Biology subjects

Costa, F. O.

Publications and source records attributed to Costa, F. O..

11 recordsLinked to original sources

bioRxiv2025

From egg to adult: comprehensive (e)DNA metabarcoding monitoring of fish diversity in a temperate estuary

Monitoring fish species through ichthyoplankton surveys provides important information for fish stock assessment and management. To test the effectiveness of DNA metabarcoding to identify fish species and to capture seasonal variations in local ichthyofauna, monthly ichthyoplankton and 2 L water samples were collected over 13 months in the lower section of the Guadiana River Estuary in southeast Portugal. Both sample types underwent high-throughput sequencing for three genetic markers (COI, 12S, 16S), with morphological identification also performed for ichthyoplankton. Bulk and water samples identified a total of 131 fish species throughout the year. DNA metabarcoding demonstrated higher taxonomic resolution and diversity detection, with 115 species recovered, while morphology identified only 23 species. Ichthyoplankton metabarcoding also detected 40% more fish than water eDNA, recovering almost the double of species despite the fact that both approaches used the same metabarcoding primers. The integration of multiple molecular markers was crucial to maximize diversity detection in both DNA-based methods. In addition, DNA metabarcoding was able to identify ichthyofaunal spawning periods and captured significant seasonal variations in fish community, with higher diversity observed during the warmer months. With this strategy, around 66% of the historically recorded ichthyoplankton taxa in the region were identified, along with several new records. The findings demonstrated the capability of (e)DNA metabarcoding to uncover seasonal variations in the regional fish community, provided new insights on the ichthyofauna of the Guadiana Estuary, and revealed the need for more in-depth studies to improve the efficiency of multiple sampling methods for fish species identification.

molecular biology↗

bioRxiv2025

Ports of Entry: Tracking Marine Invaders in Portuguese Mari-nas with DNA Metabarcoding from Coast to Archipelagos

Marine non-indigenous species (NIS), introduced primarily through shipping in marinas and ports, pose significant threats to coastal biodiversity and ecosystem functioning, requiring effective management strategies, especially for monitoring aimed at early detection. This study aimed to capture a comprehensive snapshot of the geographic variation of marine invertebrate assemblages and NIS occurrence, across 10 recreational marinas in Portugal (6 on the mainland - Viana do Castelo, Porto, Aveiro (2 marinas), and Lisbon (2 marinas) - and 2 in each archipelago, Madeira and the Azores). In each marina, hard substrates, zooplankton, and water samples (for eDNA) were collected in triplicate during summertime for metabarcoding profiling, adding up to a total of 111 samples. Using Illumina MiSeq sequencing of the mitochondrial cytochrome c oxidase subunit I (COI) gene (313 bp) and the V4 region of the small subunit 18S rRNA gene (18S, approximately 400 bp), we detected 645 species from 21 phyla, 40 of which were NIS (7 phyla). Only 5% of the species and 5% of NIS were detected in all marinas. The highest numbers of both exclusive native species and NIS were recorded in the Azores (100 and 7, respectively), and the lowest numbers of exclusive native species were recovered in Aveiro (39) while the lowest numbers of exclusive NIS were detected in the North and Aveiro regions (1 in each). A Principal Coordinate Analysis indicated a distinct separation among communities forming three main groups: 1) Viana do Castelo, Porto, and Aveiro; 2) Lisbon; and 3) Madeira and the Azores. Similarity between these groups is negatively correlated with geographic distance and overlaps with ecoregions previously identified within Lusitania. In the Azores 9 NIS were recorded for the first time, and one in Madeira. We also recorded a considerable number of potential range expansions of NIS, predominantly along the mainland coast, in both North (N) - South (S) or S-N directions, which shows promising results of the high potential of DNA metabarcoding to be included in future national marine biomonitoring campaigns.

ecology↗

bioRxiv2025

Detection of diadromous fish using environmental DNA: prospects for its use in conservation of endangered species occurring in Portugal

