Scottish Coastal Observatory monitoring site at Stonehaven in the north east of Scotland has been in operation since 1997. Temperature, salinity, nutrients, chlorophyll 'a', phytoplankton and zooplankton are measured weekly. Concentrations of total oxidised nitrogen and dissolved inorganic phosphate are often less than the limit of detection during the summer months, a time when dinoflagellates can be abundant. Two separate studies at this site have used metabarcoding techniques to examine the microbial and fungal communities present. Using the 18S and 16S small subunit rDNA, the prokaryotic and eukaryotic community sampled during 2013 -2014 showed bacterial diversity to be greater than eukaryotic with SAR 11, clade D the most dominant. Archaea were also detected in high abundances. A subsequent study focused on marine fungi at Stonehaven performed during 2014 - 2015 revealed a diverse community to be present, changing from month to month with 18S showing spikes in chytrid abundance in June of both years. Metabarcoding is generating a first description of the microbial and fungal communities at the Stonehaven monitoring site and providing information about the potential for nutrient recycling during periods when inorganic nutrients may be limiting.




Time-series analysis of zooplankton diversity of the NW Atlantic continental shelf based on 18S rRNA metabarcodes

Ann Bucklin, Jennifer M. Questel, Bo Reese, Nancy J. Copley, and Peter H. Wiebe

Presented-by:   Ann Bucklin   (ann.bucklin@uconn.edu)


The zooplankton assemblage of the NW Atlantic continental shelf has been sampled during Ecosystem Monitoring Surveys (EcoMon) of the Northeast Fisheries Science Center (US NOAA/NEFSC) since 1977. Since 2000, a subset of EcoMon samples have been preserved in alcohol for genetic analysis. Metabarcoding analysis (i.e., large-scale taxonomic identification of complex samples via analysis of one or few orthologous DNA barcode regions) was done for EcoMon samples from three regions (Gulf of Maine, Georges Bank, Mid-Atlantic Bight) over a 10-year period (2003-2012). Two genetic markers were used: 1) V9 hypervariable region of nuclear small-subunit (18S) rRNA, which shows consistent patterns of divergence across invertebrate orders, with resolution of some families and genera; and 2) barcode region of mitochondrial cytochrome oxidase I (COI), which discriminates closely-related and cryptic species, with unreliable amplification success across invertebrate phyla. Metabarcoding results were reported by classifying, counting, and identifying operational taxonomic units (OTUs) detected in each sample, with statistical comparison among regions and years. Inter-annual and inter-regional patterns of biodiversity based on metabarcodes were compared to NEFSC species data from morphological (microscopic) examination of samples.




Assessment of metabarcode as advanced tools

Li Chaolun, Wang Minxiao, Cheng Fangping and Wang Xiaodong

Presented-by:   Li Chaolun   (lcl@qdio.ac.cn)


After decades of accumulation of zooplankton barcodes, more than 5000 tags belonging to 260 species have been deposited, which covered the key species in the Yellow Sea and the East China Sea. A comprehensive database has been set up to support the efficient and accurate zooplankton identification in Chinese costal areas. On the basis of this dataset, we tested the performance of the metabarcoding in species identification of marine zooplanktons in China. A mock sample consisted of 92 species from 12 orders were prepared to compare different metabarcoding protocols including 6 amplicon schemes (3 COI-targeted, 2 18S targeted and 1 ITS targeted) and 1 shotgun scheme. Overall, highest proportion of the added species was recovered at specific level in COI targeted scheme with the primer of JgH which give a 313 bp length product. The shotgun protocol showed best performance on the abundance evaluation. Good correlation between depth coverage and biomass were given. In addition, the outcomes of the shotgun protocol may give valuable information on the ecological function. As the two sides of the same coin, both protocols have their own drawbacks. Cost (both money and time) and the lack of bioinformatic tools are two main problems for the shotgun method while inaccuracy of the richness evaluation and the taxon biased recovery are two main problems for the amplicon methods using COI. Almost all the ctenophore and chordate taxa are greatly underestimated. Prospects of metabarcoding zooplankton are proposed.




