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 last meeting

by Maria Casso

The 17th November, in Barcelona, we had the (most probably) last meeting of the ChallenGen Project. Anna and Héctor talked about DNA libraries preparation for GBS-like methods. At the end of the meeting we put in common all the publications, conferences and other outputs the group published thanks to the ChallenGen Project. You can check out our publications here.

We will have news soon about the next project!

New paper published: Genetic diversity and demography of the red starfish

A beautiful specimen of the red starfish Echinaster sepositus.

by Alex Garcia-Cisneros

One of the most wonderful starfish of the Mediterranean suffered a large demographic expansion since the last maximum glacial period. However, its demographic expansion does not prevent it to have a weakness point: a low genetic diversity that might indicate the vulnerability of the species. These and more results were recently published by the ChallenGen team in a manuscript entitled “Low genetic diversity and demographic expansion in the red starfish Echinaster sepositus (Retzius 1816)” at the Scientific Reports.

The authors used both mitochondrial and nuclear markers to resolve the phylogeography and population genetics of the commonly named “red starfish” (Echinaster sepositus). The authors analysed samples from most of the distribution range of the species, with 15 localities distributed between both Mediterranean basins and the Atlantic Ocean.

Besides to demonstrate the low genetic diversity of the species compared with other echinoderms, the species showed a weak genetic structure within marine basins despite the a priori low dispersal potential of its lecithotrophic larva. The lecithotrophic larva in this species does not live more than few days before the settlement and therefore, it makes it difficult to connect distant or isolated populations. Furthermore, we found sharp differences in two Mediterranean localities, Cartagena and Livorno, that are located close to large marine harbours and coastal areas affected by industry, although our experimental design does not allow us to assess the effects of pollutants on the genetic structure.

Bar plots of the Bayesian clustering analysis obtained using STRUCTURE for different K values and based on the combination of both mitochondrial sequences (COI) and microsatellite loci.

Crossing Borders: Temporal genetic differentiation over oceanographic fronts

by Ferran Palero

A new paper from the CHALLENGEN team entitled “Temporal and spatial genetic differentiation in the crab Liocarcinus depurator across the Atlantic-Mediterranean transition” has been recently published in the JCR journal Scientific Reports.

Median joining haplotype network. Each circle represents a haplotype and its size is proportional to its frequency. Location acronyms are DELT (Delta de l’Ebre), VALE (València), ALAC (Alacant), WALB (Málaga) CADI (Cádiz).

Proportion of individuals assigned to the MED haplogroup in each location and sampled year from 1000 pseudoreplicates.

Spatial genetic studies often require sampling broadly separated areas, difficult to access simultaneously. Although comparing localities surveyed at different time periods might result in spurious genetic differentiation, there is a general believe on the stability of genetic structure through time, particularly if sampled localities are isolated or very distant. However, should we really expect stable genetic patterns in marine species? To test this important question, Marta Pascual and collaborators have assessed the time-variation of phylogeography patterns of the portunid crab Liocarcinus depurator across the Atlantic-Mediterranean transition. A partial fragment of the cytochrome oxidase I gene was sequenced in 366 individuals collected during three time periods from localities at both sides of each of the three main oceanographic discontinuities in the area: Gibraltar Strait, Almeria-Oran Front and Ibiza Channel. Although localities showed genetic fluctuations through time, a significant gradient was detected along the coast for all sampling periods. Significant inter-annual differences identified within the Alicante area, north of the Almeria-Oran Front, were associated with shifts in the relative contribution of Atlantic and Mediterranean water masses. The authors conclude that the persistence of a clinal pattern in the Atlantic-Mediterranean transition area despite local fluctuations suggests a complex balance of dispersal and selection.

Despite the Alicante population from 2007 had a larger proportion of Atlantic haplotypes, an overall genetic cline remains stable across the 3-year sampling period.

DNA barcoding the phyllosoma of Scyllarides squammosus: New paper out!

by Ferran Palero

A new paper from the ChallenGen team entitled “DNA barcoding the phyllosoma of Scyllarides squammosus (H. Milne Edwards, 1837) (Decapoda: Achelata: Scyllaridae)” has been recently published in the JCR journal Zootaxa.

