Environmental DNA (eDNA) metabarcoding is increasingly applied for regional biodiversity assessments, yet its effectiveness for detecting mobile, low-density megafauna remains uncertain. This study employed a multi-marker eDNA metabarcoding approach (three primer pairs targeting two mitochondrial genes) across 23 sites in the Spanish Mediterranean, including the Alboran Sea, to (i) to characterise the composition of elasmobranch taxa detected by eDNA metabarcoding across sampling zones, (ii) explore spatial patterns of detection, and (iii) evaluate environmental and spatial drivers of occurrence. Ordination and permutation analyses revealed spatial structuring of overall metazoan communities, primarily driven by bathymetric gradients and proximity to marine reserves. In contrast, elasmobranch assemblages exhibited low spatial turnover. Eleven shark and ray species were detected, several of which are listed under threatened or near threatened IUCN categories, underscoring the conservation value of eDNA detections. Depth emerged as the main predictor of elasmobranch richness and composition, while read abundance showed no consistent association with environmental covariates. This work represents the first regional assessment of elasmobranch diversity in the Alboran and adjacent Mediterranean waters using eDNA metabarcoding, demonstrating its capacity to supplement traditional monitoring approaches. Overall, multi-marker eDNA effectively captures regional biodiversity gradients and detects rare, mobile taxa such as elasmobranchs. To further enhance taxonomic resolution and strengthen eDNA applications in elasmobranch conservation, we recommend three key actions: (i) increasing spatio-temporal replication, (ii) integrating targeted quantitative assays (qPCR/ddPCR), and (iii) improving reference databases.

Elasmobranchs face extinction crisis, with one third of species threatened by overfishing, habitat loss and climate change. Commercial mislabelling exacerbates this problem by masking illegal trade, undermining conservation efforts, and fueling Illegal, Unreported and Unregulated (IUU) fishing. This study assesses the extent of species mislabelling in skates and rays commercialized in primary fish markets of the Cantabrian Sea using DNA barcoding and evaluates whether science-based intervention involving species identification guides can reduce mislabelling. Between November 2024 and March 2025, tissue samples were collected at a fish market for barcoding genetic identification analysis. A total of 416 individuals from eight genetically identified species revealed a global mislabelling rate of 23.1%, based on discrepancies between the species label at sale and the species identified through genetic analyses. The most mislabelled species were Raja montagui and Raja brachyura, while Raja clavata was commonly used as a substitute label. Mislabelling also concealed the trade of protected or regulated species, including Leucoraja circularis and Raja undulata. In February 2025, an intervention was implemented through collaboration between scientists and fish market managers. Identification guides were distributed, informal training was provided to fishermen and market workers, and commercial labelling at auction was partially updated. Consequently, mislabelling decreased from 26.6% to 20.6%, indicating partial but significant effectiveness. These findings highlight the importance of combined efforts to improve traceability for long-term conservation strategies in Rajidae populations.

We report the first record of a potentially self-sustaining population of the American bloodworm Glycera dibranchiata outside its native NW Atlantic range, established for over a decade in the Villaviciosa estuary (Cantabrian Sea, Bay of Biscay). Its presence is likely linked to imports for recreational fishing bait. Morphological and genetical analyses, including mitochondrial COI sequencing, confirmed species identity and traced origins. Two haplotypes were identified, one matching Canadian populations, suggesting either long-term persistence since initial introduction (2010) or repeated introductions from similar sources. Substantial divergence between Cantabrian Sea and NW Atlantic specimens suggests a possible a cryptic species complex, highlighting limitations of traditional taxonomy and the need for integrative approaches to accurately delineate species boundaries in widely traded, ecologically impactful taxa. Additionally, ectosymbiotic ciliate protozoans and endoparasitic apicomplexan gregarines at different developmental stages were found in all examined specimens, raising concerns about ecosystem impacts, including potential public health risks, threats to endangered taxa, and mass mortalities of native hosts.

