Results of the study of invasive algae in National Parks published in Marine Pollution Bulletin

by Xavier Turon

We are happy to report that the results concerning the invaded vs non-invaded communities sampled in 2015 have now been published in the international journal Marine Pollution Bulletin: Wangensteen OS, Cebrian E, Palacín C, Turon X. 2018. Under the canopy: Community-wide effects of invasive algae in Marine Protected Areas revealed by metabarcoding. Marine Pollution Bulletin, 127:54-66.

Elsevier kindly provides a 50 days free access (starting 21 Dec) to this article at https://authors.elsevier.com/a/1WG02,ashmKxI. After this period you will need a subscription to be able to download it

In this paper we report on the effect that invasive algae have at the community level, targeting mainly the smaller size components (small macrofauna and meiofauna), using the 18S and the COI genes and the fractionation procedure and bioinformatic pipeline developed in our project. Here goes the abstract:

We analysed with multigene (18S and COI) metabarcoding the effects of the proliferation of invasive seaweeds on rocky littoral communities in two Spanish Marine Protected Areas. The invasive algae studied were Caulerpa cylindracea, Lophocladia lallemandii and Asparagopsis armata. They are canopy-forming, landscape-dominant seaweeds, and we were interested in their effects on the underlying communities of meiobenthos and macrobenthos, separated in two size fractions through sieving. A new semiquantitative treatment of metabarcoding data is introduced. The results for both markers showed that the presence of the invasive seaweed had a significant effect on the understory communities for Lophocladia lallemandii and Asparagopsis armata but not for Caulerpa cylindracea. Likewise, changes in MOTU richness and diversity with invasion status varied in magnitude and direction depending on the alga considered. Our results showed that metabarcoding allows monitoring of the less conspicuous, but not least important, effects of the presence of dominant invasive seaweeds.

Images of the communities analysed, with (left) and without (right) invasive algae. A,B: Cc (Caulerpa cylindracea) communities. C,D: Ll (Lophocladia lallemandii) communities. E,F: Aa (Asparagopsis armata) communities. Pictures: (A) from Eneko Aspillaga; (B,F) from the authors; (C,D) from Pol Capdevila; (E) from Enric Ballesteros.

Results from metabarcoding communities from the National Parks published in PeerJ Preprints

by Xavier Turon

While the article is being peer-reviewed, we have published the manuscript in Pre-print format in the repository PeerJ Preprints, where it is freely available at Wangensteen OS, Palacín C, Guardiola M, Turon X. 2017. Metabarcoding litoral hard-bottom communities: unexpected diversity and database gaps revealed by two molecular markers. PeerJ Preprints 5:e3429v1.

We report in this manuscript the astonishing diversity found in communities from Islas Cies and Cabrera. We address several technical aspects, compare the two genes studied (18S and COI), and perform ecological analyses. Here goes the abstract:

We developed a metabarcoding method for biodiversity characterization of structurally complex natural marine hard-bottom communities. Novel primer sets for two different molecular markers: the “Leray fragment” of mitochondrial cytochrome c oxidase, COI, and the V7 region of ribosomal RNA 18S were used to analyse eight different marine shallow benthic communities from two National Parks in Spain (one in the Atlantic Ocean and another in the Mediterranean Sea). Samples were sieved into three size fractions from where DNA was extracted separately. Bayesian clustering was used for delimiting molecular operational taxonomic units (MOTUs) and custom reference databases were constructed for taxonomic assignment. We found unexpectedly high values for MOTU richness, suggesting that these communities host a large amount of yet undescribed eukaryotic biodiversity. Significant gaps are still found in sequence reference databases, which currently prevent the complete taxonomic assignation of the detected sequences. Nevertheless, over 90% (in abundance) of the sequenced reads could be successfully assigned to phylum or lower taxonomical level. This identification rate might be significantly improved in the future, as reference databases are updated. Our results show that marine metabarcoding, currently applied mostly to plankton or sediments, can be adapted to structurally complex hard bottom samples, and emerges as a robust, fast, objective and affordable method for comprehensively characterizing the diversity of marine benthic communities dominated by macroscopic seaweeds and colonial or modular sessile metazoans, allowing for standardized biomonitoring of these ecologically important communities. The new universal primers for COI can potentially be used for biodiversity assessment with high taxonomic resolution in a wide array of marine, terrestrial or freshwater eukaryotic communities.

Percent of MOTUs found for the main categories of organisms in the three fractions studied with 18S (left) and COI (right) in all communities.

ChallenGen at the Pint of Science festival in Blanes

by Xavier Turon

The Pint of Science festival aims to deliver interesting talks on the latest science research in an accessible format to the public – mainly across bars and pubs (preferably with a pint of beer in hands). It is an International festival being held annually over three days at the same time in hundreds of cities across the world. Blanes, the host city of the CEAB, has participated for the first time in this year’s Pint of Science festival. The scientists of the CEAB gave nine talks three evenings (15-17 May 2017).