A step towards quantitative metabarcoding of zooplankton communities

E.A. Ershova, O.S. Wangensteen, C. Barth-Jensen, K. Praebel

Presented-by:   Elizaveta Ershova   (elizaveta.ershova@uit.no)


Although metabarcoding is a well established tool for describing diversity of pelagic communities, its quantitative value is still controversial, with poor correlations previously reported between organism abundance/biomass and sequence reads. In this study, we explore a more quantitative approach by metabarcoding whole zooplankton communities using the highly degenerate Leray-XT primer set for the mitochondrial marker COI and compare the results to biomass estimates obtained using the traditional morphological approach of processing zooplankton samples. As expected, detected species richness using the metabarcoding approach was 3-4 times higher compared to morphological processing, with the highest differences found in the meroplankton fraction. Within the taxa that were detected using both approaches, the relative numbers of sequence counts were highly and significantly correlated with their relative biomass, estimated from length-weight regressions, for a wide range of metazoan taxa. The highest correlations were found for crustaceans and the lowest for meroplanktonic larvae. The described approach, using a metabarcoding marker with improved taxonomic resolution, universal coverage for metazoans, reduced primer bias and availability of a comprehensive reference database, would allow rapid and relatively inexpensive processing of hundreds of samples at a higher taxonomic resolution than traditional zooplankton sorting, and can be widely applied for monitoring or ecological studies.




DNA barcoding the Euphausiacea: taxonomic and biogeographic analysis using COI

Alexandra Frenzel, Paola G. Batta-Lona, Peter H. Wiebe, Todd O'Brien, Ann Bucklin

Presented-by:   Alexandra Frenzel   (alexandra.frenzel@uconn.edu)


The marine crustacean zooplankton group Euphausiacea (krill) includes 87 described species, many with biogeographical distributions ranging across multiple ocean basins. Congeneric species are frequently discriminated by subtle morphological characters. The mitochondrial cytochrome oxidase I (COI) barcode region is a valuable tool for species identification and discrimination of euphausiids, and also allows evaluation of the taxonomic significance of any genetic differentiation of geographic populations. The NCBI GenBank repository currently includes a total of 1,801 COI barcode sequences for data for 66 species. This study seeks to describe the current status of euphausiid barcoding, report results of new analyses of the available COI sequence data, and identify priorities for continued effort. Our goal is to encourage determination of COI barcodes for all valid species of euphausiids, including barcoding of specimens collected throughout each species' geographic range. A taxonomically-complete and geographically-comprehensive barcode database for euphausiids will provide a valuable resource for accurate species identification and detection of cryptic and novel species, and will also be useful as a reference sequence database for metabarcoding analysis of marine zooplankton diversity.




Genetic structure and endemism in the cosmopolitan copepod Pleuromamma abdominalis in the Southeast Pacific

Carolina E. Gonzalez , Erica Goetze , Ruben Escribano

Presented-by:   Carolina E. Gonzalez   (carolgonzalez@udec.cl)


Across boundary currents, zooplankton are subject to strong oceanographic gradients and hence strong selective pressures. How such gradients interact with the speciation process of pelagic organisms is still poorly understood in the open ocean realm. Here we report the genetic diversity within the pelagic copepod Pleuromamma abdominalis in the poorly known Southeast Pacific region, with samples spanning an ocean gradient from coastal upwelling to the oligotrophic South Pacific Subtropical Gyre. We assessed variation in fragments of the mitochondrial (mt) genes cytochrome c oxidase subunit I (COI) and Cytochrome b as well as in the nuclear internal transcribed spacer (ITS) region and 28S rRNA. Phylogenetic analyses revealed the presence of 8 divergent lineages occurring across the gradient with genetic distances in the range of 0.036 and 0.95 (mt genes), and GMYC species delimitation methods support their inference as distinct (undescribed) species. Several new genetic lineages occurring across the zonal gradient showed a strong genetic structuring and presence of new lineages within the coastal upwelling zone revealed an unexpectedly high level of endemism within the Humboldt Current System. Multivariate analyses found strong correlation between genetic variation and surface Chlorophyll-a and salinity, suggesting an important role for hydrographic gradients in maintaining genetic diversity. However, the presence of cryptic lineages within the upwelling zone cannot be easily accounted for by environmental heterogeneity and poses challenging questions for understanding the speciation process for oceanic zooplankton




Evaluation of historical changes in coastal fisheries of Sri Lanka using length-based indicators

S.S. Gunasekara, R.R.P.K. Jayasinghe, J.-O. Krakstad, A. Totland, H.M.P. Kithsiri

Presented-by:   R.P.P.K. Jayasinghe   (prabath_jayasinghe@yahoo.com)


