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Background and purpose: Available data in research literature suggest that the Western Balkan countries hold a rich diversity of caddisflies. Assessment and biomonitoring of such rich diversity could be facilitated through DNA-based high-throughput approaches like DNA metabarcoding that depend on the availability of comprehensive reference libraries. Materials and methods: We assessed the status of the COI barcode sequence data for a total of 112 caddisflies species in the investigated region by determining the gaps in representative sequences in the Barcode of Life Data System (BOLD) and examining the accuracy of available records using the Barcode, Audit and Grade System (BAGS). Results: Results revealed a considerable underrepresentation of surveyed geographic region in BOLD records for the target insect group. Moreover, the large majority of the species records were rated “discordant” (72.80% grade E), and only 15.20% were classified as “consolidated concordance or basal concordance” (3.20% grade A and 12.00% B). Approximately 3.20% of the records pertaining to species occurring in multiple BINs (Barcode Index Number) and 8.80% were poorly represented (i.e., less than three specimens, grade D). A fraction of the species graded discordant were deemed concordant after detailed inspection of individual data, decreasing by 14.07%. Conclusions: The assessment of the current state of BOLD entries indicated that DNA barcoding is still not widely applied in Albania, Bosnia and Herzegovina, Montenegro, North Macedonia, Serbia, and Slovenia, emphasizing that Croatia has the most barcoded caddisflies species. The finding that available BOLD Trichopteran records for investigated countries were mainly graded as “discordant” indicates the need for better quality control of reference libraries.

Morphologically similar species are often overlooked but molecular techniques have been effective in signalling potential hidden diversity, boosting the documentation of unique evolutionary lineages and ecological diversity. Platynereis dumerilii and Platynereis massiliensis are part of a recognised species complex, where differences in the reproductive biology have mainly been highlighted to date. Analyses of DNA sequence data (COI, 16S rDNA and D2 region of the 28S rDNA) of populations of the apparent morphotype of P. dumerilii obtained from a broader sampling area along European marine waters, including the Azores and Webbnesia islands (Madeira and Canaries), provided compelling evidence for the existence of at least 10 divergent evolutionary lineages. Complementing the genetic data, morphological observations of the better represented lineages revealed two major groups with distinctive paragnath patterns. Two new Platynereis species were erected: P. nunezi sp. nov., widespread in the Azores and Webbnesia islands, and P. jourdei sp. nov., restricted to the western Mediterranean. The new combination P. agilis is also proposed for Nereis agilis, previously unaccepted for one of the lineages present both in the Northeast Atlantic and western Mediterranean. Platynereis dumerilii is redescribed based on topotypic material. However, uncertainty in the identity of P. massiliensis due to the original brief description and the absence of type and topotypic material prevents the unequivocal assignment to the lineage assumed in this and previous studies. The remaining five lineages are represented by only a few small specimens with morphological features poorly preserved and were therefore not described in this study. ZooBank: urn:lsid:zoobank.org:pub:50079615-85E5-447E-BDD7-21E81C2A6F4D

Despite a high species diversity, skates (Rajiformes) exhibit remarkably conservative morphology and ecology. Limited trait variations occur within and between species, and cryptic species have been reported among sister and non-sister taxa, suggesting that species complexes may be subject to stabilising selection. Three sibling species are currently recognised in the Raja miraletus complex: (i) R. miraletus occurring along the Portuguese and Mediterranean coasts, (ii) R. parva in the Central-Eastern Atlantic off West Africa and (iii) R. ocellifera in the Western Indian Ocean off South Africa. In the present study, the genetic variation at mitochondrial and nuclear markers was estimated in the species complex by analysing 323 individuals sampled across most of its geographical distribution area to test the hypothesis that restricted gene flow and genetic divergence within species reflect known climate and bio-oceanographic discontinuities. Our results support previous morphological studies and confirm the known taxonomic boundaries of the three recognised species. In addition, we identified multiple weakly differentiated clades in the Northeastern Atlantic Ocean and Mediterranean, at least two additional cryptic taxa off Senegal and Angola, a pronounced differentiation of ancient South African clades. The hidden genetic structure presented here may represent a valuable support to species’ conservation action plans.

The monitoring of larvae in plankton samples in recreational marinas, ports, or the close vicinities, may provide key information about non-indigenous species (NIS) introduction status or detect their presence at an earlier stage. DNA metabarcoding is a powerful method to assess biodiversity but its efficiency is dependent on the methodologies employed along the analytical chain, namely the targeted genetic markers. Therefore, we aim to analyze the ability of two genetic markers - the mitochondrial cytochrome C oxidase subunit I gene (COI) and the variable region V4 of the nuclear small subunit ribosomal gene (18S) - to assess the seasonal dynamics of zooplankton communities, including NIS, through DNA metabarcoding, and further to compare it with a compiled list of zooplanktonic species identified via morphology that occur in the Lima Estuary. To this end, we sampled zooplankton communities, spanning three consecutive seasons (spring, autumn, and winter 2020/2021) in three sampling points in the recreational marina of Viana do Castelo, located in the Lima Estuary, North of Portugal. Globally, metabarcoding recovered 157 species belonging to 19 phyla, with a dominance of Annelida, Crustacea, and Mollusca that represent 64.3% of all species. Even though our sampling area poorly represents the Lima Estuary, this approach allowed the detection of 6 NIS, which were not yet reported by traditional methods of identification. The composition of the zooplankton communities recovered was greatly affected by the markers employed, and to a lesser extent by the season. Overall 18S detected more species and fewer species were recovered in winter. NIS were predominantly detected during spring and autumn, but only 5.1% of all species were detected in all seasons. This study shows the DNA metabarcoding efficiency in assessing the dynamics of zooplankton communities and highlights the interest in using a multi-marker and seasonal approach to species detection, particularly to target NIS.