Advancements have been made in the use of DNA-based methods for the detection of single species. However, the routine application of DNA-based methods to monitor whole communities using a metabarcoding approach and derive ecosystem status continues to be limited. We undertook a structured experiment to assess factors influencing the precision and accuracy of molecular methods for freshwater macroinvertebrate community assessment. Macroinvertebrates were sorted, identified and counted from kick-net samples using standard morphometric protocols, then reconstituted. These bulk specimen samples were then homogenised. From each bulk sample, aliquots of the homogenate were distributed among seven laboratories across Europe for DNA extraction, PCR amplification, library preparation and sequencing. Additionally, each laboratory was provided with a DNA extract from the bulk homogenate to allow for amplification through to sequencing, in order to assess the influence of DNA extraction. Hierarchical nested analyses were used to identify sources of uncertainty in the data and explore factors influencing the returned community and metrics of ecosystem quality. Furthermore, comparison with morphological analysis enabled us to benchmark molecular to traditional processing approaches. Metrics derived by morphological and DNA-based methods differed substantially. The strongest methodological influences on variance in metrics derived from DNA-based methods were attributed to sequencing (read count per sample) and laboratory (strong variation within samples). Furthermore, the performance of the DNA-based method varied among samples, most likely due to differences in the composition of the invertebrate community. Logistic regression indicated that detectability did not vary among taxa, but that taxa occurring in low abundance (less than five individuals in our samples) were more likely to be missed via metabarcoding. Our findings highlight sources of uncertainty influencing DNA-based methods and, particularly, the variability of results generated from individual laboratories. Our findings underline the need for clear guidelines, standardisation and quality assurance schemes.

Abstract European Union member states are required to monitor the ecological status of their water bodies. The existing official protocols and indices for routine biomonitoring are usually based on the morphological identification of different groups of indicator organisms (biological quality indicators). However, this approach has several limitations in routine use. DNA metabarcoding is presented today as an alternative, as it is potentially able to overcome those limitations. Recent studies have applied DNA metabarcoding in wadeable river biomonitoring throughout the world, including the northwest of the Iberian Peninsula, using benthic macroinvertebrates as bioindicators. This review reanalysed previous data gathered from rivers of the northwest of the Iberian Peninsula as a case study to (a) compare the detection rates of river macroinvertebrate taxa between morphological identification and DNA metabarcoding techniques, both in bulk and water eDNA samples; (b) identify false positive and false negative detections after DNA metabarcoding, as well as other sources of variation that account for the differences in the results of both methodologies; (c) discuss the impact of these differences on DNA metabarcoding reliability for biomonitoring, and propose possible solutions to enhance it; and (d) propose a roadmap for an effective implementation of DNA metabarcoding in river biomonitoring. Several false‐positives, false‐negatives and other methodological sources of error were identified in relation to DNA metabarcoding, as well as other aspects of the sampling methodology and quality water index developments that hinder the routine use of this technique in river biomonitoring. Practical implication. This study shows that several actions need to be taken in order to ensure the effective application of metabarcoding in riverine macroinvertebrate biomonitoring: (1) the compilation of a complete list of species present in the implementation area, (2) the completion of genetic databases for relevant bioindicator species, (3) the development of metabarcoding primer pairs for those species and finally, (4) the adaptation of water quality indices to take advantage of the new taxonomic information provided by metabarcoding.

The construction of dams and reservoirs to meet increasing water and electricity demands has significantly altered many natural river flows, posing threats to the ecological integrity of freshwater ecosystems. Environmental flows (e-flows) have been advocated to mitigate the negative impacts of hydroelectric power generation and to maintain healthy river ecosystems. However, the relationship between e-flow implementation and ecological response remains poorly understudied. Here we conducted a comprehensive 2-year study monitoring macroinvertebrate biodiversity using both morphological identification and DNA metabarcoding to explore the effect of e-flow recovery after 50 years of no flow contribution (the stream was basically dry) due to hydroelectric regulation in a small stream (Valseco). Our results revealed significant changes in macroinvertebrate diversity and ecological status following e-flow restoration. Initial diversity values were low, but they progressively increased, with distinct recovery rates observed across different stream sites. Molecular analyses, which provided a higher taxonomic resolution that allowed the detection of differences in species composition, showed a slight decrease in oligochaete species and an increase in Ephemeroptera, Plecoptera, and Trichoptera species. Ecological status assessments showed a shift from bad to good status within 2 years post-restoration. Notably, the farthest downstream site (i.e. that closest to the receiving river and farthest from the dam) reached good steady status in the first year. This study highlights the effectiveness of e-flow restoration in recovering macroinvertebrate biodiversity and achieving good status of biological integrity, underscoring the importance of such management strategies in meeting the targets of the Water Framework Directive. Finally, it emphasizes the need for further research to understand early recolonization dynamics and evaluate ecological status assessment methodologies in similar restoration contexts.

