Biodiversidade e evolução da fauna dos peracarídeos costeiros da Macaronésia e Nordeste Atlântico

Doutoramento em BiologiaIn the complex and rich Northeast (NE) Atlantic and Macaronesia coasts, the superoder Peracarida (Crustacea) is one of the most abundant and commom marine invertebrate taxa with an important role in benthic communities. The study of this group is often limited to inventory lists or benthic community studies and the genetic knowledge of the group in this region is poor. The main goal of this thesis was to improve knowledge on Peracarida diversity and evolution in the NE Atlantic and Macaronesia, with particular emphasis on shallow water and rocky shore members of the orders Amphipoda, Isopoda and Tanaidacea. The thesis comprises five chapters with original research, entailing a DNA barcodebased screening of the species diversity in this group through the comparison of morphology and molecular-derived data (chapter 2), a set of two studies of the isopod genus Dynamene (chapters 3 and 4), one chapter about the amphipod family Hyalidae (chapter 5), and a multi-species analyses of the diversity and broad phylogeographic patterns of Macaronesian peracarideans (chapter 6). In the first chapter, we reported a DNA barcode reference library for the superorder Peracarida, comprising specimens from marine Atlantic coasts, mainly from Iberian Peninsula, together with additional members of the same or sister taxa from other locations. A higher number of Barcode Index Numbers (BINs) compared with the number of morphospecies was found, with some of them displaying up to six BINs. The presence of deeply divergent intraspecific lineages suggests the existence of considerable overlooked taxonomic diversity, even in one of the most well-known peracaridean faunas in the world. These findings indicate the need for a broad, comprehensive and integrated revision of the peracaridean fauna from the Southern European Atlantic coasts. In the second chapter, the commom but species-poor and controversial isopod Dynamene genus was investigated in detail by examining thousands of specimens records sampled during more than fifty years. The distribution and taxonomy of the six Dynamene species along the Northeast Atlantic-Black Sea axis was revised and updated. New distribution maps and illustrated keys to the adult males and females of the northern hemisphere species are provided. In the last three chapters, molecular delineation tools revealed extensive cryptic diversity in the genus Dynamene (3 morphospecies vs 12 molecular operational taxonomic units - MOTUs), in the amphipod family Hyalidae (7 morphospecies vs 32 MOTUs) and in twenty-five peracaridean species (25 morphospecies vs 90 MOTUs). A split between Macaronesian and continental populations was patent, and in most cases the Macaronesian populations displayed high levels of diversity. These findings suggest a much larger role of oceanic islands in the diversification of these marine invertebrates than would have been anticipated, and contributes to expose weakly explored events in the phylogeography and evolution of Macaronesia’s marine fauna. This thesis showed that marine biodiversity, as seen in peracarideans from the NE Atlantic and Macaronesia, has been considerably underestimated. The level of diversity will likely increase with the addition of different taxa, different types of habitat and distinct marine regions. It also suggests that these oceanic islands may act as drivers of evolution, diversification and endemism, just as well for marine organisms as they do for terrestrial ones.Nas costas complexas e ricas do Nordeste (NE) Atlântico e da Macaronésia, a superordem Peracarida (Crustacea) é um dos taxa de invertebrados marinhos mais abundante e comum, com um papel importante nas comunidades bentónicas. O estudo deste grupo é muitas vezes limitado a listas de inventários ou estudos de comunidade bentónicas e o seu conhecimento genético nesta região é deficiente. O objetivo principal desta tese foi o de melhorar o conhecimento sobre a diversidade e a evolução dos peracarídeos no Atlântico Nordeste e na Macaronésia, com ênfase nos membros presentes nas zonas pouco profundas e nas zonas costeiras rochosas das ordens Amphipoda, Isopoda e Tanaidacea. Esta tese compreende cinco capítulos com pesquisa original, incluindo uma biblioteca de referência de DNA barcodes neste grupo, através da comparação de dados morfológicos e moleculares (capítulo 2), um conjunto de dois estudos dedicados ao género isopode Dynamene (capítulos 3 e 4), um capítulo dedicado à família de anfípodes Hyalidae (capítulo 5), e uma abordagem multi-espécies da diversidade e dos padrões filogeográficos dos peracarídios presentes na Macaronésia (capítulo 6). No primeiro capítulo, relatamos uma biblioteca de referência de DNA barcodes para a superordem Peracarida, que inclui espécimes de costas do Atlântico, principalmente da Península Ibérica, juntamente com membros adicionais do mesmo ou semalhantes taxa de outros locais. Um maior número de Barcode Index Numbers (BINs) em comparação com o número de morfo-espécies foi encontrado, com algumas morfo-espécies exibindo até seis BINs. A presença de linhagens profundamente divergentes sugere a existência de uma considerável diversidade taxonómica anteriormente negligenciada, mesmo numa das mais conhecidas faunas de peracarídeos do mundo. Estas descobertas indicam a necessidade de uma revisão ampla, abrangente e integrada da fauna de peracarídeos das costas do Atlântico do Sul da Europa. No segundo capítulo, o abundante mas controverso género Dynamene foi investigado em detalhe, ao examinar vários milhares de individuos amostrados durante mais de cinquenta anos. A distribuição e a taxonomia das seis espécies de Dynamene ao longo do eixo Nordeste Atlântico-Mar Negro foram revistas e actualizadas. Novos mapas de distribuição e chaves ilustradas para os machos adultos e fêmeas das espécies deste género, presentes no hemisfério norte, são fornecidas. Nos três últimos capítulos, as ferramentas de delineamento molecular revelaram uma extensa diversidade críptica no género Dynamene (3 morfoespécies vs 12 unidades taxonómicas operacionais moleculares - MOTUs), na família de anfípipodes Hyalidae (7 morfoespécies vs 32 MOTUs) e em vinte e cinco espécies de peracarídeos (25 morfoespécies vs 90 MOTUs). Uma separação entre as populações presentes na Macaronésia e as presentes no continente foi visivel e, na maioria dos casos, as populações presentes na Macaronésia apresentavam maiores níveis de diversidade. Estas descobertas sugerem um papel maior das ilhas oceânicas na diversificação destes invertebrados marinhos do que se anteciparia e contribuiram para expor eventos pouco explorados na filogeografia e evolução da fauna marinha na Macaronésia. Esta tese mostrou que a biodiversidade marinha, como se observa em peracarídeos presentes no NE Atlântico e na Macaronésia, foi consideravelmente subestimada. O nível de diversidade provavelmente aumentará com a adição de diferentes taxa, diferentes tipos de habitat e de regiões marinhas distintas. Esta tese também sugere que estas ilhas oceânicas podem atuar como impulsionadoras da evolução, da diversificação e do endemismo em organismos marinhos, como acontece nos organismos terrestres