DNA metabarcoding is particularly helpful for monitoring taxonomically complex communities and hard to identify morphologically, such as several zoo and ichthyoplankton, which contain eggs and larval stages of unknown species. However, the efficiency of metabarcoding in diversity recovery is dependent on the targeted genetic markers and primers employed. In this work, we compared the performance of three different primer pairs from cytochrome oxidase subunit I (COI) genetic marker in species detection from marine mesozooplankton samples and its potential to be implemented in biomonitoring programs. We employed the mlCOIintF/LoboR1 primer combination targeting marine metazoans, and two newly designed fish-specific primer cocktails for targeting the ichthyoplankton. Mesozooplankton samples were collected at 4 locations on the Portuguese coast – 1 in the northwest (Viana do Castelo) and 3 in the south (coastal lagoons of Ria de Alvor and Ria Formosa, and in the river Guadiana estuary). Bulk community DNA was extracted using a non-destructive protocol and amplicon libraries produced for the 3 primers combinations. After quality-filtering bioinformatic steps, we obtained 3.04 x 105 usable sequences, of which 76.26% were clustered into OTUs (operational taxonomic units) and 46.30% were identified at species level - corresponding to 103 taxa from 8 different metazoan Phyla. The most diverse classes were Malacostraca, Actinopterygii, and Copepoda. As expected, the generic primer pair for marine metazoa (mlCOIintF/LoboR1) retrieved a higher number of species (94) compared with the fish-specific primer cocktails (30). Nevertheless, 9 % of the total species were identified exclusively by the cocktails, of which 42% were fish. These results confirmed the potential of metabarcoding as a tool for profiling zooplankton communities and to assess ichthyoplankton diversity. Multiple primers pairs increased species detection from different taxonomic groups, being the protocol optimization for fish-specific primer cocktails, the next step for its implementation in fish stock assessments.

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.

Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2021.151783, Environmental DNA (eDNA) metabarcoding (parallel sequencing of DNA/RNA for identification of whole communities within a targeted group) is revolutionizing the field of aquatic biomonitoring. To date, most metabarcoding studies aiming to assess the ecological status of aquatic ecosystems have focused on water eDNA and macroinvertebrate bulk samples. However, the eDNA metabarcoding has also been applied to soft sediment samples, mainly for assessing microbial or meiofaunal biota. Compared to classical methodologies based on manual sorting and morphological identification of benthic taxa, eDNA metabarcoding offers potentially important advantages for assessing the environmental quality of sediments. The methods and protocols utilized for sediment eDNA metabarcoding can vary considerably among studies, and standardization efforts are needed to improve their robustness, comparability and use within regulatory frameworks. Here, we review the available information on eDNA metabarcoding applied to sediment samples, with a focus on sampling, preservation, and DNA extraction steps. We discuss challenges specific to sediment eDNA analysis, including the variety of different sources and states of eDNA and its persistence in the sediment. This paper aims to identify good-practice strategies and facilitate method harmonization for routine use of sediment eDNA in future benthic monitoring., The work is the result of the DNAqua-Net workshop held in Rome, on June 10–12, 2019, organised by IRSA-CNR and supported by European Cooperation in Science and Technology (COST) grant CA15219 (DNAqua-Net). It largely benefited from the discussions with DNAqua-Net members during this event as well as other DNAqua-Net conferences.This study was carried out within the UNIVPM VIRIDE Project (n. 2017EKFA98) funded by the Ministry of University and Research of Italy (MUR) and the Italian National Project on the use of environmental DNA for the identification of marine non-indigenous species funded by the Istituto Superiore per la Protezione e la Ricerca Ambientale (ISPRA). JP, TC, and LAPG were supported by the Swiss National Science Foundation (grant 31003A_179125) as well as FL with an Early Postdoc.Mobility grant (P2GEP3_171829). MS-M was supported by the Ministry of Education, Youth and Sports of the Czech Republic, European Regional Development Fund-Project CZ.02.1.01/0.0/0.0/16_019/0000845 and LTC 20063. KP was supported by the European Regional Development Fund within National Programme for Addressing Socio-Economic Challenges through R&D (L190003VLVI RITA1/02-60-05) and base financed project P190250PKKH of Estonian University of Life Sciences, IM was supported by the Slovenian Research Agency (ARRS) programme P4-0085. SD was supported by the Portuguese Foundation for Science and Technology (FCT) (CEECIND/00667/2017). LC was supported by the PON AIM 1854833 – PON Ricerca e Innovazione 2014–2020 – Azione I.2 – D.D. n. 407, 27/02/2018 – Attraction and International Mobility.VK was supported by grants PUT1389 (ETAg) and Institute of Technology, University of Tartu basic funding grant.

The identification of patterns and mechanisms behind species' distribution is one of the major challenges in ecology, having important outcomes for the conservation and management of ecosystems. This is especially true for those components of biodiversity providing essential ecosystem functions and for which standard surveys may underestimate their real taxonomic diversity due to high degree of cryptic diversity and inherent diagnosis difficulties, such as meiofaunal communities. Within this context, environmental DNA (eDNA) metabarcoding may provide a fast and reliable way to refine and scale-up the characterization of biological diversity in complex environmental samples, allowing to bypass issues related to biodiversity estimation. Moreover, the possibility of integrating eDNA metabarcoding-derived data with tools and methods rooted in network theory would deepen the knowledge on the structuring processes of ecological communities in ways that cannot be predicted from studying individual species/communities in isolation. Here, a sediment eDNA metabarcoding of mitochondrial cytochrome c oxidase I (COI) and nuclear hypervariable V4 region of the 18S rDNA (18S) was used to reconstruct the bipartite networks linking intertidal meiofaunal OTUs and sampling stations from three estuaries of the North-Western Iberian Peninsula. Null models were used to identify the role of environmental and spatial constraints on the structure of COI- and 18S-derived networks and to characterize the macroecological features of surveyed phyla. Our results show the feasibility of eDNA metabarcoding to capture a fair amount of diversity hard to detect with standard survey procedures and to identify hierarchical spatial structures in intertidal meiofaunal assemblages, ultimately suggesting a restricted distributional range mostly driven by niche-based processes., This work was supported by the Norte Portugal Regional Operational Programme (NORTE 2020) project `NextSea: Next generation monitoring of coastal ecosystems in a scenario of global change' [NORTE-01-0145-FEDER-000032], under the PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund (ERDF). MF and SD were also supported, respectively, by a PhD [SFRH/BD/113547/2015] and a postdoc [SFRH/BPD/109842/2015] fellowship provided by the Fundacao para a Ciencia e a Tecnologia (FCT).

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.

