142 research outputs found
Mock samples resolve biases in diversity estimates and quantitative interpretation of zooplankton metabarcoding data
Metabarcoding is a rapidly developing tool in marine zooplankton ecology, although most zooplankton surveys continue to rely on visual identification for monitoring purposes. We attempted to resolve some of the biases associated with metabarcoding by sequencing a 313-b.p. fragment of the COI gene in 34 “mock” samples from the North Sea which were pre-sorted to species level, with biomass and abundance estimates obtained for each species and taxonomic group. The samples were preserved either in 97% ethanol or dehydrated for 24 h in a drying oven at 65 °C (the routine way of preserving samples for dry weight measurements). The visual identification yielded a total of 59 unique holoplanktonic and 16 meroplanktonic species/taxa. Metabarcoding identified 86 holoplanktonic and 124 meroplanktonic species/taxa, which included all but 3 of the species identified visually as well as numerous species of hard-to-identify crustaceans, hydrozoan jellyfish, and larvae of benthic animals. On a sample-to-sample basis, typically 90–95% of visually registered species were recovered, but the number of false positives was also high. We demonstrate robust correlations of relative sequence abundances to relative biomass for most taxonomic groups and develop conversion factors for different taxa to account for sequencing biases. We then combine the adjusted sequencing data with a single bulk biomass measurement for the entire sample to produce a quantitative parameter akin to species biomass. When examined with multivariate statistics, this parameter, which we call BWSR (biomass-weighed sequence reads) showed very similar trends to species biomass and comparable patterns to species abundance, highlighting the potential of metabarcoding not only for biodiversity estimation and mapping of presence/absence of species but also for quantitative assessment of zooplankton communities.publishedVersio
To denoise or to cluster? That is not the question. Optimizing pipelines for COI metabarcoding and metaphylogeography
Background: The recent blooming of metabarcoding applications to biodiversity studies comes with some relevant methodological debates. One such issue concerns the treatment of reads by denoising or by clustering methods, which have been wrongly presented as alternatives. It has also been suggested that denoised sequence variants should replace clusters as the basic unit of metabarcoding analyses, missing the fact that sequence clusters are a proxy for species-level entities, the basic unit in biodiversity studies. We argue here that methods developed and tested for ribosomal markers have been uncritically applied to highly variable markers such as cytochrome oxidase I (COI) without conceptual or operational (e.g., parameter setting) adjustment. COI has a naturally high intraspecies variability that should be assessed and reported, as it is a source of highly valuable information. We contend that denoising and clustering are not alternatives. Rather, they are complementary and both should be used together in COI metabarcoding pipelines. Results: Using a COI dataset from benthic marine communities, we compared two denoising procedures (based on the UNOISE3 and the DADA2 algorithms), set suitable parameters for denoising and clustering, and applied these steps in diferent orders. Our results indicated that the UNOISE3 algorithm preserved a higher intra-cluster variability. We introduce the program DnoisE to implement the UNOISE3 algorithm taking into account the natural variability (measured as entropy) of each codon position in protein-coding genes. This correction increased the number of sequences retained by 88%. The order of the steps (denoising and clustering) had little infuence on the fnal outcome. Conclusions: We highlight the need for combining denoising and clustering, with adequate choice of stringency parameters, in COI metabarcoding. We present a program that uses the coding properties of this marker to improve the denoising step. We recommend researchers to report their results in terms of both denoised sequences (a proxy for haplotypes) and clusters formed (a proxy for species), and to avoid collapsing the sequences of the latter into a single representative. This will allow studies at the cluster (ideally equating species-level diversity) and at the intra-cluster level, and will ease additivity and comparability between studies
Metabarcoding reveals high-resolution biogeographical and metaphylogeographical patterns through marine barriers
Aim: It has been predicted that there should be concordance between biogeographical and phylogeographical processes structuring multi-species regional assemblages.
We hypothesise that oceanographic barriers in the marine environment affect concomitantly the distribution and the connectivity of the marine biota, thus producing
congruent biogeographical and phylogeographical 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 (AOF) and the Ibiza Channel hydrological discontinuities.