Toxicidade de aminoácidos em peixe-zebra

Mestrado em Biologia Aplicada - Biologia Molecular e CelularProteins are synthetized through the mechanism of translation and are constituted by amino acids. Besides being the basic units of proteins, amino acids also play other important roles in the cell such as signaling or regulation of cell growth. However, in excess, amino acids can be toxic, although the mechanism of toxicity is still not clear. In this study we used zebrafish as a vertebrate model to assess the toxicity induced by different amino acids as a result of nutritional imbalance. Moreover, we evaluated the changes induced by amino acid toxicity during zebrafish development in order to understand if this toxicity could be related with wrong incorporation of mischarged amino acids during translation. The results show that some of the canonical amino acids cause high toxicity in zebrafish, namely L-tryptophan, L-glutamine, Lphenylalanine and L-arginine. To understand if this toxicity could be caused by the production of aberrant proteins, due to tRNA mischarging, result of an unbalanced amino acid pool, we analyzed the activation of protein degradation pathways. For this we did western blot analysis of the poliubiquitination state of the proteome. No differences were observed between different amino acid concentrations and the control indicating that the ubiquitin-proteasome pathway is not directly correlated with the amino acid toxicity observed.As proteínas são sintetizadas através do mecanismo de tradução e são constituídas por aminoácidos. Além de serem as unidades básicas das proteínas, os aminoácidos também desempenham outras funções importantes na célula, tais como sinalização ou regulação do crescimento celular. No entanto, em excesso, os aminoácidos podem ser tóxicos, embora o mecanismo de toxicidade não esteja claro. Neste estudo, usámos o peixezebra como modelo vertebrado para avaliar a toxicidade induzida por diferentes aminoácidos como resultado do desiquílibrio nutricional. Para tal, avaliámos as alterações induzidas pela toxicidade de aminoácidos durante o desenvolvimento do peixe-zebra, para compreender se esta toxicidade podia estar relacionada com a incorporação errada de aminoácidos durante a tradução. Os resultados mostram que alguns dos aminoácidos causam toxicidade em peixe-zebra, nomeadamente, L-triptofano, L-glutamina, Lfenilalanina e L-arginina. Para entender se esta toxicidade pode ser causada pela produção de proteínas aberrantes, devido ao carregamento errado de aminoácidos no tRNA, resultante de um excesso de aminoácidos, analisámos a activação de vias de degradação de proteínas. Para isso realizámos análises por western blot do estado de poliubiquitinação do proteoma. Não foram observadas diferenças entre as diferentes concentrações de aminoácidos e do controlo, indicando que a via da ubiquitina-proteossoma não está directamente relacionada com a toxicidade de aminoácidos observada