The polychaete genus Laeonereis (Annelida, Nereididae) occurs over a broad geographic range and extends nearly across the entire Atlantic coast of America, from the USA to Uruguay. Despite the research efforts to clarify its diversity and systematics, mostly by morphological and ecological evidence, there is still uncertainty, mainly concerning the species Laeonereis culveri, which constitutes an old and notorious case of taxonomic ambiguity. Here, we revised the molecular diversity and distribution of Laeonereis species based on a multi-locus approach, including DNA sequence analyses of partial segments of the cytochrome c oxidase subunit I (COI), 16S rRNA, and 28S rRNA genes. We examined Laeonereis specimens collected from 26 sites along the American Atlantic coast from Massachusetts (USA) to Mar del Plata (Argentina). Although no comprehensive morphological examination was performed between different populations, the COI barcodes revealed seven highly divergent MOTUs, with a mean K2P genetic distance of 16.9% (from 6.8% to 21.9%), which was confirmed through four clustering algorithms. All MOTUs were geographically segregated, except for MOTUs 6 and 7 from southeastern Brazil, which presented partially overlapping ranges between Rio de Janeiro and Sao Paulo coast. Sequence data obtained from 16S rRNA and 28S rRNA markers supported the same MOTU delimitation and geographic segregation as those of COI, providing further evidence for the existence of seven deeply divergent lineages within the genus. The extent of genetic divergence between MOTUs observed in our study fits comfortably within the range reported for species of polychaetes, including Nereididae, thus providing a strong indication that they might constitute separate species. These results may therefore pave the way for integrative taxonomic studies, aiming to clarify the taxonomic status of the Laeonereis MOTUs herein reported., This work was supported by the FAPESP (Grants n~ 2011/50317-5, 2015/25623-6, 2017/06167-5, 2018/10313-0) and CNPq through a productivity grant to A.C.Z.A (301551/2019-7). Marcos AL Teixeira was supported by a PhD fellowship (SFRH/BD/131527/2017) from FCT. Pedro Vieira was supported by a Post-Doctoral Fellowships (BPD1/next-sea/2018, NORTE-01-0145-FEDER-000032). Filipe Costa and the University of Minho's contribution was supported by the strategic program UID/BIA/04050/2013 POCI-010145-FEDER-007569. Victor C Seixas was supported by a Post-Doctoral Fellowship sponsored by CAPES-PNPD (88882.316714/2019-01). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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.

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 environments, NIS-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/150871/2021

BAGS script and associated README file can be consulted at: https://github.com/tadeu95/BAGs. To run BAGS remotely, please use https://bags.vm.ntnu.no, https://tadeu-apps.shinyapps.io/bags or any additional link available at https://github.com/tadeu95/BAGS). All the Appendixes can be found at: https://doi.org/10.5061/dryad.2rbnzs7kx., Biodiversity studies greatly benefit from molecular tools, such as DNA metabarcoding, which provides an effective identification tool in biomonitoring and conservation programmes. The accuracy of species-level assignment, and consequent taxonomic coverage, relies on comprehensive DNA barcode reference libraries. The role of these libraries is to support species identification, but accidental errors in the generation of the barcodes may compromise their accuracy. Here, we present an R-based application, Barcode, Audit & Grade System (BAGS) (https://github.com/tadeu95/BAGS), that performs automated auditing and annotation of cytochrome c oxidase subunit I (COI) sequences libraries, for a given taxonomic group of animals, available in the Barcode of Life Data System (BOLD). This is followed by implementing a qualitative ranking system that assigns one of five grades (A to E) to each species in the reference library, according to the attributes of the data and congruency of species names with sequences clustered in barcode index numbers (BINs). Our goal is to allow researchers to obtain the most useful and reliable data, highlighting and segregating records according to their congruency. Different tests were performed to perceive its usefulness and limitations. BAGS fulfils a significant gap in the current landscape of DNA barcoding research tools by quickly screening reference libraries to gauge the congruence status of data and facilitate the triage of ambiguous data for posterior review. Thereby, BAGS has the potential to become a valuable addition in forthcoming DNA metabarcoding studies, in the long term contributing to globally improve the quality and reliability of the public reference libraries., FCT - Fundação para a Ciência e a Tecnologia(PTDC/BIA‐BMA/29754/2017). European Regional Development Fund, Grant/Award Number: NORTE-01-0145-FEDER-000032; Portuguese Foundation for Science and Technology, Grant/Award Number: PTDC/BIA-BMA/29754/2017

Over the last two decades, the use of DNA barcodes has transformed our ability to identify and assess life on our planet. Both strengths and weaknesses of the method have been exemplified through thousands of peer-reviewed scientific articles. Given the novel sequencing approaches, currently capable of generating millions of reads at low cost, we reflect on the questions: What will the future bring for DNA barcoding? Will identification of species using short, standardized fragments of DNA stand the test of time? We present reflected opinions of early career biodiversity researchers in the form of a SWOT analysis and discuss answers to these questions., The Research School in Biosystematics (ForBio) is supported by a grant from the Norwegian Biodiversity Information Centre.

DNA barcoding and metabarcoding is increasingly used to effectively and precisely assess and monitor biodiversity in aquatic ecosystems. As these methods rely on data availability and quality of barcode reference libraries, it is important to develop and follow best practices to ensure optimal quality and traceability of the metadata associated with the reference barcodes used for identification. Sufficient metadata, as well as vouchers, corresponding to each reference barcode must be available to ensure reliable barcode library curation and, thereby, provide trustworthy baselines for downstream molecular species identification. This document (1) specifies the data and metadata required to ensure the relevance, the accessibility and traceability of DNA barcodes and (2) specifies the recommendations for DNA harvesting and for the storage of both voucher specimens/samples and barcode data, (undefined)