The high economic value and cultural relevance of diadromous fish make them primary targets for traditional fisheries, that need effective management to ensure the long-term survival of their populations. In Portugal, these species are experiencing a marked decline, primarily due to habitat loss and fragmentation, alongside pollution and overfishing. Accurate monitoring is therefore essential to strengthen the management of diadromous fish within Portuguese ecosystems. Advancements in monitoring methodologies could benefit from more sensitive approaches, such as environmental DNA (eDNA) analysis, which is increasingly recognized as a valuable complement to conventional fish monitoring. However, eDNA-based tools remain largely absent and untested in Portugals monitoring and management of diadromous fish. This study reviews literature on eDNA-based detection of diadromous fish species and discusses key methodological aspects influencing the detection efficiency, including sample processing (e.g., water filtration), DNA extraction methods, marker regions and primers, approach and platforms. A particular focus is placed on diadromous fishes occurring in Portugal, which includes several endangered species, and the prospects of using eDNA to monitor them. We also report a pilot study comparing specific assays and DNA metabarcoding in eDNA-based detection of sea lamprey in the Minho River watershed. By consolidating current literature, this work underscores the potential of eDNA to strengthen diadromous fish conservation in Portugal and offers insights to support the integration of eDNA-based tools into national monitoring frameworks. Although our study focuses on Portuguese species, the approaches and insights discussed are broadly applicable and can inform conservation efforts in other regions facing similar challenges. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/645525v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@d5f332org.highwire.dtl.DTLVardef@5171b0org.highwire.dtl.DTLVardef@ff1993org.highwire.dtl.DTLVardef@1e8264f_HPS_FORMAT_FIGEXP M_FIG C_FIG

ecology↗

bioRxiv2024

DNA metabarcoding-based detection of non-indigenous invertebrates in recreational marinas: influence of sample type and seasonal variation

Monitoring of marine invertebrate non-indigenous species (NIS) using DNA metabarcoding can be strongly affected by selected sample type due to life history traits, such as habitat preferences and life cycles. Two marinas in the north of Portugal were sampled to assess the impact of sample type (hard and artificial substrates, water eDNA, and zooplankton) and season (spring, autumn, winter) on species and NIS recovery. Using two molecular markers - the mitochondrial cytochrome c oxidase subunit I (COI) and the small subunit ribosomal RNA (18S) - a total of 636 species and 31 NIS were detected. Species numbers were slightly higher in the marina more exposed to maritime traffic, and the highest percentage of exclusive species was detected in zooplankton (up to 24%), as well as the highest numbers of NIS. Regarding season, the highest numbers of species and NIS were detected in the spring and autumn (varying within each marina). Taxonomic composition analysis revealed differences in species richness and community structure among seasons and sample types, particularly between hard and artificial substrates versus eDNA and zooplankton. Of the 31 NIS detected, six are potential first records for Portugal, which await morphology-based validation. No NIS were detected in all sample types nor in all sampled seasons. This highlights the need to employ different sampling approaches and markers, as well as consider seasonal variation and level of exposure to maritime-driven introductions to guarantee a comprehensive metabarcoding-based surveillance of NIS in recreational marinas.

ecology↗

bioRxiv2023

Catch me if you can: current status and topical issues on the use of eDNA-based targeted detection of rare and endangered animal species

Animal detection through DNA present in environmental samples (eDNA) is a valuable tool for detecting rare species, that are difficult to observe and monitor. eDNA-based tools are underpinned by molecular evolutionary principles, which are key to devising tools to efficiently single out a targeted species from an environmental sample, using carefully chosen marker regions and customized primers. Here, we present a comprehensive review of the use of eDNA-based methods for the detection of targeted animal species, such as rare, endangered, or invasive species, through the analysis of 460 publications (2008-2022). Aquatic ecosystems have been the most surveyed, in particular, freshwaters (75%), and to a less extent marine (14%) and terrestrial systems (10%). Vertebrates, in particular, fish (38%), and endangered species, have been the most focused in these studies, and Cytb and COI are the most employed markers. Among invertebrates, assays have been mainly designed for Mollusca and Crustacea species (22%), in particular, to target invasive species, and COI has been the most employed marker. Targeted molecular approaches, in particular qPCR, have been the most adopted (73%), while eDNA metabarcoding has been rarely used to target single or few species (approx. 5%). However, less attention has been given in these studies to the effects of environmental factors on the amount of shed DNA, the differential amount of shed DNA among species, or the sensitivity of the markers developed, which may impact the design of the assays, particularly to warrant the required detection level and avoid false negatives and positives. The accuracy of the assays will also depend on the availability of genetic data from closely related species to assess both marker and primers specificity. In addition, eDNA-based assays developed for a particular species may have to be refined taking into account site-specific populations, as well as any intraspecific variation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/544783v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@bfed8corg.highwire.dtl.DTLVardef@84acc5org.highwire.dtl.DTLVardef@6ad579org.highwire.dtl.DTLVardef@1e51bf9_HPS_FORMAT_FIGEXP M_FIG C_FIG

ecology↗

bioRxiv2022

Comparing DNA metabarcoding with morphology in the assessment of macrozoobenthos in Portuguese transitional waters in the scope of the Water Framework Directive monitoring