Assessment of species gaps in DNA barcode libraries of nonindigenous species (NIS) occurring in European coastal regions

DNA metabarcoding has the capacity to bolster current biodiversity assessment techniques, including the early detection and monitoring of non-indigenous species (NIS). However, the success of this approach is greatly dependent on the availability, taxonomic coverage and reliability of reference sequences in genetic databases, whose deficiencies can potentially compromise species identifications at the taxonomic assignment step. In this study we assessed lacunae in availability of DNA sequence data from four barcodes (COI, 18S, rbcL and matK) for NIS occurring in European marine and coastal environments. NIS checklists were based on EASIN and AquaNIS databases. The highest coverage was found for COI for Animalia and rbcL for Plantae (up to 63%, for both) and 18S for Chromista (up to 51%), that greatly increased when only high impact species were taken into account (up to 82 to 89%). Results show that different markers have unbalanced representations in genetic databases, implying that the parallel use of more than one marker can act complimentarily and may greatly increase NIS identification rates through DNA-based tools. Furthermore, based on the COI marker, data for approximately 30% of the species had maximum intra-specific distances higher than 3%, suggesting that many NIS may have undescribed or cryptic diversity. Although completing the gaps in reference libraries is essential to make the most of the potential of the DNA-based tools, a careful compilation, verification and annotation of available sequences is fundamental to assemble large curated and reliable reference libraries that provide support for rigorous species identifications.FCT - Fundação para a Ciência e a Tecnologia(PTDC/BIA-BMA/29754/2017

Current status of the DNA barcode reference library of non-indigenous marine species occurring in European coastal regions