Reference libraries of DNA sequences are the backbone of DNA-based taxonomic identification systems. The quality and accuracy of the data in reference libraries is critical to achieve reliable identifications. Faulty or inaccurate data may have detrimental impacts in various downstream applications, perpetuating errors over long-term studies and biodiversity data repositories. This risk is particularly prevalent in metabarcoding approaches, where millions of sequences are assigned to taxa in reference libraries through automated and frequently unsupervised procedures. Although quality-compliance measures have been implemented in several stages of the DNA barcode production workflow, no systematized approach has tackled the challenges of revision, curation and annotation of reference libraries. The trend for increasing detection of cryptic diversity further complicates this task. Here we outline the conclusions of the application of two distinct approaches to audit and annotate reference libraries: the hackathon on marine invertebrates hosted by the 8th IBOL conference, and the bioinformatics application “Barcode, Audit & Grade System” (BAGS; Fontes et al. 2021). The former consisted on the assembly of 18 researchers involved in marine barcoding, aiming to audit and annotate a very large number DNA barcode records available in BOLD from major marine invertebrate taxa, including all or selected groups of Annelida, Crustacea, Echinodermata and Mollusca. Discordant Barcode Index Numbers (BINs), that is, BINs including more than one species, were reviewed individually, and the respective records annotated with one of the 4 following tags: MIS-ID (misidentification); AMBIG (ambiguous, unable to resolve); COMPLEX (multiple BINs); SHARE (barcodes shared among species in the same BIN). This effort resulted in the processing of >80.000 barcodes, corresponding to >7.500 species, of which 7% were tagged MIS-ID, 17% AMBIG, 13% COMPLEX and 1% SHARE, with Gastropoda displaying particularly high levels of ambiguity. The sizeable portion of MIS-ID and AMBIG tags raises concern. Yet, part of the AMBIG tags merely reflect underlying uncertainty in species taxonomic status, rather than the deposition of erroneous data in BOLD. Hence, in addition to auditing and annotation, extensive effort should continue to be allocated to the underpinning alpha taxonomy of reference libraries. The second approach here described is BAGS, which consists on an R-based application that provides an user-friendly platform for automated auditing of user-selected metazoan cytochrome oxidase I (COI) reference libraries. BAGS sorts BOLD’s records and species into 5 grades, depending on whether they display BIN concordance (A, B) multiple BINs (C), less than two records (D) or discordant BINs (E). A WoRMS-linked filter allows to select or exclude marine taxa, and a reporting component provides a graphical overview and FASTA files assorted in different combinations of grades. Therefore, BAGS can provide a quick appraisal of the status of an user-defined reference library, allowing simultaneously to recognize the most reliable records, the incidence of cases high intraspecific divergence, gaps in representativeness, and inaccuracies of potential concern. A pilot assessment of BAGS performance in three datasets comprising marine fish, Chironomidae (Insecta) and marine Amphipoda (Crustacea) highlighted the differences in the congruence status of the respective reference libraries. In conclusion, the hackathon had and expressive contribution to the revision and annotation of a very large number of marine invertebrate records lodged in BOLD. Human-mediated revision is highly-reliable and consequential, however, it constituted a massive undertaking that can hardly be repeated without a previous refinement and substantial reduction of the datasets to be revised. This could be achieved resorting to automated revision systems, among which BAGS constitutes a first step. We intend to progress with the expansion and improvement of BAGS, namely by introducing further refinements in the analyses of grade E data, in order to automatically discard simple cases of discordance, thereby reducing the amount of data needing human-mediated revision. Recognition of the need for automated reference library auditing and curation systems is essential to raise confidence of researchers, environmental managers and governmental agencies for the adoption and implementation of DNA-based approaches in aquatic biomonitoring.

Recent studies reporting complexes of cryptic or pseudo-cryptic species with narrow geographic distributions have been challenging the cosmopolitan status of a fair number of marine benthic invertebrates. Morphologically similar species are often overlooked but molecular techniques have been extremely effective in signalling potential hidden diversity which, complemented with further detailed examination, might reveal unique morphological and ecological features. Evidence of morphological stasis, where no clear and stable morphological differences are apparent, can be exemplified by the annelids Platynereis dumerilii (Audouin & Milne Edwards, 1833) and Platynereis massiliensis (Moquin-Tandon, 1869). These sibling species, usually found among algae in marine intertidal and subtidal habitats, can only be distinguished by their different reproductive strategies and life histories. The former is gonochoric, with a single reproductive event in life (semelparous) transforming into a pelagic epitokous form called heteronereis, has free spawning synchronized by lunar periodicity and a larval stage with planktotrophic development; while the latter shows no epitokous transformation and is a protandrous hermaphrodite, characterized by egg brooding and lecithotrophic larval stages with a semi-direct development. In order to verify the possible existence of additional hidden Platynereis species within the P. dumerilii morphotype, we used a multi-locus approach to investigate 26 populations along Europe from the NE Atlantic and the Macaronesia islands (Azores, Madeira and Canaries) to the Western and Eastern Mediterranean Sea. We concatenated the mtDNA COI-5P, rDNA 16S and 28S-D2 sequences and performed a phylogenetic analysis through Bayesian inference (BI). To depict Molecular Operational Taxonomic Units (MOTUs), we applied three delimitation methods (ABGD, bPTP and GYMC) to the concatenated alignment, except for COI where we also applied the Barcode Index Number (BIN), implemented in BOLD, which is exclusive to this locus. Consensus MOTUs were defined based on the majority rule and, in case of draw, the most conservative MOTUs were chosen. We detected at least 14 MOTUs with 23.1% COI mean K2P distance (6.6 - 32.6%). The BI tree is split into three major clades (Clade A: MOTUs 1-3, Clade B: MOTUs 4-9 and Clade C: MOTUs 10-14), with MOTU 3 appearing to represent P. dumerilii sensu stricto and MOTU 9 P. massiliensis. This assumption is based on data from the Type locality and a previous study by Wage et al. (2017), which combined phylogeographic (COI barcode region), reproductive biology and life-history observations on some selected Platynereis populations thriving in the vent areas from the Italian islands of Ischia and Vulcano. Major Cade C seems to be pseudo-cryptic as some visible differences can be found in the specimens, as for example, the size of the tentacular cirri and lack of dorsal pigmentation, in contrast with Clades A and B, where only differences in pigmentation types were found so far. The Spanish archipelago of the Canary Islands and the whole Mediterranean Sea seem to be a cryptic hotspot. Five MOTUs are unique to the Macaronesia and five sympatric MOTUs are present in the Gran Canaria and La Palma islands alone. Additionally, three lineages were present exclusively in the Mediterranean with four sympatric MOTUs spotted in the southeast of Spain (Calpe) and the Greek island of Crete. Three out of four NE Atlantic MOTUs are shared with the Mediterranean with one exclusive to this part of the continent. Failure to recognise this hidden biodiversity may compromise the accuracy and the interpretation of biomonitoring data or other relevant ecological studies. Integrative taxonomy is thus essential to solve these uncertainties and to allow naming the involved undescribed species. Otherwise, most molecular data providing enough support for species hypothesis will continue to be unused, and large fractions of biodiversity will persist unnoticed.