The European catfish (Silurus glanis Linnaeus, 1758) was introduced into the Ebro Basin in Spain in 1974 for recreational fishing. Since then, the species has spread throughout the country’s river basins, reaching the Iznájar Reservoir (Guadalquivir River Basin) in 2011. This area is of great ecological and economic relevance, especially as it includes the Doñana National Park, one of the most important nature reserves in Europe. Recently, the presence of catfish has been reported in the lower reaches of the river. In this work, we used non-invasive vertical and horizontal hydroacoustic surveys and environmental DNA river sampling to unravel the actual distribution and dispersal pattern of the species in the lower Guadalquivir River. The hydroacoustic profiles and the species-specific detections by real-time PCR (qPCR) and droplet digital PCR (ddPCR) showed that these non-invasive methods allow the detection and quantification of catfish and provide valuable information on the species’ presence. We have confirmed the presence of catfish in most of the study area, including downstream areas of the Guadalquivir Basin. The results suggest the possibility of other isolated introductions and/or human-mediated movements of specimens, and imply that a coordinated catfish prevention and mitigation strategy is therefore urgently needed.

Water resources play a vital role in sustaining ecosystems, human life, food security, and biodiversity. However, these resources currently face serious threats caused primarily by human activities. Because of that, European Union have mandated that all its member states monitor the ecological status of their water bodies. One of the most commonly used bioindicator groups for routine river biomonitoring is benthic macroinvertebrates, which rely mainly on morphological identification at the family level and the use of diverse indices to estimate the ecological status of the river ecosystem. The morphological approach has certain limitations, and metabarcoding is now considered an alternative technique for biodiversity studies and biomonitoring of surface waters. However, the efficacy of this approach depends on the comparison of sequencing data with genetic databases, which are often incomplete and geographically biased. In this study, we collected 92 macroinvertebrate samples from rivers in the northern Iberian Peninsula to identify all possible species and generate new DNA sequences for the COI-5P region. The objectives were to perform region-specific enrichment of reference databases for the geographical area of northern Spain and to assess the impact, before and after this strategy, on the taxonomic assignments used for ecological status inference in metabarcoding samples. The results showed that 21% of morphospecies lacked reference sequences in the BOLD or GenBank databases, indicating that a significant number of macroinvertebrate species in the northwestern Iberian Peninsula lack reference sequences in major genetic databases. Enriching the BOLD databases with new sequences led to the detection of an average of 17.25 more BINs (species-assimilable gene cluster) and 1.54 more IBMWP taxa (river ecological status indicator macroinvertebrate taxa) per sample, causing changes in the ecological status determination for 16% of the samples, which clearly shows that improving the databases with local species sequences could enhance the detection and identification of new IBMWP taxa, potentially impacting ecological status inference from molecular data. Additionally, a historical analysis of Iberian macroinvertebrate addition in the BOLD database using public BOLD records revealed an asymptotic trend that can be partially mitigated by region-specific enrichment. These results indicate that a local biota sequencing effort can considerably improve metabarcoding results rather than waiting for a gradual improvement of genetic databases. In conclusion, we advocate for a creation an Iberian freshwater macroinvertebrate checklist and then the initiation of a coordinated effort for sequencing any unsequenced species from the region.

Rivers are crucial ecosystems supporting biodiversity and human well-being, yet they face increasing degradation globally. Traditional river biomonitoring methods based on morphological identification of macroinvertebrates present challenges in terms of taxonomic resolution and scalability. This study explores the application of DNA metabarcoding analysis in both bulk and environmental DNA (eDNA) samples for comprehensive assessment of macrozoobenthic biodiversity, detection of invasive and endangered species, and evaluation of river ecological status in northwestern Spain. DNA metabarcoding of homogenized bulk samples and water eDNA revealed a mean of 100 and 87 macrozoobenthos species per sample respectively. However, the specific composition was significantly different with only 27.3% of the total species being shared. It was not possible to identify all the OTUs to species level; only 17.43% and 49.4% of the OTUs generated could be identified to species level in the bulk and eDNA samples, respectively. Additionally, a total of 11 exotic species (two first records for the Iberian Peninsula and another three first records for Asturias region) and one endangered species were detected by molecular tools. Molecular methods showed significant correlations with morphological identification for EQR values (Ecological Quality Ratio) of IBMWP index, yet differences in inferred river ecological status were noted, with bulk samples tending to indicate higher status. Overall, DNA metabarcoding offers a promising approach for river biomonitoring, providing insights into biodiversity, invasive species, and ecological status within a single analysis. Further optimization and intercalibration are required for its implementation in routine biomonitoring programmes, but its scalability and multi-tasking capabilities position it as a valuable tool for integrated monitoring of river ecosystems.