The fish stocks of Sri Lanka are rarely surveyed, and the multi-species fishery typically carried out should be considered as 'data-poor'. The country has an extensive pelagic fishery (gillnet, hook and line, longlining). Surveys carried out with two different research vessels 'Dr. Fridtjof Nansen and in 1978, 1979 and 1980 and again in 2018 are among a few comprehensive studies and has given a unique opportunity to identify changes in community and stock size structure over time. During the survey's species composition and mean length of many species were recorded. Change in mean length of a fish stock is a key indicator to determine change in fishing pressure. Various selective fishing gears may alter the size structure in various ways and commonly in Sri Lanka, large, valuable fish species are targeted. Bottom trawl data from 20-100 m depth, (historic and 2018 survey) were compared. Mean lengths per species standardized by catch per nm were compared using StoX and R (v3.4.1). Lower mean lengths were observed in 2018 compared with historic surveys for several commercially targeted pelagic species. Unexpectedly, demersal species like Groupers, Snappers and Sweetlips showed pattern of increased mean length of fish between the periods, probably due to lower fishing pressure. This was especially evident in one region where we hypothesize that military restrictions to fishing grounds has had an added positive effect. However, overall, it's observed that catches of main stocks have declined since the first survey period and landed fish size is reduced.




Metabarcoding analysis of planktonic copepods in the Kuroshio region off the southern coast of Japan

Junya Hirai, Kiyotaka Hidaka, Satoshi Nagai, Yugo Shimizu, and Tadafumi Ichikawa

Presented-by:   Junya Hirai   (hirai@aori.u-tokyo.ac.jp)


Planktonic copepods play key roles in food web structures, functioning as important prey of fish larvae. Because morphological identifications of copepods, including cryptic species and immature stages, are challenging, we applied a 28S metabarcoding approach to characterize copepod communities in the Kuroshio region. The size-fractionated epipelagic copepods collected during 2013-2016 were analyzed, and results were compared between the Kuroshio region and the center of the subtropical gyre. Seasonal changes were evident in the Kuroshio region, with differences between winter-spring and summer-autumn. Spatial community differences were observed between the shoreward and oceanic sides of the Kuroshio Current. The copepod diversity, especially small copepods, was positively correlated with water temperature, and warm-water species intruded into the Kuroshio region during high-temperature periods, leading to high copepod diversity during the summer. Dominant middle/large-size copepods largely contributed to small copepod communities, suggesting the importance of immature stages of middle/large-size copepods as prey of fish larvae. We observed unique communities with large biomass at productive areas around the seamount in the shoreward of the Kuroshio Current. There was increasing dominance of specific large copepods around the seamount; therefore, geographical features could also be important factors to determine copepod communities in the Kuroshio region. The metabarcoding method successfully revealed sensitive responses of copepods to environmental changes, and inter-annual changes of the dominant species were observed especially on the shoreside of the Kuroshio Current. Further molecular-based monitoring of copepod communities would thus contribute to understanding the changes in the marine ecosystems of the Kuroshio region.




Building Barcode Libraries of Norwegian microcrustaceans

Hobaek, A. and Falkenhaug, T.

Presented-by:   Tone Falkenhaug   (tonef@hi.no)


Microcrustacean groups are highly diverse, and constitute important components of aquatic food webs (pelagic as well as benthic). They are also important elements in environmental monitoring as well as ecological research in both marine and fresh waters. Identification by traditional means remains time consuming and costly. Moreover, much of their life-cycle is spent in unidentifiable juvenile instars, which implies that major fractions of aquatic communities remain unknown in most samples. DNA-based methods hold promise to provide faster results at lower costs, and also substantially higher precision in environmental monitoring.
Here we summarize progress in building barcode libraries for microcrustaceans (copepods, cladocerans and ostracods) in fresh and marine waters of Norway, based on two faunal mapping projects supported by the Norwegian Biodiversity Information Centre and by NorBOL. These are included in the BOLD system as projects COCLA (Inventory of marine Copepoda and Cladocera (Crustacea) in Norway) and AHCRU (Northern freshwater microcrustacea). Our efforts are intended to contribute to barcode libraries, to expand our knowledge of species richness and distribution, and to pin-point species complexes that need further resolution.




The toxic and cryptogenic clinging jellyfish Gonionemus sp. (Hydrozoa, Limnomedusae) on the Swedish west coast

Bjorn Kallstrom, Erik Selander, Thomas Dahlgren, Annette Govindarajan, Carina Ostman

Presented-by:   Bjorn Kallstrom   (bjorn.kallstrom@gmbl.se)


We documented the presence of the cryptogenic limnomedusa Gonionemus sp. from an eelgrass bed at the Swedish west coast during the summer of 2018. The presence of these medusae were linked to several severe stings in local bathers. Using mitochondrial COI sequences, we showed that the Swedish medusae belong to the same clade as highly toxic populations previously found in the Sea of Japan and the northwestern Atlantic. We also reported detailed features of the medusa morphology using light microscopy, including details of the nematocysts. We suggested that the outbreak at the Swedish west coast is linked to the exceptionally warm summer of 2018 following either a climate-driven range shift or a direct introduction to the area via shipping activity. Given the harmful stings associated with the medusae and the high risk of additional colonization along the Swedish coast, further investigations on this species, including development of eDNA-markers for easy detection, are warranted.