Xavier Turon participated in the first day of the festival, presenting the opening talk at a nice bar (Casal la Cooperativa) on the topic: Biodiversity 2.0: genetics and computers to study the living organisms. Needless to say, his talk capitalized on results obtained during the ChallenGen project, particularly those related to biodiversity assessment via metabarcoding.

It was an enjoyable and challenging experience to try to explain in lay terms some complex analysis we do in our research. The audience was very interested and asked many questions after the talk.

ChallenGen at the meeting "Developing new genetic tools for bioassessment of aquatic ecosystems in Europe"

by Xavier Turon

ChallenGen was present at the meeting on metabarcoding held in Essen (Germany) in March 7th-9th 2017. This was the kick-off conference of the European Union COST action DNAqua-Net.

This project aims at integrating a group of researchers across disciplines with the task to identify gold-standard genomic tools and novel eco-genomic indices and metrics for routine application for biodiversity assessments and biomonitoring of European water bodies. It also envisages training and dissemination activities. The objectives of ChallenGen are fully compatible with the goals of DNAqua-Net and we wanted to be counted in and contribute.

Xavier Turon is one of the Spanish members of DNAqua-Net and attended this conference, where ca. 200 researchers gathered together to present results, discuss hot topics, and delineate the way forward for achieving a wide acceptance by management bodies of genetic methods for biodiversity assessment in the European context.

Xavier presented the communication "Under the canopy: community-wide effects of invasive algae in marine protected areas revealed by metabarcoding", by Xavier Turon, Owen Wangensteen, Emma Cebrian and Creu Palacín. In this contribution the main results concerning the effects of three invasive algae (Caulerpa cylindracea and Lophocladia lallemandii in Cabrera, and Asparagopsis armata in the Atlantic) on the benthic communities were exposed. We have also drafted and sent a manuscript for publication with these results. This contribution is also a result of the collaboration between ChallenGen and the National Park projects Metabarpark and Corclim.

A heatmap representation of the samples of communities with and without Asparagopsis armata from Cíes Islands, separated by invasion status (green: non-invaded, red: invaded) and fraction (blue: macrobenthos; turquoise: meiobenthos).

The methods developed for metabarcoding of hard bottom communities explained in an international book chapter

by Xavier Turon

Owen Wangensteen and Xavier Turon have been invited to write a chapter in the book "Marine Animal Forests. The Ecology of Benthic Biodiversity Hotspots", of the Springer International Publishing Company (ISBN: 978-3-319-17001-5), which is edited by Sergio Rossi, Lorenzo Bramanti, Andrea Gori and Covadonga Orejas. In this chapter, entitled "Metabarcoding techniques for assessing biodiversity of marine animal forests", Owen and Xavier explain the methods developed for analysing hard bottom communities using metabarcoding. We believe that it is crucial to setup standardized protocols, and that this book can be an excellent platform to share and disseminate our experience with the different steps of the metabarcoding process.

This chapter is now accepted for publication, and we foresee that it will be published in the forthcoming months.

Techniques developed in METABARPARK, such as the size fractionation of the samples, can be adopted by other teams working on metabarcoding of marine benthos.

ChallenGen at the workshop “Application of genomic tools for benthic monitoring”

by Xavier Turon

The 4-5 April 2016, the Natural History Museum of Geneva (Switzerland) hosted the workshop “Application of genomic tools for benthic monitoring of marine environment: from technology to legal and socio-economic aspects”, organised by Prof. Jan Pawlowski of the University of Geneva.

The workshop was intended as a forum for the exchange of ideas about the potential of eDNA- related techniques for assessing biodiversity and the impact of human activities on it. It was attended by environmental professionals, members of regulating bodies, and scientists. The presentations and discussion were very lively, and the overarching idea is that DNA-derived indicators may be effectively used in the field of environmental monitoring (as they are in other applied sciences). However, more development and standardization is necessary before they can be integrated with- or substitute altogether- current techniques using morphology-based indicators, which are slow, time-consuming, and reliant on a worlwide dwindling taxonomic expertise.

Xavier Turon and Owen Wangensteen contributed to the workshop with the talk “Issues in metabarcoding of marine benthos”, where they pinpointed some conflicting issues, such as the use of DNA vs RNA, the choice between 18S rDNA or COI markers, or the use of a fixed vs a variable threshold for clustering sequences into MOTUs. The talk was illustrated with results obtained in the projects CHALLENGEN and METABARPARK.

Xavier Turon and Tom Wilding, of the Scottish Association for Marine Science, at the entrance of the Natural History Museum, with a lovely pet!

Master Thesis of Manuel Orobitg presented!

by Xavier Turon

In November, 2015, Manuel Orobitg, student of the Master of Ocenagropahy and Management of the Marine Environment at the University of Barcelona, presented his MSc Thesis “Morphology vs metabarcoding. A comparison of techniques for biodiversity assessment in Maërl beds”, supervised by Xavier Turon, Creu Palacin and Owen Wangensteen. This Thesis reported results from the Metabarpark project, with which the ChallenGen project is closely collaborating.