Coastal habitats are among the most important, but also the most threatened ecosystems in the world, which may pose at risk many important services for human well-being (Solan et al. 2004). Along with global climate change, overexploitation, pollution and habitat destruction; the spread of invasive species is among the major threats to coastal ecosystems (Rilov 2009). Non-indigenous species (NIS), which can establish in areas outside their natural range and become invasive, can spread rapidly across the natural environment and displace and out-compete native species, driving to severe ecological changes that threaten ecosystem integrity (Rilov 2009). While morphology-based identification of taxa has largely contributed to appreciate the current status of NIS occurrence in coastal regions and marine environments (e.g. Chainho et al. 2015), this process is expertise-demanding, laborious and time consuming. Because of these limitations, most studies reporting bioinvasions and relying on morphotaxonomy are limited in time and space and only focus in the ecology of specific taxa or smaller groups at a time. Due to the above-mentioned reasons, it becomes a priority to develop and optimize novel detection methods capable to overcome some of these challenges and that would allow an early detection and ease the monitoring of NIS in coastal ecosystems. Particularly, the combination of DNA barcoding with high-throughput sequencing (HTS) renders metabarcoding the capacity to bolster current biodiversity assessments techniques (Cristescu 2014). Within this approach, genetic information can be obtained through the use of standardized DNA barcode markers targeting a wide taxonomic range of organisms in mixed specimens or environmental samples (Cristescu 2014). DNA-based tools promise a number of potential benefits over traditional methods, including increased sensitivity and specificity as well as greater throughput and cost effectiveness. However, the success of DNA-based methods is greatly dependent on the availability, taxonomic coverage and reliability of reference sequence databases, whose deficiencies can potentially compromise species identifications through HTS (Briski et al. 2016). In this study, we conducted a species-gap analysis on the availability of accessible DNA barcode sequences for marine NIS occurring in European coastal regions using the Barcode of Life Data System V4 (BOLD V4) (Ratnasingham and Hebert 2007). The checklist of NIS occurring in European seas was compiled by using two online databases: i) European Alien Species Information Network (EASIN) and ii) Information System on Aquatic Non-Indigenous and Cryptogenic Species (AquaNIS). The taxonomic classification of the NIS compiled in the checklist followed the World Register of Marine Species (WoRMS). All records without taxonomic assignments at the species level were removed from the list and only taxa with accepted scientific species names were maintained. The species list was submitted to BOLD v4 and the checklist progress report option implemented in BOLD was used to conduct our gap analysis (Ratnasingham and Hebert 2007). Barcoded species were considered to be those with sequences that meet the following requirements: i) minimum sequence length of 500 bp; ii) < 1% ambiguous bases; iii) presence of forward and reverse trace files; iv) minimum quality of trace files status and v) presence of a country specification, either with co-ordinates or country name. Barcode compliance flags were applied to the following markers if the sequences meet the above requirements: the mitochondrial cytochrome C oxidase gene (COI), for animals and the maturase K gene (matK) or the rubisco large subunit gene (rbcL), for plants. For Chromista, due to the high diversity within this kingdom, barcode compliance flags were applied to the following markers: COI for all Chromista groups, and, in addition to COI, matK and rbcL for Cryptophyta, Haptophyta and Ochrophyta. A list of 1,915 NIS occurring in European coastal regions was generated and uploaded to BOLD v4 (checklist: CL-NISEU). The species in the list were distributed by three kingdoms: i) Animalia (1,411 species), ii) Chromista (276 species) and iii) Plantae (228 species), comprehending 28 phyla. The most well represented phyla with NIS were Arthropoda (331 species), Mollusca (324 species) and Chordata (285 species) within Animalia; Ochrophyta (105 species), Myzozoa (79 species) and Foraminifera (70 species), within Chromista and Rhodophyta (171 species) and Chlorophyta (40 species), within Plantae. From the total species in the list, 1,109 were still missing a DNA barcode on BOLD (including private and public records), which corresponded to ca. 60% of the total NIS. In addition, within barcoded species ca. 13% were singletons (i.e. only one barcode sequence available). However, when only high-impact species were taken into account (146 species with the status of established or invasive), the gap was much lower (ca. 22% of the species). Animalia NIS were the most well represented with DNA barcodes, but still missing for ca. 49% of the species. Only 5 out of the 16 represented phyla have a % of barcoded species higher than 50% (Acanthocephala, Arthropoda, Chordata, Echinodermata and Nemertea). For Chromista and Plantae the gap of missing species was higher than 80%. Among Chromista, only 2 out of 8 phyla (Myzozoa and Ochrophyta) were represented with DNA barcodes, but the % of covered species was lower than 30%. Among Plantae phyla, the % of barcoded species varied between 50 and 100%, but for Rodophyta NIS no DNA barcodes were yet deposited on BOLD. The current study allowed us to gauge the gaps of DNA barcodes for NIS occurring in European coastal ecosystems. The few marine NIS eradications attempts that proved successful in the past owe their success to an early detection, soon after introduction. DNA-based tools would allow the detection of early developmental stages or smaller organisms, reducing the time from introduction to detection and increasing the success of NIS control and/or eradication (Holman et al. 2018). However, the gaps found in reference libraries can have strong implications for an accurate species identification through DNA-based tools. Prioritization efforts should be conducted in order to fulfil these gaps, in particular for high impact species still missing DNA barcodes.FCT – Fundação para a Ciência e a Tecnologia, I.P., in the scope of the project PTDC/BIA-BMA/29754/201

Compilation, revision, and annotation of DNA barcodes of marine invertebrate Non-Indigenous Species (NIS) occurring in european coastal regions

The script developed to create the DNA sequence reference library is available at https://github.com/tadeu95/Curated-BINs-Reference-Library (accessed on 25 January 2023). Metabarcoding datasets will be made available upon request since they belong to a study that is not published yet.The introduction of non-indigenous species (NIS) is one of the major threats to the integrity of European coastal ecosystems. DNA-based assessments have been increasingly adopted for monitoring NIS. However, the accuracy of DNA-based taxonomic assignments is largely dependent on the completion and reliability of DNA barcode reference libraries. As such, we aimed to compile and audit a DNA barcode reference library for marine invertebrate NIS occurring in Europe. To do so, we compiled a list of NIS using three databases: the European Alien Species Information Network (EASIN), the Information System on Aquatic Non-indigenous and Cryptogenic Species (AquaNIS), and the World Register of Introduced Marine Species (WRiMS). For each species, we retrieved the available cytochrome c oxidase subunit I (COI) mitochondrial gene sequences from the Barcode of Life Data System (BOLD) and used the Barcode, Audit & Grade System (BAGS) to check congruence between morphospecies names and Barcode Index Numbers (BINs). From the 1249 species compiled, approximately 42% had records on BOLD, among which 56% were discordant. We further analyzed these cases to determine the causes of the discordances and attributed additional annotation tags. Of the 622 discordant BINs, after revision, 35% were successfully solved, which increased the number of NIS detected in metabarcoding datasets from 12 to 16. However, a fair number of BINs remained discordant. Reliability of reference barcode records is particularly critical in the case of NIS, where erroneous identification may trigger action or inaction when not required.This research was funded by national funds through the Foundation for Science and Technology (FCT I.P.), grant number PTDC/BIA-BMA/29754/2017 (NIS-DNA: Early detection and monitoring of non-indigenous species (NIS) in coastal ecosystems based on high-throughput sequencing tools) and by the “Contrato-Programa” UIDB/04050/2020. Financial support granted by the FCT I.P. to S.D. (CEECIND/00667/2017) is also acknowledged. A.S.L. (UI/BD/150871/2021) and J.T.F. (UI/BD/150910/2021) are supported by the Collaboration Protocol for Financing the Multiannual Research Grants Plan for Doctoral Students with financial support from FCT I.P. and the European Social Fund under the Northern Regional Operational Program—Norte2020

Surveillance of non-indigenous invertebrate species through DNA metabarcoding in recreational marinas in the North and Center of Portugal

DNA metabarcoding has been widely used in biodiversity assessments as a complement to traditional morphology-based techniques. This technique is useful for the early detection and surveillance of non-indigenous species (NIS) in aquatic ecosystems. When introduced to new locations, NIS can establish, and become invasive, which may cause severe alterations to native ecosystems and biodiversity. As most introductions in coastal ecosystems occur by transport in ships (through ballast waters discharges and biofouling), ports and marinas are priority hubs for the early detection of NIS. The aim of this study was to survey marine invertebrate NIS in four marinas in the north and center of Portugal using (e)DNA metabarcoding and different sampling methodologies. We sampled four marinas in three locations in Portugal spanning a distance of ca. 150 km: one in Viana do Castelo (VC), one in Porto (L), inside the port of Leixões, and two in Aveiro (Av1: Costa Nova and Av2: Oudinot). Viana do Castelo, Porto, and Oudinot marinas are more sheltered, located near the coast, with a lower current flow, while Costa Nova marina is located inside a channel, more exposed to the passage of ships and to a higher current flow. Samples were taken of the organisms colonizing the marinas’ hard substrates (e.g. pontoons, buoys, ropes), as well as from water, for environmental DNA (eDNA) analysis, and zooplankton, collected using a 55-µm mesh net. We used two molecular markers: the mitochondrial cytochrome c oxidase gene (COI) and the small subunit ribosomal RNA gene (18S) to produce amplicon libraries for each sample, which were high throughput sequenced in an Illumina MiSeq platform. mBRAVE and SILVAngs were then used to process the sequence data and to generate species-level assignments for COI and 18S data, respectively. A total of 23 NIS was detected in this study: 13 using each molecular marker. Only 3 NIS were detected using both markers. The highest number of NIS was detected in the Av1 marina with COI (11), whereas in the other marinas this number ranged from 5-9 NIS depending on the molecular marker. Six NIS were common to all the marinas, amongst which Amphibalanus amphitrite and Amphibalanus eburneus , two invasive barnacle species and Eriocheir sinensis, the Chinese mitten crab, a very well-known species with invasive behavior that causes severe ecological and economic damages. Fifty percent of NIS belonged to the phyla Arthropoda: Crustacea and Chordata: Ascidiacea. The sampling method that registered the highest number of NIS (9) was the eDNA from water in Av1, using COI. Overall, 18S detected approximately 1,6 times higher numbers of OTUs (237-267), phyla (16-21) and species (179-210) than COI, and only 5-6% of species were detected using both markers. Regarding the type of sampling method, the zooplankton samples retrieved the highest number of phyla (20 in VC; COI: 12 and 18S: 20) and more species (40 to 155; COI: 90 and 18S: 155), namely from Mollusca and Annelida. Between the three methods, there were only 21 (COI) to 28% (18S) of shared species. Crustacea dominated most samples collected from hard substrates. As for marinas, VC had the highest number of recovered OTUs (267), phyla (21) and species (210), when analyzed with the 18S marker, and the lowest number of OTUs (94) and species (86) was recovered in Av1 with COI. Coincidently, this was also the marina with the highest number of NIS probably due to its more exposed location. These results show the efficiency of NIS detection using (e)DNA metabarcoding but reveal the need to employ different molecular markers and sampling methodologies to guarantee a more comprehensive surveillance and detection of NIS in these environmentsNIS-DNA: Early detection and monitoring of non-indigenous species (NIS) in coastal ecosystems based on high-throughput sequencing tools (PTDC/BIA-BMA/29754/2017). Ana S. Lavrador was supported by grant UI/BD/150871/202

Patterns of spatial and temporal variation in estuarine meiofaunal communities assessed through DNA metabarcoding: a case study in the Lima estuary (NW Portugal)

Meiobenthic organisms are key components of estuarine environments, providing invaluable functions and services. In particular, meiofaunal organisms participate actively in nutrient cycles and energy flux, supporting higher trophic levels, as well as the stabilization of sediments. Due to their small size (between 30 µm and 1mm) and high dispersal potential, meiofauna was considered to have a ubiquitous distribution. However, with the recent progress of DNA-based technologies, in particular, DNA metabarcoding, the cosmopolitan meiofauna paradox is being questioned, suggesting that meiobenthic communities can comprise also endemic and narrow-range species. Several studies on the spatial variability of meiofauna at small-scale (within meters) and at meso-scale (meters to km) have been carried out in an attempt to elucidate community structure, both using traditional approaches (such as morphological identification) and DNA-based tools. All these studies focused on the importance of environmental features (i.e., salinity, sediments’ size, percentage of organic matter) in the distribution of particular taxonomic groups, as well as of their bio-ecological characteristics, which may overall explain the spatial structure of the meiobenthic communities. However, little attention has been given to the temporal variation of meiofauna communities in estuaries, and to the best of our knowledge, no research on the spatial-temporal dynamics of Iberian estuarine meiofauna has been yet performed using DNA-based tools. Although DNA metabarcoding is assumed to i) allow a faster detection, ii) identify hard morphologically-intractable meiobenthic organisms or communities, and iii) target different taxonomic groups simultaneously, further refinement is required to better tailor this method for meiofauna profiling. A well-defined choice of marker loci and primer pairs, and the design of robust protocols for sampling effort and for the bioinformatics data processing and analysis is needed to avoid the increase of technical bias during the workflow. The main goal of the present study was to investigate spatial and temporal variations of the estuarine meiofauna communities using DNA metabarcoding. Sediments were collected in four sampling stations in the Lima estuary (North-West of Portugal), in June 2017 and June 2018, from the first 5 cm of sediment directly to 50 mL sterile falcon tubes (3 cm ø). Three samples were collected at the high and middle intertidal zones in each sampling station. For each site and yearly sampling campaign, we assessed salinity, total organic matter (TOM) and grain size. DNA was extracted from 10.0 g (± 0.50 g) of sediment from each collected sample, using the DNeasy PowerMax Isolation kit (QIAGEN®). Two different primer pairs were used for the production of amplicon libraries and their high-throughput sequencing (Illumina-MiSeq): a sub-region from the mitochondrial cytochrome c oxidase gene (COI) and the V4 hypervariable region of the nuclear ribosomal 18S rRNA gene (18S). Raw reads were quality-filtered by the removal of sequencing adapters and trimming of low-quality bases. Further de-multiplexing and read processing were carried out in mothur, using customized procedures. Reads were clustered into OTUs using a 97% similarity threshold. Then, the representative sequences of each OTU were BLASTed against the GenBank non-redundant nucleotide database and taxonomically assigned in MEGAN v.6.13. In terms of OTUs richness and turnover, our results showed that our communities globally changed along space and time. Meiobenthic structure, in general, displayed well-separated temporal and spatial clusters, with some generalist OTUs occurring along with the stations for both sampling years. Qualitatively, the two primer pairs detected different taxonomic groups attributed to meiofauna in a complementary manner. Nematoda, Platyhelminthes, Annelida, Arthropoda Crustacea, Amoebozoa and Ciliophora were the most abundant phyla, with different temporal and spatial proportions. Our results indicated that Lima estuarine meiofaunal communities are strongly influenced by the salinity, sediment grain size, and total organic matter, affecting meiofauna distribution among the sampling stations. Moreover, further differences on a time scale were found between the sampling years, probably as a consequence of greater rainfall during 2018 (https://www.pordata.pt). With a careful sampling design, laboratory and data analysis protocols, DNA metabarcoding can constitute a powerful approach for meiobenthic communities profiling, although further efforts are required for preparing dedicated meiofauna references libraryThe NextSea, under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). MF and SF benefitted from FCT Ph.D. (SFRH/BD/113547/2015) and post-doc (SFRH/BPD/109842/2015) fellowship