Metabarcoding identifies previously unknown diversity of pelagic Cyanobacteria in the Baltic Sea and the Kattegat and the Skagerrak.

Bengt Karlson, Markus Lindh, Anders F. Andersson

Presented-by:   Bengt Karlson   (bengt.karlson@smhi.se)


Cyanobacteria are important primary producers in the ocean. In the brackish water Baltic Sea, diazotrophic (nitrogen fixing) species form blooms in summer. In the Kattegat-Skagerrak, where higher salinities prevail, nitrogen fixers are not common. The picoplanktonic genus Synechococcus dominates phytoplankton cell numbers in both areas in summer. Yet, the spatiotemporal diversity of this key group of organisms in the Baltic Sea and the Kattegat-Skagerrak is not well known. Here we used DNA metabarcoding to investigate the distribution and diversity of Cyanobacteria, based on sequencing of 16S rRNA gene. Near surface (0-10 m) samples were collected monthly at approximately eight stations from February 2016 to March 2017. Now, for the first time, we are able to disentangle both spatial and temporal variation in cyanobacterial community composition in this unique marine to brackish seawater system. We identified which environmental factors that were the main influencers of cyanobacterial dynamics and coupled this information with ecosystem structure.




GenBank is a reliable resource for 21st century biodiversity research

Ryuji Machida, Matthieu Leray, Nancy Knowlton

Presented-by:   Ryuji Machida   (ryujimachida@gmail.com)


Databases of taxonomically annotated DNA sequences have revolutionized ecological and evolutionary research. The accuracy of these annotations has been questioned but never examined comprehensively. In the present study, we estimate the taxonomic accuracy with a distance-based clustering analysis of metazoan mitochondrial gene sequences in GenBank. We found, contrary to expectation, little evidence for major errors in taxonomic annotations, with fewer than 0.05% being mislabeled at the order level or above. Even at the genus level, estimates of incorrect assignments ranged from only 0.52-2.9%, with the lower estimate more likely to be accurate. This encouraging result is of particular importance in light of the rapidly growing use of high-throughput sequencing for community analyses without vouchers for characterization and monitoring of biodiversity.




HIDEA - Hidden diversity of plankton in the European Arctic

Monika Mioduchowska, Anna Iglikowska, Agata Weydmann-Zwolicka

Presented-by:   Agata Weydmann-Zwolicka   (agata.weydmann@ug.edu.pl)


The main aim of the project 'HIDEA - Hidden diversity of plankton in the European Arctic' is to assess the impact of increased transport of Atlantic water into the Arctic (called 'atlantification') on the biodiversity of Arctic plankton: from bacterioplankton, through protists, to zooplankton.

As a pilot study, designed to optimize methodology, we analysed pelagic bakterioplankton community from the Baltic Sea (Gulf of Gdansk). DNA extraction was carried out using silica membranes from a commercial kit for the universal genomic DNA isolation and a modified protocol. In order to assess bacterioplankton profile, the high throughput sequencing of the V3-V4 hypervariable regions in the bacterial 16S rRNA gene fragment was performed, which resulted in a total of 102 040 reads. 99.97% of the total reads were represented by Bacteria and next-generation reads were subsequently clustered into 282 operational taxonomic units (OTUs) at 97% similarity. Identified OTUs were classified as: Proteobacteria (40.83%), Actinobacteria (22.90%), Cyanobacteria (12.77%), Planctomycetes (8.07%) and others. Furthermore, we examined whether OTUs were represented by endosymbiotic bacteria that play important roles in the biology of their hosts and may also affect micro-evolutionary processes and population phenomena. We identified OTUs classified to cytophaga-flavobacterium-bacteroides, Polynucleobacter, Candidatus Portiera and Candidatus Xiphinematobacter.
The obtained test results gave a satisfying and promising outcome, therefore the adjusted procedure will be employed in the target Arctic samples collected during summer expeditions 2019 and 2020 around the high-Arctic Archipelago of Svalbard, which is influenced by the Atlantic water masses transported northwards with the West Spitsbergen Current.