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., This 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: Early detection and monitoring of non-indigenous species (NIS) in coastal ecosystems based on high-throughput sequencing tools” (PTDC/BIA-BMA/29754/2017).

We report on two new lineages of the Eumida sanguinea complex from Great Britain and describe one of them as a new species using a multilocus approach, including the mitochondrial DNA COI-5P and the nuclear markers ITS (ITS1, 5.8S rRNA and ITS2) and 28S rRNA. The molecular analysis placed Eumida mackiei sp. nov. in a monophyletic clade with 19.1% (COI), 10.1% (ITS) and 1.7% (28S) mean distance to its nearest neighbour. Molecular diagnoses were also applied to nine lineages within the E. sanguinea complex. This was complemented with morphometric data employing multivariate statistical analysis and the incorporation of statistical dissimilarities against three other described species from the complex. Eumida mackiei sp. nov. can be distinguished from E. notata and E. maia by the larger distance between the eyes and differences in morphometric proportions mainly in the dorsal and ventral cirri as well as in the prostomial appendages. E. sanguinea sensu stricto failed to produce a cluster of its own in the morphometric analysis, probably due to juvenile bias. Integrative taxonomy provided strong evidence to formally describe a new cryptic species that can now be used in biomonitoring or other relevant ecological research., UA - Universidade de Aveiro(70184228).

Polychaetes are common in coastal and estuarine environments worldwide and constitute one of the most complex groups of marine invertebrates. The morpho-physiology of the female reproductive system (FRS) can be understood by using histological tools to describe reproductive cycle and gametogenesis paths and, among other purposes, aiming to identify and differentiate polychaete species. However, this histology-based approach is rarely combined with molecular tools, which is known to accurately delimitate species. In the same way, the description and understanding of oogenesis and vitellogenesis paths within polychaetes are lacking for most families, narrowing the range of its utility. Therefore, the present study aims to describe the oogenesis in three polychaete species common and abundant on the South American Atlantic coast (Laeonereis culveri, Scolelepis goodbodyi and Capitella biota) and investigate the utility of reproductive features and gametogenesis as a relevant associate knowledge to discriminate species, particularly useful for putative cryptic species, integrated with morphological and molecular data. In a first attempt, the results obtained herein allow the authors to describe two new subtypes of oogenesis, dividing it in extraovarian oogenesis type I and II and intraovarian type I and II. The results also demonstrate that the following histological characters of the FRS can be relevant for the separation of related species: a) oogenesis type, b) occurrence or absence of a true ovary, c) ovary tissue organization, d) type of accessory cells present, and e) oocyte morphology. Additionally, these histological features of FRS, when compared with correlated species studied under this scope, converge with the genetic data. The analysis of cytochrome oxidase I (COI) barcode sequences differentiates between North and South American Atlantic populations of L. culveri (16.78% genetic distance), while in S. goodbodyi and C. biota it discriminates them from their congeneric species. These results highlight the, Os poliquetas são comuns em ambientes costeiros e estuarinos em todo o mundo e constituem um dos grupos mais complexos de invertebrados marinhos. A morfo-fisiologia do sistema reprodutor feminino (FRS) pode ser compreendida por meio de ferramentas histológicas para identificar e diferenciar estes anelídeos. No entanto, essa abordagem histológica raramente é combinada com ferramentas moleculares, amplamente conhecidas por delimitar espécies congenéricas ou crípticas com maior precisão. Do mesmo modo, a descrição e o entendimento da oogênese e vitelogênese dentre os poliquetas, para a maioria das famílias, é ainda limitado. Portanto, o presente estudo tem como objetivo descrever a oogênese em três espécies de poliquetas comuns e abundantes na costa sul-americana (Laeonereis culveri, Scolelepis goodbodyi e Capitella biota) e investigar a utilidade das características reprodutivas e da gametogênese como um conhecimento associado relevante para discriminar espécies, particularmente útil para espécies crípticas putativas, integradas a dados morfológicos e moleculares. Os resultados aqui obtidos permitiram descrever dois novos subtipos de oogênese, dividindo-a em oogênese extra-ovariana dos tipos I e II e intra-ovariana dos tipos I e II. Os resultados também demonstram que os seguintes caracteres histológicos do FRS podem ser relevantes para a separação de espécies relacionadas: a) tipo de oogênese, b) presença ou ausência de um ovário verdadeiro, c) organização tissular ovariana, d) tipo de células acessórias presentes e, e) morfologia do ovócito. Além disso, essas características histológicas do FRS, quando comparadas às espécies correlatas estudadas sob esse escopo, convergem com os dados genéticos separando espécies putativas e congenéricas. As análises com DNA barcode demonstraram que em L. culveri é possível diferenciar as populações atlânticas Norte e Sul-americanas (16,78% de distância genética), enquanto para S. goodbodyi e C. biota fica evidente sua distinção com espécies congenéricas. Esses resultados destacam a importância da abordagem com múltiplas ferramentas e mostram que tanto a histologia quanto a histo-fisiologia do FRS e o DNA barcode podem ser usados para identificar e discriminar espécies crípticas e potencialmente crípticas, o que geralmente não é possível quando se utilizam apenas caracteres morfológicos. Além disso, esses caracteres também podem ser úteis na diferenciação de espécies relacionadas e / ou populações geograficamente distintas desses poliquetas., The authors would like to thank IB/UNICAMP, IO/USP and CEBIMar/USP for providing logistic support. In addition, the authors would like to thank the CBMA and the IB-S for the technical support. This work was supported by the FAPESP (Grants no 2011/50317-5, 2015/25623-6, 2017/06167-5) and CNPq through a productivity grant to A.C.Z.A (306534/2015-0). M.A.L.T was supported by a PhD fellowship (SFRH/BD/131527/2017) from FCT. P.E.V. was supported by a Post-Doctoral Fellowships (BPD1/next-sea/2018, NORTE-01-0145-FEDER-000032). F.O.C. and the University of Minho contribution was supported by the strategic programme UID/BIA/04050/2013 POCI-01-0145-FEDER-007569.