Despite the growing use and potential of DNA metabarcoding to improve and expedite macrozoobenthos monitoring, its employment in Water Framework Directive (WFD) monitoring of transitional ecosystems still remains largely unexplored and pending proof-of-concept studies. In the current study, we addressed this research gap by building upon the biomonitoring network program of the Portuguese Environmental Agency (APA) to benchmark metabarcoding against the morphology-based approach for characterizing macrozoobenthic communities. We assessed the ecological condition of 20 sites from four major transitional ecosystems in the west coast of Portugal, namely Minho, Lima, Vouga and Mondego estuaries. A total of 154 marine invertebrate species were detected with both methodologies, distributed by 11 phyla. In the majority of the sites, metabarcoding returned a higher number of species and phyla than the morphology-based approach (up to 2.5 times higher). In parallel, the proportion of species detected concurrently by both methods was low (35 species, 23%). The use of a multi-locus strategy increased recovered diversity through metabarcoding, since 37 species were detected exclusively with COI and 46 with 18S. For about 61% of the species recovered through morphology, metabarcoding failed detection, among which 20% was due to the lack of reference sequences in genetic databases. For the remaining, we did not find any plausible reason for only 10%, which could be due either to inefficient DNA extraction or PCR failure. Although morphological and metabarcoding-derived biotic indices did not match completely, similar responses to the environmental gradient were obtained in morphology and metabarcoding based-datasets. We anticipate that metabarcoding can increase the throughput and quality of the assessments, allowing faster assessments with greater spatial-temporal density and robust identifications of all specimens in a sample including larval stages, juveniles, and cryptic lineages, as well as smaller taxonomic groups that cannot be identified to species level using the traditional approach.

ecology↗

bioRxiv2021

Molecular evidence for sweeping discontinuity between peracarid (Crustacea) fauna of Macaronesian islands and nearby continental coasts: over fifty candidate endemic species

Oceanic islands are recognized evolutionary hotspots for terrestrial organisms, but little is known about their impact on marine organisms evolution and biogeography. The volcanic archipelagos of Macaronesia occupy a vast and complex region which is particularly suitable to investigate marine island biogeography. In this study, we used mitochondrial DNA sequences to investigate the genetic diferentiation between the populations from Webbnesia (i.e. Madeira, Selvagens and Canaries) and adjacent coasts, of 23 intertidal peracarid species. All species had unexpectedly high intraspecific genetic distances, reaching more than 20% in some cases. Between 79 and 95 Molecular Operational Taxonomic Units (MOTUs) were found in these species. Webbnesia populations displayed an impressive genetic diversity and high endemicity, with 83% of the MOTUs being private to these islands, particularly La Palma and Madeira. Network analyses suggested higher similarity between Webbnesia and Azores than with adjacent continental coasts. These results reveal an unanticipated and sweeping biogeographic discontinuity of peracaridean fauna between Webbnesia and the Iberian Peninsula, raising suspicion about the possible occurrence of identical patterns in other groups of marine invertebrates in the region. We emphasize the unique genetic heritage hosted by these islands, underlining the need to consider the fine scale endemicity in marine conservation efforts.

ecology↗

bioRxiv2021

Revision and annotation of DNA barcode records for marine invertebrates: report of the 8th iBOL conference hackathon

The accuracy of the identification of unknown specimens using DNA barcoding and metabarcoding relies on reference libraries containing records with reliable taxonomy and sequence quality. A rampant growth in barcode data led to a stringent need for data curation, especially in taxonomically difficult groups such as marine invertebrates. A major effort in curating marine barcode data deposited in the Barcode of Life Data Systems (BOLD) has been undertaken during the 8th International Barcode of Life Conference (Trondheim, Norway, 2019). For practical reasons, only major taxonomic groups were reviewed and annotated (crustaceans, echinoderms, molluscs, and polychaetes). The congruence of Linnean names with Barcode Index Numbers (BINs) was investigated, and the records deemed uncertain were annotated with four tags: a) MIS-ID (misidentified, mislabeled or contaminated records), b) AMBIG (ambiguous records unresolved with the current data), c) COMPLEX (species occurring in multiple BINs), and d) SHARE (barcodes shared between species). A total of 83,712 specimen records corresponding to 7,576 species were reviewed and 39% of the species were tagged (7% MIS-ID, 17% AMBIG, 14% COMPLEX, and 1% SHARE). High percentages (>50%) of AMBIG tags were recorded in gastropods, whereas COMPLEX tags dominated in crustaceans and polychaetes. This high proportion of tagged species reflects either flaws in the barcoding workflow (e.g., misidentification, cross -contamination) or taxonomic difficulties (e.g., synonyms, undescribed species). Although data curation is crucial for barcode applications, such manual efforts of reviewing large datasets are not sustainable and the implementation of automated solutions to the furthest possible extent is hi ghly desirable.

genetics↗

bioRxiv2020

Status and prospects of marine NIS detection and monitoring through (e)DNA metabarcoding

In coastal ecosystems, non-indigenous species (NIS) are recognized as a major threat to biodiversity, ecosystem functioning and socio-economic activities. Here we present a systematic review on the use of metabarcoding for NIS surveillance in marine and coastal ecosystems, through the analysis of 42 publications. Metabarcoding has been mainly applied to environmental DNA (eDNA) from water samples, but also to DNA extracted from bulk organismal samples. DNA extraction kits have been widely used and the 18S rRNA and the COI genes the most employed markers, but less than half of the studies targeted more than one marker loci. The Illumina MiSeq platform has been used in >50% of the publications. Current weaknesses include potential occurrence of false negatives due to the primer-biased or faulty DNA amplification and the incompleteness of reference libraries. This is particularly concerning in the case of NIS surveillance, where proficiency in species level detection is critical. Until these weaknesses are resolved, ideally NIS metabarcoding should be supported by complementary approaches, such as morphological analysis or more targeted molecular approaches (e.g. qPCR, ddPCR). Even so, metabarcoding has already proved to be a highly sensitive tool to detect small organisms or undifferentiated life stages across a wide taxonomic range. In addition, it also seems to be very effective in ballast water management and to improve the spatial and temporal sampling frequency of NIS surveillance in marine and coastal ecosystems. Although specific protocols may be required for species-specific NIS detection, for general monitoring it would be vital to settle on a standard protocol able to generate comparable results among surveillance campaigns and regions of the globe, seeking the best approach for detecting the broadest range of species, while minimizing the chances of a false positive or negative detection.

ecology↗

bioRxiv2019

DNA barcode reference libraries for the monitoring of aquatic biota in Europe: Gap-analysis and recommendations for future work

Effective identification of species using short DNA fragments (DNA barcoding and DNA metabarcoding) requires reliable sequence reference libraries of known taxa. Both taxonomically comprehensive coverage and content quality are important for sufficient accuracy. For aquatic ecosystems in Europe, reliable barcode reference libraries are particularly important if molecular identification tools are to be implemented in biomonitoring and reports in the context of the EU Water Framework Directive (WFD) and the Marine Strategy Framework Directive (MSFD). We analysed gaps in the two most important reference databases, Barcode of Life Data Systems (BOLD) and NCBI GenBank, with a focus on the taxa most frequently used in WFD and MSFD. Our analyses show that coverage varies strongly among taxonomic groups, and among geographic regions. In general, groups that were actively targeted in barcode projects (e.g. fish, true bugs, caddisflies and vascular plants) are well represented in the barcode libraries, while others have fewer records (e.g. marine molluscs, ascidians, and freshwater diatoms). We also found that species monitored in several countries often are represented by barcodes in reference libraries, while species monitored in a single country frequently lack sequence records. A large proportion of species (up to 50%) in several taxonomic groups are only represented by private data in BOLD. Our results have implications for the future strategy to fill existing gaps in barcode libraries, especially if DNA metabarcoding is to be used in the monitoring of European aquatic biota under the WFD and MSFD. For example, missing species relevant to monitoring in multiple countries should be prioritized. We also discuss why a strategy for quality control and quality assurance of barcode reference libraries is needed and recommend future steps to ensure full utilization of metabarcoding in aquatic biomonitoring.

ecology↗

bioRxiv2017

DNA Metabarcoding For High-Throughput Monitoring Of Estuarine Macrobenthic Communities

Benthic communities are key components of aquatic ecosystems biomonitoring. However, morphology-based species identifications remain a low-throughput, and sometimes ambiguous, approach. Despite metabarcoding methodologies have been applied for above-species taxa inventories in marine meiofaunal communities, a comprehensive approach providing species-level identifications for estuarine macrobenthic communities is still lacking. Here we report a combination of experimental and field studies demonstrating the aptitude of COI metabarcoding to provide robust species-level identifications within a framework of high-throughput monitoring of estuarine macrobenthic communities. To investigate the ability to recover DNA barcodes from all species present in a bulk DNA extract, we assembled 3 phylogenetically diverse communities, using 4 different primer pairs to generate PCR products of the COI barcode region. Between 78 and 83% of the species in the tested communities were recovered through HTS. Subsequently, we compared morphology and metabarcoding-based approaches to determine the species composition from four distinct sites of an estuary. Our results indicate that the species richness would be considerably underestimated if only morphological methods were used. Although further refinement is required for improving the efficiency and output of this approach, here we show the great aptitude of COI metabarcoding to provide high quality and auditable species identifications in macrobenthos monitoring.

genetics↗