Environmental DNA metabarcoding enables a data-driven approach for fish community research in large spatial and temporal scales

Masaki Miya

Presented-by:   Masaki Miya   (miya@chiba-muse.or.jp)


Recent studies demonstrated the utility of eDNA for detecting fishes from various aquatic environments, including ponds, rivers, streams and seawaters. Most of the earlier eDNA studies focused on detection of a single or a few invasive and rare or threatened species, while a number of recent studies attempted simultaneous detection of multiple species in local fish communities and mesocosms. The latter approach is called 'metabarcoding' and eDNA metabarcoding uses one or multiple sets of PCR primers to coamplify a gene region across taxonomically diverse samples. This is followed by library preparation with indexing and adapter addition, and the indexed libraries are analyzed by a high-throughput parallel sequencing platform. Recently Miya et al. (2015) developed universal PCR primers for metabarcoding eDNA from fishes (called 'MiFish'). The MiFish primers target a hypervariable region of the mitochondrial 12S rRNA gene (163-185 bp), which contains sufficient information to identify fishes to taxonomic family, genus and species except for some closely related congeners. With the use of MiFish primers in eDNA metabarcoding (MiFish metabarcoding), my research group conducted a nation-wide study based on 528 samples from a length of the Japanese archipelago extending over 3,000 km along the northeastern coast of the Eurasia continent My research group has also conducted 50 biweekly samplings in Boso Peninsula located along the Pacific coast of Japan from August 2017 to August 2019. I will briefly introduce the latest results from these two projects and show the usefulness of these approaches to fish community research.




ITS SEQUENCES IN MARINE INVERTEBRATES METABARCODING

Tatiana Neretina, Glafira Kolbasova, Anna Zhadan, Nikolai Neretin, Ksenia Kosobokova, Alexander Tzetlin

Presented-by:   Tatiana Neretina   (nertata@wsbs-msu.ru)


Metabarcoding studies of marine invertebrates have used various regions of nuclear small- (18s) and large-subunit (28s) rRNA gene, which allow accurate classification of novel sequences and reliable amplification with conservative primers. However, this may underestimate species diversity in a community under investigation due to their low nucleotide distance (if any) in closely related species. To assess faunistic diversity at species level, more variable genes are needed. Widely used Folmer DNA barcoding region (COI) is not often suitable because of common pseudogen amplification or pure primer match. We propose to use the ITS region for metabarcoding of invertebrates in Arctic Seas. For some taxa ITS has been used quite often as a nuclear marker in recent years, in particular, for representatives of Cnidaria. The use of this marker for the representatives of this group is rather successful but in many cases it is limited due to intragenomic polymorphism associated with its multicopy, and as a result it can not be used in the Sanger sequencing. Amplicon sequencing by NGS with relatively long reads (such as offered by MiSeq chemistry) makes the application of this marker highly effective. We obtained sequences for more than 300 species from different taxa ranging from





Marine zooplankton studies in the Philippines: an evaluation of current status and future prospects from descriptive to molecular approaches

Mary Mar Noblezada-Payne, Ephrime Metillo and Wilfredo L. Campos

Presented-by:   Mary Mar Noblezada-Payne   (mmpnoblezada@gmail.com)


The Philippine marine waters contain some of the world's highly diverse and richest ecosystems characterized by extensive coral reefs, sea-grass beds, mangrove forest, and deep and shallow seas. It is known as the biodiversity hotspot and has been recognized as the global epicenter of marine biodiversity. Knowledge on the biodiversity of marine zooplankton in Philippine waters would further reinforce the claim, and may further strengthen sustained management and conservation efforts in the country which, ironically, is also known as the hotspot of hotspots for biodiversity loss attributed mainly to various harmful anthropogenic activities and more recently climate change.

This study reviews the current state of marine zooplankton studies in the Philippines and evaluate the scientific development achieved to date in this field. Sporadic research has been undertaken into the diversity, distribution and ecology of marine zooplankton over the past 100 years, the first observations conducted came from the earliest oceanographic expeditions done in the country. More recent researches include the application of molecular tools in taxonomy however, very few. While the use of next generation sequencing has been limited due mainly to financial and technical difficulty coupled with critical need to developed skills and expertise.




MZGdb: The MetaZooGene Database

Todd D. O'Brien

Presented-by:   Todd D. O'Brien   (Todd.OBrien@noaa.gov)


One of the legacy products of the MetaZooGene SCOR working group, and its MetaZooGene Barcode Review paper, will be a geo-referenced, taxonomic database of COI barcode sequences for marine zooplankton. This MetaZooGene database, hereafter called MZGdb, will be an add-on to the GenBank and Barcode of Life Data (BOLD) databases, not duplicating their content but linking to their zooplankton-COI content and providing additional features and capabilities needed by MetaZooGene and its community. At a most basic level, MZGdb can provide a 'cross-database duplicates-resolved' listing of all publicly available zooplankton COI data found in GenBank or BOLD or both, and then offer a variety of summary and access options for acquiring and/or working with these data. At a more advanced level, through its cross-integration with the Coastal & Oceanic Plankton Ecology, Production, and Observation Database (COPEPOD), MZGdb can provide listings of commonly sampled zooplankton taxa (e.g., in the North Atlantic, the Antarctic, or world-wide) that currently do not have a barcode in either GenBank or BOLD. For those familiar with GenBank and BOLD, neither of these two tasks is trivial or frustration free. This is just the beginning, as MZGdb's development and capabilities will continue as the MetaZooGene working group progresses. This presentation will summarize the current capabilities and future goals of MZGdb.




