from genes to communities
Published March 25 , 2024
In January, 22nd, Carles Galià-Camps presented his PhD dissertation “An omics world: colonisation and adaptation processes of Styela plicata”. He got the highest mark “excellent cum Laude”, congratulations!
This thesis has been done within the previous project of the group (PopCOmics) an the MARGECH project. The supervisors have been Marta Pascual, Carles Carreras, and Xavier Turon.
This is the abstract of the Thesis:
In the last decades biological invasions have emerged as a global concern, as they impact local biodiversity by disrupting ecosystem functioning and ultimately affect human health, economy and wellness. In a climate change scenario, catalyzed by globalization, the issue of invasive species is expected to become magnified in the near future. However, little is known about the intrinsic mechanisms allowing invasive species to overcome new conditions and become a major threat worldwide. This PhD thesis aims to study the colonization and adaptive processes of the invasive tunicate S. plicata worldwide using ‘-omic’ approaches, including population genomics and microbiome. By combining multiple sequencing techniques such as PacBio Long Reads, Illumina Whole Genome Sequencing (WGS), Illumina RNAseq and Illumina amplicon sequencing, we have adopted multiple approaches to the study of this species. First, by using 80 publicly available genomes, we evaluated general output trends when using the 2b-RADseq technique depending on the genomic architecture of target species, demonstrating that overall the technique does not generate biases although it slightly enriches exonic regions. Furthermore, by empirically testing the effect of 2b-RADseq base-selective adaptors on genotyping using four S. plicata individuals, we paved the way for cost-effective population studies on this species. Next, we built and annotated a “de-novo” reference genome for S. plicata, and combined it with Illumina WGS of 24 individuals worldwide. The resulting pangenome revealed the presence of 4 potentially adaptive inversions, the first evidence of population structure, SNPs related to local adaptation, and mito-nuclear interactions. These methodological improvements and the generation of a reference genome assembly allowed us to analyze 87 individuals from 18 localities worldwide using 2b-RADseq to further reveal its population genomic structure, finding clear signals of local adaptation and recent demographic bottlenecks related to invasive events. Finally, using Illumina sequencing of the V4 region of the 16S gene of several tissues of juvenile and adult individuals and water samples from three harbors, in addition to adult samples on two of these harbors over two years, we shed light on the dynamics of the microbiome hosted by S. plicata. We revealed that Methylocaeanibacter sp. ASV0 is the most important bacteria in the tunic, whereas Gammaproteobacteria ASV1 and the unidentified bacteria ASV2 are so in the gill for holobiont survival. Moreover, we found evidence of abundance shifts through adulthood for these bacteria, suggesting active enrichment by S. plicata towards its symbiotic bacterial community. Finally, we revealed potential adaptive bacteria responding to seasonality and trace elements such as Candidatus Hepatoplasma sp. ASV11 and Endozoicomonas sp. ASV50, among others. This multilevel approach provides a solid basis to properly understand the mechanisms of Styela plicata and other invasive marine species to colonize and quickly adapt to new habitats.
​
​
​
​
​
​
​
​
​
​
​
​
Amazing art cover of the Thesis, by Jagoba Malumbres-Olarte
​
Published March 19 , 2024
We have published our first paper on the microbiome of the introduced ascidian Styela plicata. We found that multiple factors determine the structure of the microbiome, such as compartment studied (tunic, gill, gut), ontogeny (juvenile vs adult), and space (comparing populations of different ports). We hope you will enjoy reading!
Galià-Camps C, Baños E, Pascual M, Carreras C, Turon X
Multidimensional variability of the microbiome of an invasive ascidian species
Published in iScience 26:107812, 2023
Here you have the old-style abstract and the graphical abstract:
Animals, including invasive species, are complex entities consisting of a host and its associated symbionts (holobiont). The interaction between the holobiont components is crucial for the host’s survival. However, our understanding of how microbiomes of invasive species change across different tissues, localities, and ontogenetic stages, is limited. In the introduced ascidian Styela plicata, we found that its microbiome is highly distinct and specialized among compartments (tunic, gill, and gut). Smaller but significant differences were also found across harbors, suggesting local adaptation, and between juveniles and adults. Furthermore, we found a correlation between the microbiome and environmental trace element concentrations, especially in adults. Functional analyses showed that adult microbiomes possess specific metabolic pathways that may enhance fitness during the introduction process. These findings highlight the importance of integrated approaches in studying the interplay between animals and microbiomes, as a first step toward understanding how it can affect the species’ invasive success.
