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With the on-going efforts in digitising museum collections, increased participation of citizen scientists, and greater accessibility to research data, accurately determining global patterns of diversity has become more achievable. Here, we used occurrence records from the Global Biodiversity Information Facility, with annotation of authoritative taxonomy, to evaluate the taxonomic richness of marine gastropods and to identify global species hotspots for this group. We also reviewed the availability of genetic resources within hotspots to detect potentially important regions where reference sequences for identifying these organisms are wanting. We find 33,268 unique and valid species under 3291 genera belonging to 380 gastropod families that have been recorded from 1662 to 2023. Globally, only 12.1% of reported species are linked to a COI barcode, whilst 27.1% of the families are represented with complete mitogenomes. Georeferenced records show the wide albeit disparate distribution of observations and species counts. The compiled dataset, published by organisations that are restricted to 55 countries, reveal a seeming lack of local submissions, but nonetheless demonstrates the growing contribution of citizen science platforms. We present 28 marine provinces across the globe as putative gastropod species hotspots and call for further work and stronger involvement, particularly within COI barcoding cold spots, to address the observed genetic reference inequity. Lastly, we highlight the important and relevant role of open and inclusive science to biodiversity monitoring and research.

Over the past decades the sampling of environmental DNA (eDNA) — encompassing the DNA of all organisms present in an environmental sample1 — has emerged as a technique for biodiversity monitoring and discovery in a diversity of environments. Avoiding the physical collection and identification of biota, this approach is praised for its independence of taxonomic expertise and has changed the way biologists study biodiversity. However, a common result in eDNA studies is the finding of unexpected taxa which are often removed by conservative bioinformatic filters or disregarded, since the authors are uncertain about the result and rarely have the interest, time, skills, and/or resources to return to the field and confirm with actual specimens2. Here, we report a case in which an eDNA discovery led to the physical localization of a member of the Micrognathozoa (Figure 1B) — a rare group of limnic micrometazoans, and the animal phylum to be discovered last3, which is the sister group to rotifers4,5. To this day, Micrognathozoa still comprises only a single named species from Greenland and a few additional disparate places

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.

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. We focused 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 co

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.

Traditional morphological methods for species identification are highly time consuming, especially for small organisms, such as Foraminifera, a group of shell-building microbial eukaryotes. To analyze large amounts of samples more efficiently, species identification methods have extended to molecular tools in the last few decades. Although a wide range of phyla have good markers available, for Foraminifera only one hypervariable marker from the ribosomal region (18S) is widely used. Recently a new mitochondrial marker cytochrome oxidase subunit 1 (COI) has been sequenced. Here we investigate whether this marker has a higher potential for species identification compared to the ribosomal marker. We explore the genetic variability of both the 18S and COI markers in 22 benthic foraminiferal morphospecies (orders Miliolida and Rotaliida). Using single-cell DNA, the genetic variability within specimens (intra) and between specimens (inter) of each species was assessed using next-generation sequencing. Amplification success rate was twice as high for COI (151/200 specimens) than for 18S (73/200 specimens). The COI marker showed greatly decreased intra- and inter-specimen variability compared to 18S in six out of seven selected species. The 18S phylogenetic reconstruction fails to adequately cluster multiple species together in contrast to COI. Additionally, the COI marker helped recognize misclassified specimens difficult to morphologically identify to the species level. Integrative taxonomy, combining morphological and molecular characteristics, provides a robust picture of the foraminiferal species diversity. Finally, we suggest the use of a set of sequences (two or more) to describe species showing intra-genomic variability additionally to using multiple markers. Our findings highlight the potential of the newly discovered mitochondrial marker for molecular species identification and metabarcoding purposes.

In a recent paper, “Environmental DNA: What's behind the term? Clarifying the terminology and recommendations for its future use in biomonitoring,” Pawlowski et al. argue that the term eDNA should be used to refer to the pool of DNA isolated from environmental samples, as opposed to only extra-organismal DNA from macro-organisms. We agree with this view. However, we are concerned that their proposed two-level terminology specifying sampling environment and targeted taxa is overly simplistic and might hinder rather than improve clear communication about environmental DNA and its use in biomonitoring. This terminology is based on categories that are often difficult to assign and uninformative, and it overlooks a fundamental distinction within eDNA: the type of DNA (organismal or extra-organismal) from which ecological interpretations are derived.