In recent decades, worldwide cetacean species have been protected, but they are still threatened. The bottlenose dolphin (Tursiops truncatus) is a vulnerable keystone species and a useful bioindicator of the health and balance of marine ecosystems in oceans all over the world. The genetic structure of the species is shaped by their niche specialization (along with other factors), leading to the classification of two ecotypes: coastal and pelagic. In this study, the genetic diversity, population structure, and ecotypes of bottlenose dolphins from the Canary Islands were assessed through the analysis of 49 new samples from biopsies and from stranded animals using the 636 bp portion of the mitochondrial control region and 343 individuals from databases (n = 392). The results reveal high genetic diversity in Canarian bottlenose dolphins (Hd = 0.969 and π = 0.0165) and the apparent lack of population genetic structure within this archipelago. High genetic structure (Fst, Φst) was found between the Canary Islands and coastal populations, while little to no structure was found with the pelagic populations. These results suggest that Canarian bottlenose dolphins are part of pelagic ecotype populations in the North Atlantic. The studied Special Areas of Conservation in the Canary Islands may correspond to a hotspot of genetic diversity of the species and could be a strategic area for the conservation of the oceanic ecotype of bottlenose dolphins.

Early detection of invasive species is crucial to deal effectively with biological invasions in ports, which are hotspots of species introductions. In this study, a simplified end-time PCR methodology conducted on eDNA from water samples was developed for rapid detection of the invasive seaweed Asparagopsis armata (four hours from water collection to result visualization). It was tested dockside in four international Spanish ports in presence of stakeholders, whose feedback was obtained to explore the real applicability of this biotechnology. Although biological invasions were not a main concern for them, results indicate a unanimous approval of the methodology by the stakeholders, having detected the presence of A. armata in three of the ports. Stakeholders suggested further developments for easier application of the tool and multiple species detection, to be adopted for the control of invasive species in ports.

ABSTRACTAn eco‐monitoring program to assess the biodiversity of insects affected by yellow‐legged hornet (Vespa velutina) trapping in the north of the Iberian Peninsula (Spain) revealed the first occurrence of the southern giant hornet Vespa soror (Hymenoptera, Vespidae) on the European continent. We present a detailed characterization, combining morphological characteristics and molecular tools for genetic identification, as well as key information on its identification with respect to other hornets found on the Iberian Peninsula. We discuss the most plausible pathways and vectors of introduction, its potential invasiveness, and subsequent impacts on host localities. Our preliminary results raise concerns about the potential threat of V. soror to human health and ecosystem dynamics, as it is a highly predatory species on other insects and even small vertebrates. Finally, this study confirms once again the usefulness of studying insects trapped in such traps for rapid response and early detection of inland invasive species. We also propose a common Spanish name for the species, “avispón sóror”.

Recent changes in climate and oceanographic conditions have been affecting the dynamics and composition of species communities worldwide, increasing the occurrence of tropical-subtropical species within a specific biogeographic area. This phenomenon defined as tropicalization is occurring in the Cantabrian Sea (Bay of Biscay), a transitional zone between two different regions of the North Atlantic. In this study we present new records of the tropical-subtropical gastropod species Plicopurpura patula and Monoplex parthenopeus in the European Atlantic, which constitute their northernmost distribution in the Atlantic Ocean to date. We provide a detailed characterization of both species, combining morphological and molecular features. Finally, supported by provided evidence and justification, we propose both species as two new potential bioindicators of the tropicalization phenomenon in the North Atlantic waters.

Octopus vulgaris is one of the most harvested octopus species in the world. In the Iberian Peninsula, there are several small-scale fisheries that have a long-term tradition of harvesting octopus. The Asturias fleet (in Northern Spain) has an internationally recognized MSC label for its exploitation. Of concern, genetic assessments of exploited stocks are currently scarce, which could prevent the implementation of adequate managing strategies. We use two mitochondrial regions (cytochrome oxidase subunit 1 and control region) to analyze the genetic status and evolutionary events that conditioned octopus populations' characteristics in the Northeastern Atlantic. A total of 90 individuals were sampled from three different localities in the Iberian Peninsula as well as a location in Macaronesia. Temporal genetic analyses on Asturias and Algarve populations were also performed. Results indicated the absence of fine spatial genetic structuring but showed the Canary Islands (in Macaronesia) as the most distinct population. Our analyses detected two distinct clades, already described in the literature, but, for the first time, we confirmed the presence of the α-southern haplogroup in the Northern Iberian Peninsula. This result indicates a more continuous cline for the distribution of these two haplogroups than previously reported. Temporal changes in the distribution of both haplogroups in contact zones were also detected.