As shown by their fierce-looking mouthparts, phyllosoma larvae rank high on the planktonic food-chain as specialized predators. Luckily for us, they generally do not grow above a few centimeters in total length!

Despite being the slipper lobster genera with the largest number of species with commercial importance, little is known of the unique long-lived planktonic phyllosoma stages of Scyllarides. Recently, a large and diverse collection of Scyllaridae phyllosoma from the Coral Sea was analysed by members of our team. DNA-barcoding and phylogenetic analyses allowed Ferran Palero and colleagues to identify several S. squammosus phyllosoma larvae, including stages that were previously undescribed or poorly known. From a combination of adult and larval morphology with molecular data, we could reveal inconsistencies with regard to the affinities among species assigned to Scyllarides. This new evidence will contribute to future studies addressing the phylogenetic relationships within the genus.

Furthermore, this new paper represents the first contribution of Rebeca Genis-Armero, a MSc. student recently graduated from the University of Valencia and a name to keep in mind for the future of scientific drawing and phyllosoma larvae.

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.

New paper published: Spatio-temporal patterns of genetic diversity of Styela plicata in harbour populations

by Xavier Turon

We add another paper to the Styela plicata history: “Stochasticity in space, persistence in time: genetic heterogeneity in harbour populations of the introduced ascidian Styela plicata”, by Mari-Carmen Pineda, Beatriz Lorente, Susanna López-Legentil, Creu Palacín and Xavier Turon, which has been published today (23rd June) in PeerJ.

In this study, we analysed genetic structure of the populations of Styela plicata in 9 harbours along the Iberian Mediterranean coast and adjacent Atlantic waters (>1,200 km range) at two time points 5 years apart (2009 and 2014). Using COI sequence data of ca. 400 specimens, we found strong spatial genetic structure, with significant differences among many populations, but no significant differences among years. Our results revealed spatial genetic heterogeneity and temporal homogeneity in S. plicata, suggesting a limited role of recurrent, vessel-mediated transport of organisms among small to medium-size harbours. Our study area is representative of many highly urbanized coasts with dense harbour networks. In these environments, the episodic chance arrival of colonisers appears to determine the genetic structure of populations and the genetic composition of these early colonising individuals persists in the respective harbours, at least over moderate time frames (five years) that encompass ca. 20 generations of S. plicata.

This article is yet another instance of the importance of the temporal component in studies of introduced species to unravel processes occurring during secondary dispersal of non-indigenous species.

Map of the Iberian Peninsula (NW Mediterranean) showing the sampling sites of Styela plicata. Pie charts represent haplotype frequencies for the COI gene in each population analysed in 2009 and 2014.

Measuring settlement rates of sea urchins. New paper out!

by Owen Wangensteen

Team members of the ChallenGen Project at the University of Barcelona, in collaboration with scientists from the University of La Laguna (Canary Islands) have just published a new paper on Scientia Marina journal, describing and testing new designs of artificial collectors for quantitative assessment of settlement rates in sea urchins.

The study of population dynamics of sea urchins is crucial for understanding the ecology of Mediterranean and temperate Atlantic rocky shores, since these echinoderms are often the main herbivore species in shallow benthic ecosystems and they are responsible of linking the primary production from seaweeds to other higher levels of the trophic chains. Many sea urchin populations may be limited by the settlement rates (the process by which the planktonic sea urchin larvae settle and become juvenile sea urchins). Methods for measuring these settlement rates were not standardized and yielded non-comparable results. The work presented in this paper, carried out in parallel at Canary Islands and Costa Brava (Spain) shows that three-dimensional collectors made from “bioballs” (a biofilm-friendly, high surface material often used as filters in aquaria) are the most suitable device for this assessment, allowing for an accurate and repeatable measurement of this elusive, yet important, ecological process.

The best part is that these bioballs collectors are useful not only for sea urchins settlers. They could be suitable for assessing settlement and colonization rates for a wide-range of marine invertebrates and can help marine biologists to get a more accurate view of the population dynamics of many species present in benthic ecosystems.

The three types of collector tested in this work: plastic biofilter ball (A, C), vertical brush (B, D) and horizontal triangular mat of coconut fibre (B, E).