Sampling of the Maërl community in Islas Cíes.

Assortment of crustaceans from the 1 mm fraction.

In this study, Manuel analysed morphologically samples collected at the same time as the ones for the genetic studies in the detritic bottoms of Islas Cies and Cabrera Archipelago. He focused his analyses on three major groups: polychaetes, crustaceans, and molluscs. He identified at the lowest level possible, with the help of taxonomists (a big thanks goes to Daniel Martin and Lídia Delgado) the species present in the 10 mm and 1 mm filtrates, and obtained biomass values by weight. The morphological analyses found 77, 45, and 44 morphospecies of polychaetes, crustaceans, and molluscs, respectively, while the genetic analyses yielded 182, 245, and 70 MOTUs, respectively, for the same size fractions. Thus, metabarcoding allowed the determination of a higher diversity, but the taxonomic precision attained was higher with morphology. On the other hand, all structural parameters (biomass dominance curves, alpha- and beta-diversity patterns) were markedly different between the metabarcoding and the morphology datasets, indicating that both methods capture different aspects of the biodiversity structure present. The morphological datasets tended to be quantitatively dominated by a few species, while metabarcoding found a more even distribution of biomass of the dominant MOTUs.

Seminar: DNA Metabarcoding

by Maria Casso

Yesterday, Xavier Turon and Owen Wangensteen participated at the seminar "DNA Metabarcoding", organised by the Biodiversity Research Institute (IRBio). DNA metabarcoding is a method to assess biodiversity which combines DNA based identification and next generation sequencing, and it can have several applications. The aim of the seminar was to show the potencial and limitations of the method.

The first lecture was Xavier's and it was an introduction about the impediments of taxonomy and how can metabarcoding methods contribute to assess biodiversity. After him, Owen talked about the specific case of metabarcoding of hard bottom marine communities. He also explained the few but critical stages of the pipeline were there isn't a consensus among scientists. It is really awesome to watch live how a new method is developing and be part of it.

The seminar took place at the School of Biology (UB). Many researchers and students attended, and some interesting questions came out. It was dense but also clarifying and motivating.

New paper published: Deep-sea, deep-sequencing: metabarcoding extracellular DNA from sediments of marine canyons

Getting sediment samples from a multicorer device.

by Xavier Turon

Another article with results from the ChallenGen Project is now published:

Guardiola M, Uriz MJ, Taberlet P, Coissac E, Wangensteen OS, Turon X. 2015. Deep-sea, deep-sequencing: metabarcoding extracellular DNA from sediments of marine canyons. PLoS One 10(10): e0139633. doi:10.1371/journal.pone.0139633

This is our first paper about the metabarcoding of marine communities. In this article we focus on deep-sea sedimentary communities. Samples were collected during the DOSMARES and INDEMARES projects from canyons and adjacent areas in the Catalan and Balearic Islands coasts.

This is the abstract of the article:

Marine sediments are home to one of the richest species pools on Earth, but logistics and a dearth of taxonomic work-force hinders the knowledge of their biodiversity. We characterized α- and β-diversity of deep-sea assemblages from submarine canyons in the western Mediterranean using an environmental DNA metabarcoding. We used a new primer set targeting a short eukaryotic 18S sequence (ca. 110 bp). We applied a protocol designed to obtain extractions enriched in extracellular DNA from replicated sediment corers. With this strategy we captured information from DNA (local or deposited from the water column) that persists adsorbed to inorganic particles and buffered short-term spatial and temporal heterogeneity. We analysed replicated samples from 20 localities including 2 deep-sea canyons, 1 shallower canal, and two open slopes (depth range 100-2,250 m). We identified 1,629 MOTUs, among which the dominant groups were Metazoa (with representatives of 19 phyla), Alveolata, Stramenopiles, and Rhizaria. There was a marked small-scale heterogeneity as shown by differences in replicates within corers and within localities. The spatial variability between canyons was significant, as was the depth component in one of the canyons where it was tested. Likewise, the composition of the first layer (1 cm) of sediment was significantly different from deeper layers. We found that qualitative (presence-absence) and quantitative (relative number of reads) data showed consistent trends of differentiation between samples and geographic areas. The subset of exclusively benthic MOTUs showed similar patterns of β-diversity and community structure as the whole dataset. Separate analyses of the main metazoan phyla (in number of MOTUs) showed some differences in distribution attributable to different lifestyles. Our results highlight the differentiation that can be found even between geographically close assemblages, and sets the ground for future monitoring and conservation efforts on these bottoms of ecological and economic importance.

Multidimensional Scaling (MDS) plots showing the differentiation found between the zones studied (BC, Blanes Canyon; OS, Blanes Open Slope; CC, Cap de Creus Canyon; MC, ;Menorca Canal; ST, Serra de Tramuntana Slope).