Diversity and evolution of pteropods along latitudinal transects in the Atlantic Ocean

Katja T.C.A. Peijnenburg, Le Qin Choo, Alice Burridge, Lisette Mekkes, Deborah Wall-Palmer, Erica Goetze

Presented-by:   Katja Peijnenburg   (K.T.C.A.Peijnenburg@uva.nl)


Shelled pteropods, an important group of calcifying zooplankton, are widely regarded as bio-indicators of ocean acidification. However, their species diversity, abundance and evolution are still poorly characterised, in part because of difficulties to rigorously sample across diverse ocean habitats, the lack of easily accessible identification keys and incomplete DNA barcode reference databases. Here, we will present an overview of the research carried out in the Naturalis zooplankton team focussing on pteropod diversity along latitudinal transects (~35 N to 40 S) sampled across the Atlantic Ocean. We use a combination of approaches including state-of-the-art imaging, experimental, population genomics, DNA barcoding, and metabarcoding. Our aim is to link phenotypic, genetic and oceanographic information to gain insight into the adaptive potential of shelled pteropods, and more generally, of marine zooplankton.




Mobilizing sequence-based observations into Biodiversity Atlas Sweden: An Open-Science effort to make metabarcoding data easier to process, publish and re-use

Maria Prager 1,2,3, Diego Brambilla 4, Daniel Lundin 4,5, Anna Rosling 6, Jeanette T

1 SciLifeLab, Dept. Ecology, Environment and Plant Sciences (DEEP), Stockholm University
2 Dept. Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet
3 Gothenburg Global Biodiversity Centre (GGBC), University of Gothenburg
4 Centre for Ecology and Evolution in Microbial model Systems (EEMiS), Linnaeus University
5 Dept. Biochemistry and Biophysics, Stockholm University
6 Dept. Ecology and Genetics, Uppsala University
7 Dept. Molecular Biology, Ume
8 SciLifeLab, Dept. Gene Technology, KTH Royal Institute of Technology

Presented-by:   Maria Prager   (maria.prager@scilifelab.se)


Massively parallel sequencing methods have revolutionized the collection of biodiversity data from environmental samples, such as pelagic sea water. Data from these metabarcoding and metagenomics studies are not, however, easily accessible for re-use in integrated ecosystem analyses. Biodiversity Atlas Sweden (BAS), part of the Swedish Biodiversity Data Infrastructure (SBDI), is currently working towards solving these problems in collaboration with the Global Biodiversity Information Facility (GBIF) and the international Living Atlas communities. We will mobilize Swedish metabarcoding data to make them freely available and usable in the online BAS portal, together with other biodiversity data. While our initial focus is on bacterioplankton, we will subsequently accommodate sequence-based observations of protists and metazoans. We aim to do this by simplifying processing and publishing of molecular biodiversity data. Recognizing data archiving in public repositories as a potential bottleneck for publication, we have thus compiled a step-by-step guide to aid submission of raw reads to the European Nucleotide Archive (ENA). By the end of this year, we will also provide an automated pipeline for denoising, quantification and taxonomic annotation of Illumina and PacBio amplicon sequence reads. Another objective of the project is to set up a Swedish Amplicon Sequence Variant (ASV) database, and to complement GBIF taxonomy with ASV identifiers, in order to store metabarcoding data at the highest possible resolution. Subsequent work will focus on developing and adapting web-based graphical front ends, as well as APIs, to provide data access, analysis and visualization tools that are useful to the metabarcoding and metagenomics communities.