Understanding public attitudes toward invasive species is crucial to curtail the reasons for their introduction and to increase the effectiveness of control measures. A questionnaire was distributed in three European countries (Italy, Spain and United Kingdom) to evaluate public attitudes on the problems posed by invasive species, their perception of the impacts and their willingness to introduce and support management actions. People whose occupations are not nature related or who practice gardening as a main outdoor activity, represent the highest risk groups relating to the introduction of invasive species. Ecosystem damage and species extinctions were the main concerns for people, and signal crayfish and zebra mussel were the species of most concern. People firstly supported control and eradication followed by awareness resulting in increasing public awareness as management measures. This information can feed into educational, prevention and eradication campaigns promoting the necessary socio-cultural changes to prevent the negative impacts of invasive species.

The fishing and aquaculture sectors are an important source of development around the globe. In Asturias (Spain), the diversity and richness of the fishing grounds of the Cantabrian Sea favored the historical settlement of a large number of communities closely linked to the marine environment and fishing resources, forming an integral part of the region’s cultural and natural heritage. However, aquatic ecosystems are facing, nowadays, important threats from anthropogenic activities. To address these problems and avoid their impact on fishing activities, it is essential to know the ecological and genetic status of the species. Despite this, the application of genetic tools is still incipient in many species of commercial interest; however, its use can help to generate data that allow better regulation and fisheries planning. Here, the use of genetic markers and educational strategies in the management of some shellfish species of great commercial and cultural value in Asturias are reviewed. Moving toward sustainable fisheries management is a priority that can only be achieved through R + D + i, educational strategies, and the development and implementation of a regional strategy oriented toward the sustainable management and exploitation.

Early detection of alien species is crucial to deal effectively with biological invasions in maritime ports, which are hotspots of non-indigenous species. In this study, a simplified end-time PCR methodology conducted on eDNA from water samples was applied dockside for rapid detection (four hours from water collection to result visualization) of the invasive seaweed Asparagopsis armata in four international Spanish ports. Feedback from port authorities was obtained in a focus group to explore the real applicability of this biotechnology. The stakeholders’ feedback was analyzed from relevant terms clustering. Although biological invasions are not a main concern for them, results indicate a unanimous approval of the methodology by the ports, having detected the presence of A. armata in three of them. Port stakeholders suggested further developments for easier application, especially in ballast water, and for multiple species detections, to be adopted for the control of invasive species in maritime ports.

Octopus vulgaris (Cuvier, 1797) is a cephalopod species with great economic value. In western Asturias (northwest of Spain), O. vulgaris artisanal fisheries are relatively well monitored and conditionally eco-labeled by the Marine Stewardship Council (MSC). Despite this, the Asturian octopus stocks have not been genetically assessed so far. In order to improve the current fishery plan and contrast the octopus eco-label validity in Asturias, 539 individuals from five regions of the O. vulgaris geographic distribution, including temporal samplings in Asturias, were collected and genotyped at thirteen microsatellite loci. All the samples under analysis were in agreement with Hardy–Weinberg expectations. Spatial levels of genetic differentiation were estimated using F -statistics, multidimensional scaling, and Bayesian analyses. Results suggested that the O. vulgaris consists of at least four genetically different stocks coming from two ancestral lineages. In addition, temporal analyses showed stability in terms of genetic variation and high N E (> 50) for several generations in different localities within Asturias, pointing out to indeed sustainable fishery exploitation levels. Even though, the current Asturias fishery plan shows no significant genetic damages to the stocks, the regional-specific management plans need systematic genetic monitoring schemes as part of an efficient and preventive regional fishery regulation strategy.

Anthropogenic litter is considered a potential vector for the dispersal of non-indigenous species (NIS) in marine ecosystems. Using the bay of Gijon (Southwestern Bay of Biscay) as a case study, we studied the composition and potential transfer of the communities inhabiting three different environment components: 1) natural and artificial substrates from the international port of Gijon, 2) six proximate rocky beaches and 3) floating litter collected in the adjacent coast. A total of 717 organisms were morphologically identified and DNA barcoded using COI and 18S genes. In total 23 NIS were detected, six of them considered invasive in the area. The taxonomic profiles of the three environment components were significantly different, flotsam containing higher proportions of Hexanauplia and less mollusks, echinoderms and polychaetes than ports and beaches. Contrary to expectations, floating litter showed higher densities of native and exotic species than beaches or port surfaces. This and shared haplotypes between port, flotsam and beaches in some invasive species may indicate that marine litter could represent a new habitat for species to disperse into new areas.