Global environmental changes may have a profound impact on ecosystems. In this context, it is crucial to gather biological and ecological information of the main species in marine communities to predict and mitigate potential effects of shifts in their distribution, abundance, and interactions. Using genotyping by sequencing (GBS), we assessed the genetic structure of a keystone species in the Mediterranean shallow littoral ecosystems, the black sea urchin Arbacia lixula. This bioengineer species can shape their communities due to its grazing activity and it is experiencing an ongoing expansion with increasing temperatures. The population genomic analyses on 5,241 loci sequenced in 240 individuals from 11 Mediterranean sampled populations revealed that all populations were diverse and showed significant departure from equilibrium. Albeit genetic differentiation was in general shallow, a significant break separated the western and eastern Mediterranean populations, a break not detected in previous studies with less resolutive markers. Notably, no clear effect of the Almería-Oran front, an important break in the Atlanto-Mediterranean transition, could be detected among the western basin populations, where only a slight differentiation of the two northernmost populations was found. Despite the generally low levels of genetic differentiation found, we identified candidate regions for local adaptation by combining different genomic analysis with environmental data. Salinity, rather than temperature, seemed to be an important driver of genetic structure in A. lixula. Overall, from a population genomics standpoint, there is ample scope for A. lixula to continue thriving and adapting in the warming Mediterranean.

In a recent paper, “Environmental DNA: What's behind the term? Clarifying the terminology and recommendations for its future use in biomonitoring,” Pawlowski et al. argue that the term eDNA should be used to refer to the pool of DNA isolated from environmental samples, as opposed to only extra-organismal DNA from macro-organisms. We agree with this view. However, we are concerned that their proposed two-level terminology specifying sampling environment and targeted taxa is overly simplistic and might hinder rather than improve clear communication about environmental DNA and its use in biomonitoring. This terminology is based on categories that are often difficult to assign and uninformative, and it overlooks a fundamental distinction within eDNA: the type of DNA (organismal or extra-organismal) from which ecological interpretations are derived.

Reproductive rates of copepods are temperature-dependent, but poorly known for small copepods at low temperatures, hindering the predictions of population dynamics and secondary production in high-latitude ecosystems. We investigated egg hatching rates, hatching success and egg production of the small copepods Oithona similis and Microsetella norvegica (sac spawners) and Microcalanus pusillus (broadcast spawner) between March and August. Incubations were performed at ecologically relevant temperatures between 1.3 and 13.2°C, and egg production rates were calculated. All egg hatching rates were positively correlated to temperature, although with large species-specific differences. At the lowest temperatures, M. pusillus eggs hatched within 4 days, whereas the eggs from sac spawners took 3–8 weeks to hatch. The egg hatching success was ≤25% for M. pusillus, >75% for O. similis and variable for M. norvegica. The maximum weight-specific egg production rate (μg C μg−1 C d−1) of M. pusillus was higher (0.22) than O. similis (0.12) and M. norvegica (0.06). M. norvegica reproduction peaked at 6–8°C, the prevailing in situ temperatures during its reproductive period. The difference in reproductive rates indicates species-specific thermal plasticity for the three copepods, which could have implications for present and future population dynamics of the species in arctic fjords.

In the marine realm, biomonitoring using eDNA of benthic communities requires destructive direct sampling or the setting-up of settlement structures. Comparatively much less effort is required to sample the water column, which can be accessed remotely. In this study we assess the feasibility of obtaining information from the eukaryotic benthic communities by sampling the adjacent water layer. We studied two different rocky-substrate benthic communities with a technique based on quadrat sampling. We also took replicate water samples at distances from a few centimetres to 20 m from the benthic habitat. Using as marker a fragment of the Cytochrome c oxidase subunit I gene with universal primers, we obtained a total of 3,543 molecular operational taxonomic units (MOTUs) from the samples. The structure obtained in the two environments was markedly different, with Metazoa, Archaeplastida Rhodophyta and Stramenopiles being the most diverse group in benthic samples, and HacrobiaAlveolata, Metazoa and Alveolata Rhizaria in the water. Only 265 MOTUs (7.5%) were shared between benthos and water samples, and of these 180 MOTUs (5.1%) were identified as benthic MOTUs that left their DNA in the water. Most of them were found immediately adjacent to the benthos, and their number decreased and The distribution of these benthic shared MOTUs showed a decrease both in number of MOTUs and in number of reads as we moved apart from the benthic habitat. It was concluded that water eDNA, even in the close vicinity of the benthos, was a poor proxy for the analysis of benthic structure, and that direct sampling methods are required for monitoring these complex benthic communities via metabarcoding.