​
​
​
​
​
​
​
​
​
​
​
​
Microbial community scheme and concentration of accumulated pollution in Styela plicata.
​
PAPER LINK: https://www.cell.com/iscience/pdf/S2589-0042(23)02665-2.pdf
​
Published December 20 , 2023
Metabarcoding techniques are revolutionizing studies of marine biodiversity. They can be used for monitoring non-indigenous species (NIS) in ports and harbors. However, they are often biased by inconsistent
sampling methods and incomplete reference databases. Logistic constraints in ports prompt the development of simple, easy-to-deploy samplers. We tested a new device called polyamide mesh for ports organismal monitoring (POMPOM) with a high surface-to-volume ratio. POMPOMS were deployed inside a fishing and recreational port in the Mediterranean alongside conventional settlement plates. We also compiled a curated database with cytochrome oxidase (COI) sequences of Mediterranean NIS. COI metabarcoding of the communities settled in the POMPOMs captured a similar biodiversity than settlement plates, with shared molecular operational units (MOTUs) representing ca. 99% of reads. 38 NIS were detected in the port accounting for ca. 26% of reads. POMPOMs were easy to deploy and handle and provide an efficient method for NIS surveillance.
​
​
​
​
​
Results obtained from invasive species present in the port of Blanes using two methods of sample collection.
​
PAPER LINK: https://www.cell.com/iscience/pdf/S2589-0042(23)02665-2.pdf
​
Published April 12, 2023​
Our last paper, with the results of the metabarcoding study performed on the Posidonia oceanica beds in Cabrera, has been published in Frontiers of Marine Science.
This is the reference:
Turon X, Zarcero J, Antich A, Wangensteen OS, Ballesteros E, Cebrian E, Marco-Méndez C, Alcoverro T. 2023. Metabarcoding the eukaryotic community of a threatened, iconic Mediterranean habitat: Posidonia oceanica seagrass meadows. Frontiers in Marine Science, 10:1145883. DOI: 10.3389/fmars.2023.1145883.
Is is an open access paper and here is the link:
https://www.frontiersin.org/articles/10.3389/fmars.2023.1145883/full
We hope you will find it interesting and will enjoy reading, here is the abstract:
Against the accelerating pace of worldwide species extinction, reliable biodiversity assessments are critical, both as baselines and to track potential declines. DNA metabarcoding techniques allow for fast and comprehensive assessment of biodiversity in both terrestrial and marine habitats. However, these methods need to be adapted and standardised for each ecosystem in order to be effective. Seagrass meadows are among the most diverse marine habitats and are irreplaceable in terms of the ecosystem services they provide, yet metabarcoding has never been implemented for these systems. In this study, we developed and tested a protocol for metabarcoding the eukaryotic community of meadows of the iconic species, Posidonia oceanica L. (Delile). This seagrass is the main habitat-forming species in Mediterranean coastal waters and is known for its high diversity due to the structural complexity of its canopy and rhizome structures. This habitat is experiencing a range-wide retreat, and there is an urgent need for fast and efficient methods for its biomonitoring and detection of early changes. Our proposed method involves direct sampling of the community, collecting and processing the leaves and rhizome strata separately. To test the utility of the method in distinguishing between different meadow conditions, we sampled two distinct meadows that differ in their prevailing wind and surge conditions, and a nearby rocky reef for comparison. We then adapted a method and pipeline for COI metabarcoding using generalist primers that target the eukaryote diversity present. We detected a high diversity in the two meadows analysed (3,350 molecular operational taxonomic units, dominated by Metazoa and Archaeplastida) and a clear differentiation of the seagrass samples from those of the nearby rocky reefs. The leaves and rhizomes harboured clearly distinct assemblages, and differences were also detected between the two meadows sampled. This new tool has the potential to deliver big biodiversity data for seagrass habitats in a fast and efficient way, which is crucial for the implementation of protection and management measures for this key coastal habitat.
.jpeg)
Image of a Posidonia oceanica meadow in Cabrera Island (photo Kike Ballesteros)
Heatmap and cluster of the Posidonia oceanica samples. Colour codes indicate stratum (horizontal bars) or meadow (vertical bars).