Meiofauna organisms play an important role in ecological and sedimentary processes in estuarine ecosystems. Recently, the application of environmental DNA (eDNA) for investigating meiofauna in different environments, improved the accessibility to its diversity and composition in a scale, frequency and depth previously unattainable. Nevertheless, little attention has been given to the description of baseline patterns of coupled spatial and temporal dynamics of meiobenthic communities. In an earlier study conducted in Lima estuary (NW Portugal), using eDNA metabarcoding of sediment samples, high levels of meiofauna Operational Taxonomic Units (OTUs) turnover were recorded, between sampling points only a few metres apart, and among sampling stations along the estuary. In order to verify the consistency of these patterns, in the current study we re-assessed Lima estuary's meiofauna communities approximately 1 year after, applying the same methodological approach (targeting segments of the COI and 18S rRNA genes), and expanding HTS-data analyses through the use of association networks. A high degree of spatial turnover was found both within and between sampling stations and this was consistent for both markers and years. As a consequence, most of the beta-diversity was accounted by OTU replacement with only a minor contribution from OTU richness. Despite the high levels of OTU replacement, relatively stable network properties were found in meiofaunal communities, irrespective of the sampled year. Network properties appear to shift sharply from the downstream/high salinity area of the estuary to the mesohaline medium-upstream areas, suggesting high resilience and redundancy of meiofaunal communities along the estuarine gradient. The recognition of meiofauna's networks features may improve the understanding of the ecology and dynamics of these communities that apparently hold large portions of variable elements, thereby making difficult their analyses solely based on the OTU/species composition., This study was funded by the project "The NextSea: Next generation monitoring of coastal ecosystems in a scenario of global change'' (operacao NORTE-01-0145-FEDER-000032), supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). Maria Fais and Sofia Duarte were supported, respectively by a Ph.D. (SFRH/BD/113547/2015) and a post-doc fellowship (SFRH/BPD/109842/2015) from FCT. The authors would like to thank Prof. Jesus Troncoso (University of Vigo) and Prof. Pedro Gomes (University of Minho) for their availability and hospitality for the lab processing; Dr. Bruno Sampieri and Barbara Leite (University of Minho) for the great support during the implementation of the practical stage of the research. The authors would like also to thank the anonymous reviewers for critically reading the manuscript and suggesting substantial improvements.

In electromagnetism, the concept of Poynting vector as measured by an observer is well known. A mathematical analogue in general relativity is the super-Poynting vector of the Weyl tensor. Observers for which the (super-)Poynting vector vanishes are called principal. When, at a given point, the electromagnetic field is non-null, or the gravitational field is of Weyl-Petrov type I or D, principal observers instantaneously passing through that point always exist. We survey characterizations of such observers and study their relation to arbitrary observers. In the non-null electromagnetic case it is known that, given any observer, there is a principal observer which moves relative to the first in the direction of his Poynting vector. Replacing Poynting by super-Poynting yields a possible gravitational analogue; we show that this analogy indeed holds for any observer when the Petrov type is D, but only for a one-dimensional variety of observers when the Petrov type is I. We provide algorithms to obtain the principal observers directly from the electric and magnetic fields (in the electromagnetic case) or electric and magnetic parts of the Weyl tensor (in the gravitational case) relative to an arbitrary observer. It is found that in Petrov type D doubly aligned non-null Einstein-Maxwell fields (which include all classical charged black hole solutions) the Poynting and super-Poynting vectors are aligned, at each point and for each observer, and the principal observers coincide. Our results are illustrated in simple examples., Comment: 42 pages, 6 figures. Minor improvements, typos corrected, references added. Matches the final published version

Meiofaunal organisms play a key role in estuarine ecosystems, being responsible for significant ecological processes. However, meiofauna constitutes a particularly difficult community to be monitored through conventional morphology-based approaches. New emerging tools, such as DNA metabarcoding, facilitate the access to these communities and provide an opportunity to develop routine monitoring programs. In the present study, the small-scale spatial variation of meiofaunal communities in the Lima estuary (NW Portugal) was investigated using DNA metabarcoding. The first stage of the study aimed to establish the amount of sediment sample to be used for DNA extraction and to test six primer pairs, three of them amplifying fragments from the mitochondrial cytochrome c oxidase gene (COI) and three other the nuclear ribosomal 18S rRNA gene (18S). In a subsequent stage, sediment samples were collected in four stations along an estuarine gradient (salinity ranging between 9 and 28), in which six sampling points about 4–5 m apart were considered: three in the high intertidal and three in the mid intertidal. After the DNA extraction from sediments, COI and 18S amplicon libraries were produced and sequenced in an Illumina MiSeq platform. OTUs (operational taxonomic units) recovered by either COI or 18S displayed generally high turnover in occurrence among sampling points within a station and tidal horizon, among tidal horizons within a station, and among stations of distinct salinity (approx. 60–93%). Both markers recorded little variation among stations in OTU richness and in the taxonomic composition of the most dominant groups. However, the meiofauna detected differed qualitatively between the two markers used; Amoebozoa and Cnidaria were mostly detected with COI while Ciliophora and Platyhelminthes with 18S. In addition, the structure of the meiofauna community diverged significantly among stations and was strongly influenced by salinity and sediment features. Globally, results indicated a highly patchy distribution of meiofauna taxa in the Lima estuary, revealed by the high OTU turnover even between sampling points only a few meters apart. Hence, eDNA-based meiofauna surveys require consideration of the necessary sampling effort on relatively small spatial scales, as well as an appreciation of the tidal level-induced variation of these communities., This study was funded by the project “The NextSea: Next generation monitoring of coastal ecosystems in a scenario of global change” (operação NORTE-01-0145-FEDER-000032), supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). Maria Fais and Sofia Duarte were supported, respectively by a PhD (SFRH/BD/113547/2015) and a post-doc fellowship (SFRH/BPD/109842/2015) from FCT. The authors would like to thank Prof. Jesús Troncoso (University of Vigo) and Prof. Pedro Gomes (University of Minho) for their availability and hospitality for the lab processing, as well as Barbara Leite (University of Minho) and Manuel Mendoza (University of Vigo) for their help during the sampling and data analysis phases, respectively.