Sponges have recently been proposed as ideal candidates to act as natural samplers for environmental DNA due to their efficiency in filtering water. However, validation of the usefulness of DNA recovered from sponges to reveal vertebrate biodiversity patterns in Marine Protected Areas is still needed. Additionally, nothing is known about how different sponge species and morphologies influence the capture of environmental DNA and whether biodiversity patterns obtained from sponges are best described by quantitative or qualitative measures. In this study, we amplified and sequenced a vertebrate specific 12S barcode with a set of universal PCR primers (MiFish) for metabarcoding environmental DNA from fishes, to unveil fine-scale patterns of fish communities from natural-sampler DNA retrieved from 64 sponges (16 species) located in eutrophic and well-preserved coral reefs in Nha Trang Bay (central Vietnam). Ninety tropical fish species were identified from the sponges, corresponding to one third of the total local ichthyofauna reported from previous extensive conventional surveys. Significant differentiation in fish communities between eutrophic and well-preserved environments was observed, albeit eutrophication only explained a modest proportion of the variation between fish communities. Differences in efficiency of capturing fish environmental DNA among sponge species or morphologies were not observed. Overall, the majority of detected fish species corresponded to reef-associated small-sized species, as expected in coral reefs environments. Remarkably, pelagic, migratory, and deep-sea fish species were also recovered from sponge tissues, pointing out the ability of sponge natural sampled DNA to detect fishes that were not permanently associated to the biomes where the sponges were sampled. These results highlight the suitability of natural samplers as a cost-effective way to assess vertebrate diversity in hyper-diverse environments.

In the marine realm, biomonitoring using environmental DNA (eDNA) of benthic communities requires destructive direct sampling or the setting-up of settlement structures. Comparatively much less effort is required to sample the water column, which can be accessed remotely. In this study we assess the feasibility of obtaining information from the eukaryotic benthic communities by sampling the adjacent water layer. We studied two different rocky-substrate benthic communities with a technique based on quadrat sampling. We also took replicate water samples at four distances (0, 0.5, 1.5, and 20 m) from the benthic habitat. Using broad range primers to amplify a ca. 313 bp fragment of the cytochrome oxidase subunit I gene, we obtained a total of 3,543 molecular operational taxonomic units (MOTUs). The structure obtained in the two environments was markedly different, with Metazoa, Archaeplastida and Stramenopiles being the most diverse groups in benthic samples, and Hacrobia, Metazoa and Alveolata in the water. Only 265 MOTUs (7.5%) were shared between benthos and water samples and, of these, 180 (5.1%) were identified as benthic taxa that left their DNA in the water. Most of them were found immediately adjacent to the benthos, and their number decreased as we moved apart from the benthic habitat. It was concluded that water eDNA, even in the close vicinity of the benthos, was a poor proxy for the analysis of benthic structure, and that direct sampling methods are required for monitoring these complex communities via metabarcoding.

Information about the genomic processes underlying responses to temperature changes is still limited in non-model marine invertebrates. In this sense, transcriptomic analyses can help to identify genes potentially related to thermal responses. We here investigated, via RNA-seq, whole-transcriptomic responses to increased and decreased temperatures in a thermophilous keystone sea urchin, Arbacia lixula, whose populations are increasing in the Mediterranean. This species is a key driver of benthic communities’ structure due to its grazing activity. We found a strong response to experimentally induced cold temperature (7°C), with 1,181 differentially expressed transcripts relative to the control condition (13°C), compared to only 179 in the warm (22°C) treatment. A total of 84 (cold treatment) and three (warm treatment) gene ontology terms were linked to the differentially expressed transcripts. At 7°C the expression of genes encoding different heat shock proteins (HSPs) was upregulated, together with apoptotic suppressor genes (e.g., Bcl2), genes involved in the infection response and/or pathogen-recognition (e.g., echinoidin) and ATP-associated genes, while protein biosynthesis and DNA replication pathways were downregulated. At 22°C neither HSPs induction nor activation of the previously mentioned pathways were detected, with the exception of some apoptotic-related activities that were upregulated. Our results suggest a strong transcriptional response associated with low temperatures, and support the idea of low water temperature being a major limitation for A. lixula expansion across deep Mediterranean and northern Atlantic waters.