Taxon: 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 biogeographical and phylogeographical 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 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 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 Ibiza Channel. The MOTU dataset provided more
clear-cut patterns than the ESVs. The metaphylogeographical approach provided the
highest resolution in terms of differentiating localities and identifying geographical
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
characterisation 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 geographical distribution of different components of biodiversity for basic and
applied research
Using DNA Metabarcoding to Characterize the Prey Spectrum of Two Co-Occurring Themisto Amphipods in the Rapidly Changing Atlantic-Arctic Gateway Fram Strait
The two congeneric hyperiids Themisto libellula and T. abyssorum provide an important
trophic link between lower and higher trophic levels in the rapidly changing Arctic marine ecosystem.
These amphipods are characterized by distinct hydrographic affinities and are hence anticipated
to be impacted differently by environmental changes, with major consequences for the Arctic food
web. In this study, we applied DNA metabarcoding to the stomach contents of these Themisto species,
to comprehensively reveal their prey spectra at an unprecedented-high-taxonomic-resolution and
assess the regional variation in their diet across the Fram Strait. Both species feed on a wide variety
of prey but their diet strongly differed in the investigated summer season, showing overlap for only
a few prey taxa, such as calanoid copepods. The spatially structured prey field of T. libellula clearly
differentiated it from T. abyssorum, of which the diet was mainly dominated by chaetognaths. Our
approach also allowed the detection of previously overlooked prey in the diet of T. libellula, such as
fish species and gelatinous zooplankton. We discuss the reasons for the differences in prey spectra
and which consequences these may have in the light of ongoing environmental changes
DNA metabarcoding reveals the importance of gelatinous zooplankton in the diet of Pandalus borealis, a keystone species in the Arctic
Information about the dietary composition of a species is crucial to understanding their position and role in the food web. Increasingly, molecular approaches such as DNA metabarcoding are used in studying trophic relationships, not least because they may alleviate problems such as low taxonomic resolution or underestimation of digestible taxa in the diet. Here, we used DNA metabarcoding with universal primers for cytochrome c oxidase I (COI) to study the diet composition of the northern shrimp (Pandalus borealis), an Arctic keystone species with large socio-economic importance. Across locations, jellyfish and chaetognaths were the most important components in the diet of P. borealis, jointly accounting for 40%–60% of the total read abundance. This dietary importance of gelatinous zooplankton contrasts sharply with published results based on stomach content analysis. At the same time, diet composition differed between fjord and shelf locations, pointing to different food webs supporting P. borealis in these two systems. Our study underlines the potential of molecular approaches to provide new insights into the diet of marine invertebrates that are difficult to obtain with traditional methods, and calls for a revision of the role of gelatinous zooplankton in the diet of the key Arctic species P. borealis, and in extension, Arctic food webs
Spatio-temporal patterns of eukaryotic biodiversity in shallow hard-bottom communities from the West Antarctic Peninsula revealed by DNA metabarcoding
Aim: We studied molecular eukaryotic biodiversity patterns in shallow hard-bottom
Antarctic benthic communities using community DNA metabarcoding. Polar ecosystems are extremely exposed to climate change, and benthic macroinvertebrate communities have demonstrated rapid response to a range of natural and anthropogenic
pressures. However, these rich and diverse ecosystems are poorly studied, revealing
how little is known about the biodiversity of the Antarctic benthos associated with
hard-bottom habitats.
Location: West Antarctic Peninsula and South Shetland Islands.
Methods: Using data collected in seven localities along the western Antarctic
Peninsula, we calculated spatial patterns of alpha and beta diversities. Furthermore,
we analysed temporal changes in benthic composition in one location (Deception
Island) over 3 years. We calculated the temporal alpha and beta diversities to reveal
changes in this community over time.
Results: We obtained a final list of 2057 molecular operational taxonomic units. We
found significant differences in benthic community composition between localities
and among years. Our dataset revealed a total of 10 different kingdom-level lineages
and 34 different phyla in the samples. The most diverse phylum was Arthropoda, followed by Bacillariophyta, and Annelida, while the highest relative read abundances
belonged to Annelida, Porifera and Echinodermata. Benthic community compositions
changed between 2016 and 2018 in Deception Island, and decreasing species richness was the main component of temporal beta diversity.
Main Conclusions: Direct sampling methods are required for monitoring these complex communities. Informative biodiversity patterns can be retrieved even though
most of the benthic biodiversity found in Antarctic habitats is yet to be taxonomically
described and barcoded. Hard-bottom assemblages exhibit high spatial variability
and heterogeneity, not related to depth, which represent a huge challenge for largescale studies in the Southern Ocean. Local patchiness and structure within the communities are probably a consequence of a combination of several biotic and abiotic factors (i.e. ice disturbance, food supply and competition)
For all audiences : Incorporating immature stages into standardised spider inventories has a major impact on the assessment of biodiversity patterns
Although arthropods are the largest component of animal diversity, they are traditionally underrepresented in biological inventories and monitoring programmes. However, no biodiversity assessment can be considered informative without including them. Arthropod immature stages are often discarded during sorting, despite frequently representing more than half of the collected individuals. To date, little effort has been devoted to characterising the impact of discarding nonadult specimens on our diversity estimates. Here, we used a metabarcoding approach to analyse spiders from oak forests in the Iberian Peninsula, to assess (1) the contribution of juvenile stages to local diversity estimates, and (2) their effect on the diversity patterns (compositional differences) across assemblages. We further investigated the ability of metabarcoding to inform on abundance. We obtained 363 and 331 species as adults and juveniles, respectively. Including the species represented only by juveniles increased the species richness of the whole sampling in 35% with respect to those identified from adults. Differences in composition between assemblages were greatly reduced when immature stages were considered, especially across latitudes, possibly due to phenological differences. Moreover, our results revealed that metabarcoding data are to a certain extent quantitative, but some sort of taxonomic conversion factor may be necessary to provide accurate informative estimates. Although our findings do not question the relevance of the information provided by adult-based inventories, they also reveal that juveniles provide a novel and relevant layer of knowledge that, especially in areas with marked seasonality, may influence our interpretations, providing more accurate information from standardised biological inventories.Peer reviewe
DNA metabarcoding of littoral hard-bottom communities: high diversity and database gaps revealed by two molecular markers
Biodiversity assessment of marine hard-bottom communities is hindered by the high diversity and size-ranges of the organisms present. We developed a DNA metabarcoding protocol for biodiversity characterization of structurally complex natural marine hard-bottom communities. We used two molecular markers: the "Leray fragment" of mitochondrial cytochrome c oxidase (COI), for which a novel primer set was developed, and the V7 region of the nuclear small subunit ribosomal RNA (18S). Eight different shallow marine littoral communities from two National Parks in Spain (one in the Atlantic Ocean and another in the Mediterranean Sea) were studied. 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. Despite applying stringent filters, we found high values for MOTU richness (2,510 and 9,679 MOTUs with 18S and COI, respectively), 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 assignment of the detected sequences. In our dataset, 85% of 18S MOTUs and 64% of COI MOTUs could be identified to phylum or lower taxonomic level. Nevertheless, those unassigned were mostly rare MOTUs with low numbers of reads, and assigned MOTUs comprised over 90% of the total sequence reads. The identification rate might be significantly improved in the future, as reference databases are further completed. Our results show that marine metabarcoding, currently applied mostly to plankton or sediments, can be adapted to structurally complex hard bottom samples. Thus, eukaryotic metabarcoding emerges as a robust, fast, objective and affordable method to comprehensively characterize the diversity of marine benthic communities dominated by macroscopic seaweeds and colonial or modular sessile metazoans. The 18S marker lacks species-level resolution and thus cannot be recommended to assess the detailed taxonomic composition of these communities. Our 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
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