Gaps in DNA sequence libraries for macaronesian marine macroinvertebrates imply decades till completion and robust monitoring

Aim: DNA metabarcoding has great potential to improve biomonitoring in island's marine ecosystems, which are highly vulnerable to global change and non-indigenous species (NIS) introductions. However, the depth and accuracy of the taxonomic identifications are mainly dependent on reference libraries containing representative and reliable sequences for the targeted species. In this study, we evaluated the gaps in the availability of DNA sequences and their accuracy for macroinvertebrates inhabiting Macaronesia's shallow marine habitats. Location: Macaronesia (Azores, Madeira, Selvagens, Canaries). Methods: Checklists of marine invertebrates occurring above 50 m depth were compiled using public databases and published checklists. The availability of cytochrome c oxidase subunit I (COI) and 18S rRNA (18S) gene sequences was verified in BOLD and GenBank. Finally, COI data were audited to check the congruence between morphospecies and Barcode Index Numbers (BINs). Results: The taxonomic coverage of different phyla was greater for COI but unbalanced and variable among archipelagos. NIS were better represented in genetic databases (up to 73% and 59%, for COI and 18S, respectively) than native species (up to 47% and 31%, for COI and 18S, respectively). NIS displayed a higher number of discordant records, and native species, a higher quantity of cases of multiple BINs. Notably, DNA sequences generated from specimens collected from Macaronesia were found in less than 10% of the species. Projection of the rates of accretion of DNA sequences suggests that decades will be needed to complete these reference libraries. Main conclusions: The level of completion of reference libraries for Macaronesia's marine macroinvertebrates is generally poor. Without a solid effort to speed up the production of sequence data (i.e. generate more DNA barcodes), the ability to employ DNA-based biomonitoring of such vulnerable fauna is compromised. The high levelsof suspected hidden diversity further deepen the expected gaps and reinforce the vulnerability of this endemism-rich fauna.Fundacao para a Ciencia e a Tecnologia, Grant/Award Number: EECIND/00667/2017, POCI-01-0145-FEDER-006821, PTDC/BIA-BMA/29754/2017, UID/BIA/50027/2019-2020 and UIDB/04050/202

Gap-analysis and annotated reference library for supporting macroinvertebrate metabarcoding in Atlantic Iberia