Temporal genetic structure in the introduced ascidian Styela plicata

by M.Carmen Pineda

It’s been almost three years since the last paper on Styela plicata was published by our group, but the saga is not over yet!! The article entitled “Stable populations in unstable habitats: temporal genetic structure of the introduced ascidian Styela plicata in North Carolina”, by M.Carmen Pineda, Xavier Turon, Rocio Perez-Portela and Susanna Lopez-Legentil, has been just published online in the journal Marine Biology.

The solitary ascidian Styela plicata.

Styela plicata is a solitary ascidian, which has been introduced by ship fouling to harbours and marinas around the globe. This species has been extensively studied during the last years, based on its potential to become a plague. However, this is the first time that its genetic composition is studied on a temporal scale. On this recent study, a combination of nuclear and mitochondrial markers (7 microsatellites and a fragment of the gene Cytochrome Oxidase subunit I, COI) were used to analyse the genetic composition of one population subjected to environmental fluctuations and periodical die-offs, every 2 months and over 2.5 years. Overall, our results indicate that the investigated population is stable over time and relies on a periodic arrival of larvae from other populations, maintaining high genetic diversity and a complex interplay of allele gains and losses.

Temporal analyses exploring genetic trends over time are a great tool to predict the likelihood of long time survival of an introduced population in a new habitat and its invasive potential. This kind of information is particularly relevant when deciding which introduced species are more detrimental, and should help resource managers to focus their control and eradication efforts.

New paper published: Clavelina oblonga in the Ebro Delta

by Xavier Turon

The article “When invasion biology meets taxonomy: Clavelina oblonga (Ascidiacea) is an old invader in the Mediterranean Sea” by Víctor Ordóñez, Marta Pascual, Margarita Fernández and Xavier Turon is now published online in Biological Invasions.

An oyster culture rope totally covered by colonies of Clavelina oblonga.

In this paper we report the taxonomic status of a marine pest that has recently proliferated in bivalve cultures of the Ebro Delta, smothering mussel spat and causing concern among fishermen. The species was initially identified as Clavelina phlegraea, described in 1929 in Italy and endemic of the Mediterranean. Further morphological and genetic studies, however, have concluded that C. phlegraea is a synonym of the Atlantic species C. oblonga, which has been introduced into the Mediterranean at least 80 years ago, but was known under a different name there. This study illustrates the problems of traditional taxonomy to cope with the global-scale distribution of introduced species.

We have also monitored growth and reproductive cycles of C. oblonga in the Ebro Delta, which were markedly seasonal, with peak abundance and reproduction in summertime, followed by regression in winter. Avoidance of mussel and oyster seeding during late summer and early autumn can readily reduce the damage caused by this species.

New paper published

by Xavier Turon

New paper published in Frontiers in Zoology: “Feeding cessation alters host morphology and bacterial communities in the ascidian Pseudodistoma crucigaster”, by Susanna López-Legentil, Xavier Turon and Patrick M. Erwin.

This article is the result of yet another collaboration between the projects ChallenGen and MarSymbiOmics. We have coupled electron microscopy and 16S rRNA gene tag pyrosequencing to investigate the bacterial communities associated with the colonial ascidian Pseudodistoma crucigaster, a species endemic to the Mediterranean Sea that has a life cycle with two phases: actively-filtering (active) and non-filtering (resting) forms.

Active (A) and resting (B) colonies of Pseudodistoma crucigaster. The active form has functional siphonal apertures for filtering water, while the resistance form has all apertures sealed by a glassy cuticle.

Resting colonies exhibited a reduced branchial sac (feeding apparatus) and a thickened cuticle. Electron microscope images also suggested higher abundance of colonizing microorganisms on surfaces of resting colonies. Accordingly, bacterial sequences associated with environmental sources (sediment and biofilms, >99 % similarity) were detected exclusively in resting colonies. Bacterial communities of P. crucigaster colonies (active and resting) were dominated by 3 core taxa affiliated (>94 % similarity) with previously described symbiotic Alphaproteobacteria in marine invertebrates. Shifts in rare bacteria were detected when ascidians entered the resting phase, including the appearance of strictly anaerobic lineages and nitrifying bacterial guilds.

These findings suggest that physical (thickened cuticle) and metabolic (feeding cessation) changes in host ascidians have cascading effects on associated bacteria, where modified oxygen concentrations and chemical substrates for microbial metabolism may create anaerobic microhabitats and promote colonization by environmental microorganisms.

Network of bacterial OTUs in three active and three resting samples of Pseudodistoma crucigaster, with edges coded by specificity.

Starfish telomeres on a radio interview

by Alex Garcia

The starfish Coscinasterias tenuispina has the ability to reproduce sexually and asexually by fission, and many populations are even maintained by only asexual reproduction. One of our issues was to investigate if asexual populations can be maintained indefinitely. Our findings revealed that the starfish elongates their telomeres after fission, regenerating their DNA. Telomeres are repetitive sequences at the caps of chromosomes and are correlated with the age of individuals, therefore, in some point of view; the starfish have a mechanism to rejuvenate themselves.

Different media has been interested in this finding after its publication in Heredity. Among them, the radio program “Al otro lado del espejo” in which I participated last week.

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).

New article of ChallenGen accepted!

by Xavier Turon

Another article including results from ChallenGen has been accepted for publication in Frontiers in Microbiology. The title of the paper is “Temporal stability of bacterial symbionts in a temperate ascidian” by López-Legentil S, Turon X, Espluga R, Erwin PM.

This article is the result of collaborative work between the projects ChallenGen and MarSymbiOmics (CTM2013-43287). The goal of MarSymbiOmics is the study of symbioses of invertebrates from a molecular perspective. It is only natural that we coordinate efforts… We are more focused on the genetics of the host, while the MarSymbiOmics team is interested in the genetics of the microbiome.

A colony of Didemnum fulgens in the monitoring site at L’Escala (Catalan coast, NW Mediterranean).

In this particular study we have monitored the population of an ascidian species, Didemnum fulgens, over an annual cycle. Its pattern of growth and reproduction has been published a while ago (López-Legentil S, Erwin PM, Velasco M, Turon X. 2013. Growing or reproducing in a temperate sea: optimization of resource allocation in a colonial ascidian, Invertebrate Biology 132:69-80).

Now we publish the results of the temporal changes in microbiome composition of that same population using terminal restriction fragment length polymorphism (T-RFLP) and clone library analyses based on the16S rRNA gene. The overall bacterial community in D. fulgens had a distinct signature from the surrounding seawater and was stable over time and across seasonal fluctuations in temperature. Bacterial symbionts were also observed in the inner tunic of larvae of D. fulgens. Our results suggest that, as seen for sponges and corals, some species of ascidians host stable and unique bacterial communities that are at least partially inherited by their progeny by vertical transmission.

Soon available in Open Access!!!

New paper published: Corridors for aliens but not for natives: effects of marine urban sprawl at a regional scale

"Marine urban sprawl creates corridors for invasives

It is well established that corridors can, in certain cases, help spread non-native species in terrestrial ecosystems. In a new study by Airoldi et al., the authors expand on this theme with a look at the spread of non-native species at a regional scale. This study compares artificial marine infrastructure (ex. harbors, dikes, piers, breakwaters, etc.) and natural reefs. Their extensive look at 500 kilometers of shoreline in the North Adriatic Sea provides clues as to how marine corridors contribute to the spread of native and non-native species alike."

Read more here.

Paper Reference: Airoldi, L., X. Turon, S. Perkol-Finkel, and M. Rius. 2015. Corridors for aliens but not for natives: effects of marine urban sprawl at a regional scale. Diversity and Distributions 21: 755-768.

New paper published: Applications of next generation sequencing to the study of biological invasions

"Genomics holds the key to understanding ecological and evolutionary processes
Scientists call for a gap between genomics and invasion biology to be bridged

Scientists at the University of Southampton think that Next-Generation Sequencing (NGS) of invasive organisms holds the key to furthering our understanding of ecological and evolutionary processes."

Read more at the University of Southampton website.

Paper Reference: Rius M, Bourne S, Hornsby HG, Chapman MA. 2015. Applications of next generation sequencing to the study of biological invasions. Current Zoology 61: 488–504