Published February 22, 2023​
Time passes fast, and we are already in the mid-term of the project. On this occasion, we held a general meeting in February, the 21st, hosted by the University of Barcelona team at the Faculty of Biology.
It was an enjoyable experience attended by the two teams (UB and CEAB) of the project. We were updated on the ongoing research by talks given by Mathilde, Carles G., Marta, Eli, Adrià and Jesús.
New projects related to MarGech and granted to members of the teams were presented by Carles C., Marc, and Xavier.
Xavier and Carles C. presented progress reports on the two subprojects, with already a lot of interesting results and 16 papers published in 18 months!
Finally, there was a wrap-up presentation and an open discussion on several aspects of the project unfolding and its research, outreach and dissemination activities.
A nice coffee break plus a lunch at the Biology Cafeteria completed the event and contributed to generate a good atmosphere for interaction, socialization, and feed-back between the team members.
We are already looking forward for having the next general meeting of MarGech!
A group photo of the attendant MarGech members in front of the Faculty of Biology at the University of Barcelona
Metabarcoding reveals high-resolution biogeographical and metaphylogeographical patterns through marine barriers
By Adrià Antich, Creu Palacín, Jesús Zarcero, Owen Simon Wangensteen, Xavier Turon
Journal of Biogeography, 2023, 50:515-527
Published February 13, 2023​
Aim Biogeographic and phylogeographic processes are linked over evolutionary time. We hypothesize that oceanographic barriers in the marine environment affect concomitantly the distribution and the connectivity of the marine biota, resulting in congruent biogeographic and phylogeographic structures. We also predict that macro- and meio-eukaryotes will be differentially affected by hydrological features.
Location The Atlanto-Mediterranean transition along the E Iberian coast marked by the Almeria-Oran Front and the Ibiza Channel hydrological discontinuities.
Taxa Eukaryotes
Methods A new analytical framework based in the metabarcoding of community DNA and a hypervariable marker is presented. This framework allows the simultaneous detection of multispecies biogeographic and phylogeographic structures. Shallow hard-bottom communities were sampled at 12 sites over the littoral zone and community-DNA metabarcoding was performed using the cytochrome c oxidase I (COI) marker. The resulting dataset was analysed at several levels: beta diversity of Molecular Operational Taxonomic Units (MOTUs) as surrogate for species, and Exact Sequence Variants (ESVs) as surrogate for haplotypes. We also assessed genetic differentiation within MOTUs (metaphylogeography). Analyses were performed for the combined dataset and separately for macro- and meio-eukaryotes.
Results Both hydrological discontinuities had a detectable effect, more marked at all levels for the AOF than for the IC. The MOTU dataset provided more clear-cut patterns than the ESVs. The metaphylogeographic approach provided the highest resolution in terms of differentiating localities and identifying geographic barriers. The separate analyses of macro- and meio-eukaryotes showed a higher differentiation of the latter, both in terms of beta diversity and genetic differentiation.
Main conclusions Metabarcoding coupled with metaphylogeography allowed the characterization of the heterogeneity in community composition and population genetic structure along the Atlanto-Mediterranean transition, coherent with known hydrological discontinuities. This methodology unlocks a vast amount of information on the geographic distribution of different components of biodiversity for basic and applied research.
Keywords: Benthos, Biogeography, Cytochrome c oxidase I, Eukaryotes, Marine Barriers, Metabarcoding, Metaphylogeography
Results of the network analysis performed using genetic distances within MOTUs (metaphylogeography)
Adrià Antich got an “Excellent cum laude” in his PhD dissertation.
In October, 24th, Adrià Antich defended his PhD dissertation entitled “Biodiversity assessment of marine benthic communities with COI metabarcoding: methods and applications”, and supervised by Owen Wangensteen, Creu Palacin and Xavier Turon.
​
Published October 28, 2022​
The thesis work has been done in part within the framework of the project MARGECH, and the candidate and their supervisors are members of the research team of the project. The thesis abstract follows:
Ecosystem biomonitoring is crucial for proper management of natural communities during the Anthropocene era. With the advent of new sequencing technologies, DNA metabarcoding has been proposed as a game-changing tool for biomonitoring. In this Thesis we plead for the use of metabarcoding of a highly variable marker to infer not only the interspecies but also the intraspecies variability to assess both biogeographic, at the species level, and metaphylogeographic patterns, at the haplotype level. Our focus was on highly complex hard-substratum benthic littoral communities. The term "Metaphylogeography", coined in this Thesis, refers to the study of phylogeographic patterns of many species at the same time using metabarcoding data. However, as of the start of this Thesis, only a few studies had tested the metabarcoding method to directly characterize the whole eukaryotic community in highly diverse benthic ecosystems. This required to set up and calibrate methods for these communities as a prior step.
We first evaluated both the sampling methods and the bioinformatic pipelines. We assessed the viability of detecting the environmental DNA released from the benthic community into the adjacent water layer using metabarcoding of COI with highly degenerated primers targeting the whole eukaryotic community. We sampled water from 0 to 20m from shallow rocky benthic communities and compared the DNA signal with the results obtained from metabarcoding directly the benthic communities by traditional quadrat sampling. We also designed a pipeline combining clustering and denoising methods to treat metabarcoding data of COI. We considered the entropy of each codon position of this coding fragment both to improve the detection of spurious sequences and to calibrate the best performing parameters of the software used. In addition, we created our own denoising program, DnoisE, to incorporate information on the codon position. This new code and parameter calibration were required as the commonly used bioinformatic pipelines had been designed and tested mostly for less variable ribosomal fragments and, particularly, in prokaryotes.
Results showed that the DNA signal from the benthos decreased with the distance but was too weak for a correct assessment of benthic biodiversity. The proportion of eukaryotic DNA sequenced was also very low in water samples due to the amplification of prokaryotic DNA. We thus concluded that the benthos must be sampled directly to properly assess its biodiversity composition. The new bioinformatic developments allowed us to propose new methods for processing metabarcoding reads, combining clustering and denoising steps, and to set optimal values for the parameters used at each step. These contributions effectively expanded the field to the novel analysis of inter- and intraspecies genetic variability with metabarcoding data.
Finally, we applied this methodology to 12 localities of the Western Iberian Coast along two well studied fronts, the Almeria-Oran Front (AOF) and the Ibiza Channel (IC). We analysed the species and haplotypes using the COI barcode. From a biogeographical perspective, the AOF had a strong effect in separating regions, while the IC effect was less marked, but still half of the MOTUs were found in only one side of this divide. For the metaphylogeographic analysis, only 10% of the MOTUs could be used. However, they showed a good separation between populations of the three regions with a strong effect of the AOF break. The IC, on the other hand, seemed to be more a transitional zone than a fixed break.
This Thesis laid the ground for the efficient use of metabarcoding in the biomonitoring of benthic reef habitats, allowing community composition, $\beta$-diversity, and biogeographic patterns to be analysed in a fast, repeatable, and cost-efficient way. We also developed the metaphylogeography approach as a new tool to assess population genetic structure at the community-wide level.
​Adrià got an “Excellent cum laude”, the highest mark possible. Congratulations Dr. Antich!
​
​
​
​
​
​
​
​
​
​
​
​
​
​
​
​
​
​
​
​
Adrià during his dissertation at the “Aula Magna” of the Faculty of Biology
​
New Margech paper
​
Abstract
​
DNA metabarcoding is broadly used in biodiversity studies encompassing a wide range of organisms. Erroneous amplicons, generated during amplification and sequencing procedures, constitute one of the major sources of concern for the interpretation of metabarcoding results. Several denoising programs have been implemented to detect and eliminate these errors. However, almost all denoising software currently available has been designed to process non-coding ribosomal sequences, most notably prokaryotic 16S rDNA. The growing number of metabarcoding studies using coding markers such as COI or RuBisCO demands a re-assessment and calibration of denoising algorithms. Here we present DnoisE, the first denoising program designed to detect erroneous reads and merge them with the correct ones using information from the natural variability (entropy) associated to each codon position in coding barcodes. We have developed an open-source software using a modified version of the UNOISE algorithm. DnoisE implements different merging procedures as options, and can incorporate codon entropy information either retrieved from the data or supplied by the user. In addition, the algorithm of DnoisE is parallelizable, greatly reducing runtimes on computer clusters. Our program also allows different input file formats, so it can be readily incorporated into existing metabarcoding pipelines.
Figure 1: Scheme of the workflow of DnoisE.
The Margech team gets underway!