DNA metabarcoding provides a rapid and effective identification tool of macroinvertebrate species. The accuracy of species-level assignment, and consequent taxonomic coverage, relies on comprehensive DNA barcode reference libraries, which, due to incompleteness, are currently a recognized limitation for metabarcoding applications. In this study, we assembled a comprehensive reference library of DNA barcodes for Atlantic Iberia marine macroinvertebrate species, assessed gaps in species coverage and examined data ambiguities. Initially, an Iberian species checklist for the three dominant groups of marine macroinvertebrates was compiled, comprising 2827 species (926 Annelida, 638 Crustacea and 1263 Mollusca). A total of 18162 DNA sequences of the cytochrome c oxidase subunit I barcode region (COI-5P) matching the species checklist were compiled in a BOLD dataset, where taxonomic discordances were evaluated and cases of deep intraspecific divergence flagged. Gap-analysis showed that 63% of the Iberian macroinvertebrate species still lack a DNA barcode. Coverage gaps varied considerably across taxonomic groups with Mollusca displaying the highest sequence representation in the dataset (427 species, 49% of the total number of sequences), and Crustacea the highest species coverage with 338 species barcoded (53% of the checklist). In contrast, Polychaeta displayed the lower levels of completion (288 species, 16% of the total number of sequences). In total, 1545 Barcode Index Numbers (BINs) were assigned to 1053 barcoded species, of which 66% were taxonomically concordant, 26% displayed multiple BINs and 8% were discordant. Overall, results show that there is still a large portion of marine invertebrate taxa in this region of Europe pending barcode coverage, even considering only the dominant groups. However, the most notable finding was the relevant proportion of species flagged for significant intraspecific divergence and possible hidden diversity. The annotated reference library and gap-analysis here provided can thereThis study was supported by the project The NextSea [NORTE-01-0145-FEDER-000032], under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This work was supported by the "Contrato-Programa'' UIDB/04050/2020 funded by national funds through the FCT I.P. SD and PEV work was supported by national funds through the Portuguese Foundation for Science and Technology (FCT, I.P.) in the scope of the project NIS-DNA [PTDC/BIA-BMA/29754/2017]. BRL benefitted from an FCT fellowship PD/BD/127994/2016. MALT benefitted from an FCT fellowship co-financed by European Social Fund (ESF) SFRH/BD/131527/2017

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

The accuracy of specimen identification through DNA barcoding and metabarcoding relies on reference libraries containing records with reliable taxonomy and sequence quality. The considerable growth in barcode data requires stringent data curation, especially in taxonomically difficult groups such as marine invertebrates. A major effort in curating marine barcode data in the Barcode of Life Data Systems (BOLD) was undertaken during the 8th International Barcode of Life Conference (Trondheim, Norway, 2019). Major taxonomic groups (crustaceans, echinoderms, molluscs, and polychaetes) were reviewed to identify those which had disagreement between Linnaean names and Barcode Index Numbers (BINs). The records with disagreement were annotated with four tags: A) MIS-ID (misidentified, mislabeled, or contaminated records), b) AMBIG (ambiguous records unresolved with the existing data), c) COMPLEX (species names 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. The 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 essential for barcode applications, such manual attempts to examine large datasets are unsustainable and automated solutions are extremely desirable.The hackathon was organized with financial support from the European Union COST Action DNAqua-Net (CA 15219 https://dnaqua.net/) in the scope of the 8th International Barcode of Life Conference in Trondheim, Norway on 16 June 2019. DNAqua-Net is acknowledged for the funding provided and the local conference organizers for all the logistical support that ensured a successful event. Tyler Elliot and the rest of the BOLD team are acknowledged for their help with data queries and analytics. The authors also thank the hackathon participants for vibrant discussions during and after the event: Berry van der Hoorn, Katrine Konsghavn, Guy Paz, Mouna Rifi, Malin Strand, Anne Helene Tandberg, Adam Wall, and Endre Willassen. Marcos A. L. Teixeira was supported by a PhD grant from the Portuguese Foundation for Science and Technology (FCT I.P.) co-financed by ESF (SFRH/BD/131527/2017). Financial support granted by FCT to Sofia Duarte (CEECIND/00667/2017) and to Pedro E. Vieira (project NIS-DNA, PTDC/BIA-BMA/29754/2017) is also acknowledged. Sanna Majaneva was financially supported by the Norwegian Taxonomy Initiative (project no. 70184235). The authors thank the five reviewers who provided valuable input into the earlier version of the manuscript