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)

The Lewis solutions describe the exterior gravitational field produced by infinitely long rotating cylinders, and are useful models for global gravitational effects. When the metric parameters are real (Weyl class), the exterior metrics of rotating and static cylinders are locally indistinguishable, but known to globally differ. The significance of this difference, both in terms of physical effects (gravitomagnetism) and of the mathematical invariants that detect the rotation, remain open problems in the literature. In this work we show that, by a rigid coordinate rotation, the Weyl class metric can be put into a "canonical" form where the Killing vector field $\partial_{t}$ is time-like everywhere, and which depends explicitly only on three parameters with a clear physical significance: the Komar mass and angular momentum per unit length, plus the angle deficit. This new form of the metric reveals that the two settings differ only at the level of the gravitomagnetic vector potential which, for a rotating cylinder, cannot be eliminated by any global coordinate transformation. It manifests itself in the Sagnac and gravitomagnetic clock effects. The situation is seen to mirror the electromagnetic field of a rotating charged cylinder, which likewise differs from the static case only in the vector potential, responsible for the Aharonov-Bohm effect, formally analogous to the Sagnac effect. The geometrical distinction between the two solutions is also discussed, and the notions of local and global staticity revisited. The matching in canonical form to the van Stockum interior cylinder is also addressed., 43 pages, 6 Figures. Minor re-write/simplifications to the text, new section 5.2.4 added discussing the spinning cosmic string limit, titles of subsections 5.4 and 5.4.2 changed, references added, typos corrected. Matches the final published version

Originally introduced in connection with general relativistic Coriolis forces, the term $\textit{frame-dragging}$ is associated today with a plethora of effects related to the off-diagonal element of the metric tensor. It is also frequently the subject of misconceptions leading to incorrect predictions, even of nonexistent effects. We show that there are three different levels of frame-dragging corresponding to three distinct gravitomagnetic objects: gravitomagnetic potential 1-form, field, and tidal tensor, whose effects are independent, and sometimes opposing. It is seen that, from the two analogies commonly employed, the analogy with magnetism holds strong where it applies, whereas the fluid-dragging analogy (albeit of some use, qualitatively, in the first level) is, in general, misleading. Common misconceptions (such as viscous-type "body-dragging") are debunked. Applications considered include rotating cylinders (Lewis-Weyl metrics), Kerr, Kerr-Newman and Kerr-dS spacetimes, black holes surrounded by disks/rings, and binary systems., Comment: 20+8 pages, 4 figures. Invited feature paper for Universe special issue on "Frame-Dragging and Gravitomagnetism"; matches the final published version

Amphipods of the genus Gammarus are a vital component of macrozoobenthic communities in European inland and coastal, marine and brackish waters of the Mediterranean and the Black Sea. Exceptional levels of cryptic diversity have been revealed for several widespread freshwater Gammarus species in Europe. No comprehensive assessment has yet been made for brackishwater counterparts, such as Gammarus aequicauda and G. insensibilis, which are among the most widely dispersed members of the so-called “G. locusta group” in the Mediterranean and in the Black Sea. Here we probe the diversity of these morphospecies examining the partitioning of mtDNA and nDNA across multiple populations along their distribution range and discuss it within the regional paleogeographic framework. We gathered molecular data from a collection of 166 individuals of G. aequicauda and G. insensibilis from 47 locations along their distribution range in the Mediterranean including the Black Sea. They were amplified for both mitochondrial COI and 16S rRNA as well as the nuclear 28S rRNA. All five MOTU delimitation methods (ABGD, BIN, bPTP, GMYC single and multiple threshold models) applied revealed deep divergence between Black Sea and Mediterranean populations in both G. aequicauda and G. insensibilis. There were eight distinct MOTUs delimited for G. aequicauda (6–18% K2P) and 4 MOTUs for G. insensibilis (4–14% K2P). No sympatric MOTUs were detected throughout their distribution range. Multimarker time-calibrated phylogeny indicated that divergence of both G. aequicauda and G. insensibilis species complexes started already in the late Oligocene/early Miocene with the split between clades inhabiting eastern and western part of the Mediterranean occurring in both species at the similar time. Our results indicate a high cryptic diversity within Mediterranean brackishwater Gammarus, similar to that observed for freshwater counterparts. Moreover, the phylogenetic history combined with the current geographic distribution indicate that the evolution of bot, This work was supported by Polish National Science Center (projects no. 2014/15/B/NZ8/00266 and 2015/17/N/NZ8/01628) and partially by the statutory funds of the Department of Invertebrate Zoology and Hydrobiology of University of Lodz. F. Costa and the University of Minho contribution was supported by the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI). There was no additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Diversification and speciation of terrestrial organisms are anticipated in oceanic islands such as Macaronesia, a group of Atlantic islands that have remained unconnected to continental landmasses. Hitherto, the diversification of marine organisms in oceanic islands, especially those with low vagility, has received little direct empirical analysis using molecular markers. Here, we focus on such a case study, through applying a multilocus molecular approach to investigate the diversity and evolution of a group that lacks a planktonic larval stage, the isopod genus Dynamene, in Macaronesia and Northeast Atlantic. Sequences of two mitochondrial (cytochrome c oxidase subunit I and 16S rRNA) and two nuclear (18S rRNA and 28S rRNA) loci were obtained from specimens of Dynamene edwardsi (Lucas, 1849), Dynamene magnitorata Holdich, 1968 and Dynamene bidentata (Adams, 1800) collected along the Northeast Atlantic and Macaronesia. Although no major phylogeographic structure was detected in D. bidentata and D. magnitorata, from five to nine deeply divergent lineages were evident within D. edwardsi. The divergent lineages displayed genetic distances comparable to those found among established species of peracarids. D. edwardsi exhibits a long, rich and complex phylogeographic history in Macaronesia, where the geodynamics of the islands possibly associated with founder effects and subsequent lack of gene flow among populations confounds patterns based on geographic proximity of targeted populations. Our findings collectively suggest a much larger role of oceanic islands in the diversification of marine invertebrates than previously anticipated. The work provides insights into the origins and dynamics of ongoing geographic segregation and associated deep divergence among sister evolutionary lineages in Macaronesia., UA - Universidade de Aveiro(141565/2017-9). Fundação para a Ciência e Tecnologia (FCT) under the COMPETE program, supported by the European Regional Development Fund. FCT also supported a Ph. D. grant to P.E.V. (SFRH/BD/86536/2012). Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) provided a Ph. D. grant to A.D. (process No 141565/2017-9). CBMA (P.E.V., P.S. and F.O.C.) contribution was supported by the strategic programme UID/BIA/04050/2019, funded by national funds through the FCT I.P.