Seed banks as reservoirs of diversity driving evolutionary dynamics and persistence of Baltic phytoplankton in a changing environment - the RESERVOIR project

Henna Savela(1), Sanna Suikkanen(1), Anke Kremp(2)

1 Finnish Environment Institute, Marine Research Centre
2 Leibniz Institute for Baltic Sea Research Warnemunde

Presented-by:   Henna Savela   (henna.savela@ymparisto.fi)


Multiple factors brought on by climate change, including increased temperatures and pCO2 levels as well as reduced salinity are predicted to profoundly affect Baltic Sea phytoplankton populations. Community composition changes are already taking place, most notably an overall increase of the relative proportion of dinoflagellates e.g. in the spring bloom. Blooms of toxin-producing dinoflagellates are also becoming more commonplace. The production of dormant resting stages is a strategy which provides an organism with better chances of survival during unfavorable environmental conditions, and which makes it possible for a species to accumulate a reservoir of genetic and physiological diversity, important for adaptation during environmental change. Among the species that are becoming more dominant in the Baltic Sea, and/or are toxin-producing, some are known to be able to produce resting stages and seed banks. However, it is unclear how widespread this trait is, as only a few key species have been documented extensively and shown to be capable of dormancy. In an ongoing project, we investigate the diversity of eukaryotic phytoplankton resting stages using 18S metabarcoding in the southern Baltic Sea and the Gulf of Finland. Additionally, we evaluate sample preparation methods aiming to extract genomic DNA exclusively from dormant cells in marine sediments. The aim of the project is to increase the knowledge on the diversity of resting stage producing phytoplankton species for a better understanding of potential future changes and evolution of Baltic Sea phytoplankton communities.




A DNA metabarcoding approach to diet comparison in two sympatric pelagic fish in the Adriatic Sea - European sardine (Sardina pilchardus) and European anchovy (Engraulis encrasicolus)

David Stankovic, Diego Borme, Valentina Tirelli, Alberto Pallavicini, Sergio Stefanni

Presented-by:   David Stankovic   (david.stankovic@nib.si)


Foodwebs are at the core of ecosystem models with small pelagic fish being the link between planktonic production and higher trophic levels. However, as diet analyses typically rely on visual identification of stomach contents, they are often coarsely characterized. Advances in DNA metabarcoding promise a simultaneous identification of even from semi-digested prey items. Here, we are presenting preliminary results on diet composition of anchovy and sardine in the Adriatic Sea using a DNA metabarcoding approach based on the COI marker. For both species, diet was dominated by medium-size copepod and cladoceran species (Paracalanus parvus, Pleopis polyphemoides, Diaxis sp., Temora stylifera, Acartia clausi, Oncea sp.). Strong abundance of other crustaceans was also identified - Jaxea nocturna was common in some sardines and absent in anchovies, while Solenocera membranacea and Liocarcinus vernalis were common in some anchovies and rare in sardines. Besides crustaceans, mollusks also formed a noticeable share of diet in both species. Furthermore, our analysis revealed an essential presence of gelatinous plankton in diet of both species. Namely, an unidentified sequence belonging to a Narcomedusae was the second most abundant OTU recovered in both species and Nanomia bijuga the fourth most abundant species detected in anchovies. Finally, our results reveal moderate levels of sardine eggs predation by anchovy in some station, while predation of anchovy eggs was almost absent. Our data indicate that the differences in prey selection is more limited by the available zooplankton than by a specific diet preference by fish species, resulting in an overall high diet overlap.




A 'cold case' of metabarcoding analysis unravel hidden mesozooplankton diversity at two Azorean seamounts and neighboring island slopes (NE Atlantic)

Sergio Stefanni, Diana Catarino, Elio Biffali, Raimondo Pannone, Andrea Tarallo, Clara Loureiro, Ana Martins, David Stankovic

Presented-by:   Sergio Stefanni   (sergio.stefanni@szn.it)


Biodiversity of zooplankton is central to the functioning of ocean ecosystems. In the Azores, the mesozooplankon is poorly studied, particularly in relation to Seamounts although most of them are considered biodiversity hotspots. In this study, zooplankton communities at the Condor and Princess Alice seamounts area (Azores) collected during 2009-2010. Metabarcoding analysis of a fragment of COI was sequenced for 45 zooplankton samples on the Condor and Princess Alice Seamounts and neighboring areas during Spring, Summer and Fall. Catches were taken during both day and night with a Bongo net of 200 mm mesh that towed obliquely within the first 100 m of the water column.
A total of 15 055 664 sequences (reduced to 4 833 982 after trimming for quality and length) provided an output of 4927 unique reads that were identified and classified into 193 marine taxa belonging to 95 families.
Comparative analysis of molecular and morphological from previous studies highlight strength and weakness of the two complementary approaches and the potential of metabarcoding for time-series analysis of zooplankton biodiversity, ocean ecosystem assessment, and fisheries management.




Cryptic species in open ocean holozooplankton - are they real?

Joelle van der Sprong, Katja T.C.A. Peijnenburg

Presented-by:   Joelle van der Sprong   (joelle.vandersprong@student.uva.nl)


Marine zooplankton play a key role in open ocean ecosystems. Although their biodiversity is considered to be crucial for understanding ecosystem functioning, we still know little about their genetic diversity.

Molecular techniques, such as DNA sequencing and metabarcoding, promise detailed and accurate biodiversity assessments and are therefore considered as novel tools to overcome shortcomings inherent to traditional morphology-based taxonomy. However, with the application of these DNA-based methods, the cryptic diversity of these holozooplankton communities has been uncovered. In this review, we investigated how many cryptic species were reported for different holozooplankton groups and ask- are they real? We summarized what genes were used for their discovery, whether there is evidence for morphological divergence, and whether cryptic species occurred in sympatry or not.

We found that the majority of cryptic species were reported for copepods and that the other groups are highly underrepresented. Most cryptic species were discovered with the mitochondrial gene marker COI. However, COI appears to be of limited usefulness for groups that have high mitogenomic variation, such as medusozoa and chaetognaths. In addition, the use of both mitochondrial genes and nuclear genes provided often more convincing evidence for cryptic diversity. In many studies, however, only COI was applied. Furthermore, we found that the lack of taxonomic knowledge has often lead to inconclusive results. As a result many species are likely incorrectly labelled as cryptic. Hence, we argue that it is necessary to close the gap between traditional and DNA-based taxonomy, as both are indispensable for uncovering cryptic diversity.




Raising awareness of the atlantid heteropods (Gastropoda, Pterotracheoidea) - calcifying holoplanktonic predators.

Deborah Wall-Palmer, Katja T.C.A. Peijnenburg

Presented-by:   Deborah Wall-Palmer   (dmwallpalmer@gmail.com)


The atlantid heteropods are holoplanktonic gastropods living in the epipelagic zone from tropical to temperate latitudes. The Atlantidae is the most diverse family of heteropods with at least 24 species. Atlantids are small (<14mm) and have remarkable adaptations for a planktonic life, including complex eyes, a long proboscus, a swimming fin and a disc-shaped shell used as a second swimming appendage. These swimming snails appear to be sensitive to environmental change, while being the only fully calcified holoplanktonic predators and as such, they are uniquely suited to give us a new perspective on the effects of ocean warming and ocean acidification. However, only a handful of people currently work on atlantids, which means a general lack of identification skills and overall awareness of this group, and thus a shortage of abundance, distribution and behavioural data. An important goal of our research is to address these issues by promoting the identification and recording of this little-known but fairly common group of zooplankton. To achieve this we have developed the Shelled Heteropod Identification Portal (planktonic.org) to aid visual identification. Metabarcoding also holds great potential to improve our knowledge of atlantid distributions, and we have produced a new CO1, 28S and 18S dataset for all atlantid clades. It is hoped that this will facilitate identification and improve the quantity and quality of atlantid data gathered in modern studies. Only with a community wide effort to collect data, will it be possible to determine temporal patterns of distribution and the effects of ocean changes.




Discovering food web interactions in the ocean twilight zone: metabarcoding analysis of the diet of mesopelagic fishes

Melissa Wojcicki, Sarah Glancy, Joel K. Llopiz, Ann Bucklin

Presented-by:   Melissa Wojcicki   (melissa.wojcicki@uconn.edu)


The ocean twilight zone or mesopelagic (200m - 1,000m depth) holds the largest biomass of organisms in the ocean and supports the largest diurnal migration on the planet. The food web dynamics of these deep ocean regions are central to the functioning of the ecosystem, determining responses to climate change, and resilience to commercial fishing. Mesopelagic fishes were collected using mid-water trawls from the Slope Water of the Northwest Atlantic Ocean during summer 2018 and spring 2019. Fishes were removed from samples immediately and frozen in liquid nitrogen. Selected fish specimens were partially thawed for species identification and dissection of gut contents. Specimens were paired by species and size for comparative analysis of gut contents by either morphological (microscopic) taxonomic identification or metabarcoding analysis. Extracted DNA was sequenced for the V4 hypervariable region of the 18S rRNA gene using an Illumina MiSeq sequencer. Results were analyzed using custom scripts and reference databases to classify sequences and operational taxonomic units (OTUs) into major groups of organisms. Additional analysis with more variable markers (including COI) is planned to identify species of the major groups for direct comparison with morphological taxonomic results. This study will contribute to new understanding of the role of mesopelagic fishes in the pelagic food web and the ecosystem dynamics of the ocean twilight zone.






Last Updated:   Mon Aug 19 08:14:30 EDT 2019