Coastal regions are widely recognised for their ecologic and socio-economic importance. The spread of invasive species, along with global climate change, overexploitation, pollution and habitat destruction, is a major threat to coastal ecosystems (Solan et al. 2004). Some of these species, in particular macroalgae and invertebrates, can spread quickly and become invasive, causing severe ecologic and economic impacts. When prevention fails, the early detection and surveillance of NIS could be essential for coordinating a timely and effective response to invasions. Marine ecosystems on oceanic islands, such as Azores, can be especially prone to invasions by non-indigenous species since they are characterized by a small number of native species and by a high availability of empty niches, and thus, the list of NIS is relatively long (Micael et al. 2014). There are no doubts that traditional morphology-based methods have highly contributed to the current knowledge about NIS communities inhabiting Portuguese coastal waters, including the Portuguese oceanic islands (Micael et al. 2014, Chainho et al. 2015). However, the hurdles associated with morphological methods may compromise the early detection and monitoring of the most risky species. On the other hand, DNA-based tools promise advantages over traditional ones, particularly the identification of individuals and life stages that cannot be recognized by using morphological approaches (Darling and Mahon 2011). Plus, most surveys typically target specific species or taxonomic groups, which may neither accurately represent overall ecosystems’ condition nor allow an early detection of NIS. 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 of DNA barcode sequences available for marine NIS occurring in the Azores archipelago in the Barcode of Life Data System V4 (BOLD V4) (Ratnasingham and Hebert 2007) and in the GenBank (https://www.ncbi.nlm.nih.gov/genbank/). The list of NIS occurring in Azores was compiled from a wide variety of sources, mainly from literature searches through scientific papers and reports, including recent field and taxonomic studies, and supplemented with existing databases and this is the list that was submitted to the 2019 interim ICES WGITMO report (International Council for the Exploration of the Sea; Working Group on Introductions and Transfers of Marine Organisms). The taxonomic classification of the compiled NIS followed the AlgaeBase and World Register of Marine Species (WoRMS) databases. A checklist of 80 marine NIS occurring in Azores archipelago was compiled and the species in the list were checked for the presence of public sequences on both BOLD v4 and GenBank databases. The species in the list were distributed by three kingdoms: i) Animalia (48 species), ii) Plantae (30 species) and iii) Chromista (2 species), comprehending 10 phyla. The most well represented phyla with NIS were Chordata (15 species), Mollusca and Bryozoa (both with 8 species) within Animalia, and Rhodophyta within Plantae (26 species); while the only represented phyla within Chromista was Ochrophyta with 2 species. In total, 3,884 sequences belonging to 25 different markers were found for the 80 species, published in BOLD and GenBank. Among these published sequences, the COI-5P is the most well represented loci, with 3,224 records belonging to 44 species. However, from the total species in the list, 25 were still missing a DNA sequence, which corresponded to ca. 31% of the total NIS. In addition, within sequenced species ca. 14% were singletons (i.e. only one sequence available). Animalia NIS were the most well represented with sequences, missing only for ca. 27% of the species, while for Chromista and Plantae the gap of missing species was higher than 35%. The current study allowed us to characterize the gaps in available sequences in public repositories for marine NIS occurring in the Azores archipelago. Actions developing DNA-based tools should be a priority for detection and effective management of biological invasions. DNA-based tools would allow the detection of early developmental stages or smaller organisms, reducing the time from introduction to discovery 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, FCT – Fundação para a Ciência e a Tecnologia, I.P., in the scope of the project PTDC/BIA-BMA/29754/2017 and by FEDER funds through the Operational Programme for Competitiveness Factors - COMPETE and by National Funds through FCT - Foundation for Science and Technology under the UID/BIA/50027/2013 and POCI-01-0145-FEDER-006821

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 for future collaborative programs. 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 utilisation of metabarcoding in aquatic biomonitoring., This paper is a deliverable of the European Cooperation in Science and Technology (COST) Action DNAqua-Net (CA15219) Working Group 1, led by Torbjørn Ekrem and Fedor Čiampor. Thanks to the University of Minho and University of Pécs for hosting workshops and working group meetings. We also thank staff at National Environment Agencies and others that provided national checklists of taxa used in biomonitoring, and otherwise assisted with checklist proof-reading: Jarmila Makovinská and Emília Mišíková Elexová (Slovakia); Steinar Sandøy and Dag Rosland (Norway); Mišel Jelič (Croatia); Marlen Vasquez (Cyprus); Adam Petrusek (Czech Republic); Kristel Panksep (Estonia); Panagiotis Kaspiditis (Greece); Matteo Montagna (Italy); Marija Katarzyte (Lithuania); Ana Rotter (Slovenia); Rosa Trabajo (Spain); Florian Altermatt (Switzerland); Kristian Meissner (Finland), Rigers Bakiu (Albania), Valentina Stamenkovic and Jelena Hinic (Macedonia); Patricia Mergen (Belgium); Gael Denys & the French Biodiversity Agency (France); Mary Kelly-Quinn (Ireland); Piotr Panek and Andrzej Zawal (Poland); Cesare Mario Puzzi (Italy); Carole Fitzpatrick (United Kingdom); Simon Vitecek (Austria); Ana Filipa Filipe (Portugal); Peter Anton Stæhr & Anne Winding (Denmark); Michael Monaghan (Germany); Alain Dohet, Lionel L'Hoste, Nora Welschbillig & Luc Ector (Luxembourg), Lujza Keresztes, (Romania). The authors also want to thank Dirk Steinke for providing the original European ERMS list for marine taxa and Florian Malard for comments on the manuscript. The preparation of the AMBI checklist was carried out in the scope of a Short-term Scientific Mission (ECOST-STSM-CA15219-150217- 082111) granted to SD visiting AZTI, Spain. ZC was supported by grants EFOP-3.6.1.-16-2016-00004 and 20765-3/2018/FEKUTSTRAT. TE was supported by the NorBOL-grant (226134/F50) from the Research Coun cil of Norway. BR, FL and MFG contributed through support from the GBOL project, which is generously funded by the German Federal Min istry of Education and Research (FKZ 01LI1101 and 01LI1501). MG contributed through support of the Polish National Science Centre, grants N N303 5794 39 and 2014/15/B/NZ8/00266. SF was funded by the project PORBIOTA - Portuguese E-Infrastructure for Information and Research on Biodiversity (POCI-01-0145-FEDER-022127), supported by Operational Thematic Program for Competitiveness and Internationalization (POCI), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER).