Phyllodocida (Annelida, Polychaeta) of the North East Atlantic as a model for the investigation of cryptic species

Tese de doutoramento em Biology (especialização em Integral Management of the Sea)Molecular techniques have been effective in signalling potential hidden diversity in species displaying similar morphology and presumed widespread distribution. In this study, members of Phyllodocida collected along European coasts were used as a model taxon to investigate this topic, by employing a combination of multi-locus molecular data (mtCOI-5P, 16SrRNA, ITS regions and 28SrRNA), together with morphological and morphometric examination. This work identified a large number of undescribed cryptic lineages within 6 morphospecies, namely: Eumida sanguinea (22 Molecular Operational Taxonomic Units - MOTUs); Eulalia clavigera (9 MOTUs); Hediste diversicolor (5 MOTUs); Platynereis dumerilii (10 MOTUs); Perinereis cultrifera (14 MOTUs) and Trypanosyllis zebra (10 MOTUs). In total 70 lineages were uncovered, of which 43 are unique to this work. Five of these morphospecies have a dedicated chapters where an integrative approach allowed the description of 13 new species to science and the clarification of ambiguities regarding previously descriptions. The Macaronesian islands and especially, the western part of the Mediterranean Sea, are hotspots of cryptic diversity, with a total of 10 and 30 unique lineages for each region, respectively. Mediterranean MOTUs appear to be genetically closer to the ones from Macaronesia islands, instead of the NE Atlantic lineages. A total of 2171 new sequences (1012 COI, 307 16S, 320 ITS and 532 28S) were added to the reference libraries (GenBank and BOLD systems) and will be publicly available upon publication in peer-reviewed journals. Upon minute morphological examination of the specimens, it become apparent that several lineages with obvious morphological differences have been overlooked in the literature, being commonly misidentified to the morphologically closer described species. Morphological stasis was challenged, since it appears that the older the ancestral split resulting from the different geological event periods, the higher is the probability of finding slight phenotypic disparities in cryptic lineages, previously thought to be morphological identical. Evidence for this can be seen in the deep divergence between major phylogenetic clades within some of the analysed species complex, and the perfect match of each clade to the specific morphological variation (e.g. complexes within Perinereis, Platynereis and Eulalia). In spite of these contributes, the analyses indicated that only 11% of the existing Phyllodocida species have DNA barcodes publicly available. Naming molecular lineages which lacked enough specimens with structural integrity, further sampling in subtidal regions and additional bio-informatic tools to explore the cryptic phenomena from an evolutionary and phylogeographic point of view is desirable in future works.Técnicas moleculares têm vindo a ser eficazes na sinalização de diversidade oculta em espécies com uma ampla distribuição geográfica. Nesta tese, membros dos Phyllodocida coletados ao longo das costas europeias foram utilizados como um táxon modelo para investigar espécies crípticas, usando uma combinação de dados moleculares multi-locus (mtCOI-5P, rRNA16S, regiões ITS e rRNA28S), morfológicos e morfométricos. Este estudo identificou um grande número de linhagens crípticas não descritas em 6 morfo-espécies distintas: Eumida sanguinea (22 Unidades moleculares taxonómicas operacionais - MOTUs); Eulalia clavigera (9 MOTUs); Hediste diversicolor (5 MOTUs); Platynereis dumerilii (10 MOTUs); Perinereis cultrifera (14 MOTUs) e Trypanosyllis zebra (10 MOTUs). No total, foram descobertas 70 linhagens, das quais 43 aparentam ser exclusivas deste trabalho. Cinco dessas morfo espécies têm nesta tese um capítulo dedicado, onde uma abordagem integrativa permitiu a descrição de 13 novas espécies para a ciência e a remoção de ambiguidades em relação a descrições anteriores. As ilhas da Macaronésia e a parte ocidental do Mar Mediterrâneo, são hotspots de especiação críptica, tendo-se encontrado um total de 10 e 30 linhagens únicas para cada região, respetivamente. MOTUs mediterrâneos aparentam ser geneticamente mais próximos das ilhas da Macaronésia, com as linhagens do Nordeste Atlântico aparentando ser mais distantes. Um total de 2171 novas sequencias (1012 COI, 307 16S, 320 ITS e 532 28S) foram adicionadas às bibliotecas de referência (GenBank e BOLD) e estarão disponíveis publicamente após publicação. Ao examinar mais detalhadamente o grau real de semelhança morfológica entre algumas destas supostas linhagens crípticas, fica claro que um numero considerável possui diferenças morfológicas que foram negligenciadas e erroneamente identificadas. A estase morfológica foi desafiada, uma vez que parece que quanto mais antiga a divisão ancestral resultante dos diferentes períodos geológicos, maior é a probabilidade de encontrar pequenas disparidades fenotípicas em linhagens que inicialmente aparentavam ser morfologicamente idênticas. A evidência disso pode ser vista na divergência profunda entre os principais clados filogenéticos em alguns dos complexos aqui analisados e a combinação perfeita de cada clado com uma variação morfológica específica (por exemplo, nos complexos Perinereis, Platynereis e Eulalia). Além do mais, verificou-se neste estudo que apenas 11% das espécies existentes na ordem dos Phyllodocida têm códigos de barra de ADN disponíveis ao público. Linhagens moleculares por nomear, mais amostragens em regiões subtidais e ferramentas bioinformáticas adicionais são necessárias para continuar a explorar este fenômeno críptico do ponto de vista evolutivo e filogeográfico.I am grateful to the Portuguese Foundation for Science and Technology (FCT) for supporting my PhD grant SFRH/BD/131527/2017, hosted by the University of Minho (Portugal), the University of Aveiro (Portugal) and University of Gothenburg (Sweden) in the scope of the PhD programme in Marine Science, Technology and Management (Do*Mar), specialization in Integral Management of the Sea. I also received financial support from the DNAqua-Net STSM grant "Rich and hidden biodiversity not yet barcoded in the Canary archipelago (Spain) as an opportunity to enrich the DNA barcode reference library for European polychaetes", under the EU Cost action CA15219 - Developing new genetic tools for bioassessment of aquatic ecosystems in Europe. The research leading to these results also received partial funding, from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 730984, ASSEMBLE Plus project (application n. 8229, 4th CALL, "Crypticism in the marine realm: DNA barcode-based outlook into selected invertebrate taxa of the Eastern Mediterranean"). I would like to thank the project “The NextSea: Next generation monitoring of coastal ecosystems in a scenario of global change” (NORTE-01-0145-FEDER-000032), supported by NORTE 2020 (Norte Portugal Regional Operational Programme), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and the project River2Ocean – Socio-ecological and biotechnological solutions for the conservation and valorisation of aquatic biodiversity in the Minho Region, with the reference NORTE-01-0145-FEDER-000068, co-financed by the European Regional Development Fund (ERDF), through Programa Operacional Regional do Norte (NORTE 2020), for supporting this work. Financial support to Arne Nygren from the Norwegian Taxonomy Initiative [http://www.biodiversity.no/Pages/135523] (Cryptic polychaete species in Norwegian waters, knr 49- 13, pnr 70184228), the Swedish Taxonomy Initiative [https://www.artdatabanken.se/en/the-swedish taxonomy-initiative/] (Polychaete species complexes in Swedish waters, dnr 140/07 1.4 and 166/08 1.4), and Kungliga Fysiografiska sällskapet Nilsson-Ehle donationerna [https://www.fysiografen.se/sv/]

Taxonomy, distribution and ecology of the order Phyllodocida (Annelida, Polychaeta) in deep-sea habitats around the Iberian margin

The polychaetes of the order Phyllodocida (excluding Nereidiformia and Phyllodociformia incertae sedis) collected from deep-sea habitats of the Iberian margin (Bay of Biscay, Horseshoe continental rise, Gulf of Cadiz and Alboran Sea), and Atlantic seamounts (Gorringe Bank, Atlantis and Nameless) are reported herein. Thirty-six species belonging to seven families – Acoetidae, Pholoidae, Polynoidae, Sigalionidae, Glyceridae, Goniadidae and Phyllodocidae, were identified. Amended descriptions and/or new illustrations are given for the species Allmaniella setubalensis, Anotochaetonoe michelbhaudi, Lepidasthenia brunnea and Polynoe sp.. Relevant taxonomical notes are provided for other seventeen species. Allmaniella setubalensis, Anotochaetonoe michelbhaudi, Harmothoe evei, Eumida longicirrata and Glycera noelae, previously known only from their type localities were found in different deep-water places of the studied areas and constitute new records for the Iberian margin. The geographic distributions and the bathymetric range of thirteen and fifteen species, respectively, are extended. The morphology-based biodiversity inventory was complemented with DNA sequences of the mitochondrial barcode region (COI barcodes) providing a molecular tag for future reference. Twenty new sequences were obtained for nine species in the families Acoetidae, Glyceridae and Polynoidae and for three lineages within the Phylodoce madeirensis complex (Phyllodocidae). A brief analysis of the newly obtained sequences and publicly available COI barcode data for the genera herein reported, highlighted several cases of unclear taxonomic assignments, which need further study.Thanks are due to the chief-scientists, scientific parties and crews of the 17 cruises that originated the material examined herein. We would like to thank Clara F. Rodrigues who participated in most of the cruises, often assisting with sample collection and sorting, and also provided the map with the location of sampling sites, and João Gil for his very useful comments and indispensable help with bibliography. This work was funded by Fundação para a Ciência e a Tecnologia (FCT) under the strategic programmes PEst-C/MAR/LA0017/2013 and UID/AMB/50017/2013, and the projects CHEMECO (EURODEEP/0001/2007, ESF EuroDEEP programme) SWIMGLO; the European Commission under the projects HERMES (EC contract GOCE-CT-511234 FP6) and HERMIONE (EC contract 226354, FP7). The first author was supported by the postdoctoral grants BPD/UI88/2911/2013 (Universidade de Aveiro, project MARES (CENTRO-07-ST24 FEDER-002033) co-funded by QREN Mais Centro (Programa Operacional do Centro) and EU structural funds (European Regional Development Funds)), and SFRH/BPD/112408/2015 (FCT). Sequencing at the Biodiversity Institute of Ontario was supported by funding of the International Barcode of Life Project (iBOL) through the Canadian Centre for DNA Barcoding, from the Ontario Genomics Institute, Genome Canada, the Ontario Ministry of Research and Innovation, and the Natural Sciences and Engineering Research Council of Canada. CBMA researchers were 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).info:eu-repo/semantics/publishedVersio

How oogenesis analysis combined with dna barcode can help to elucidate taxonomic ambiguities: A polychaete study-based approach

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 theOs 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

Revealing the diversity of the green Eulalia (Annelida, Phyllodocidae) species complex along the European coast, with description of three new species

The green phyllodocids Eulalia clavigera and E. viridis are a known European pseudo-cryptic complex, but questions about its distribution and evidence of additional lineages in previous studies call for an investigation of the real diversity within the complex. We analyze DNA sequences (mtCOI-5P, ITS, and 28S rRNA) of different populations of E. clavigera from intertidal and subtidal marine waters along the North East Atlantic, Mediterranean Sea, the Azores and Webbnesia (Madeira, Savage islands and Canaries), and populations of E. viridis from the Scandinavia. This provided compelling evidence for the existence of six additional divergent evolutionary lineages, three of the most abundant being described here as new species: Eulalia feliciae sp. nov., intertidal and unique to the Western Mediterranean, Eulalia madeirensis sp. nov., subtidal and unique to the Madeira Island (Portugal), and Eulalia xanthomucosa sp. nov., mostly subtidal and occurring in the British Isles and southern France. Complementary morphometric analyses showed that E. feliciae sp. nov. and E. madeirensis sp. nov. formed two independent morphometric clusters, while E. xanthomucosa sp. nov. often overlapped with E. clavigera sensu stricto (s. s.), although being unique in showing a yellow coloration and parapodial cirri on median segments larger in relation to its body size. Recent biotechnological findings based on “E. clavigera” specimens highlight the importance of formally describing cryptic complexes, since each lineage chemistry might be unique and may have a range of distinct effects and applications.This study was supported by the project ATLANTIDA–Platform for the monitoring of the North Atlantic Ocean and tools for the sustainable exploitation of the marine resources, with the reference NORTE-01–0145-FEDER-000040, co-financed by the European Regional Development Fund (ERDF), through Programa Operacional Regional do Norte (NORTE 2020). Thanks are due, for the financial support to CESAM (UIDB/50017/2020 + UIDP/50017/2020), to Portuguese Foundation for Science and Technology and Ministry of Education and Science (FCT/MEC) through national funds, and the co-funding by the FEDER, within the PT2020 Partnership Agreement and Compete 2020. Marcos A. L. Teixeira was supported by a PhD grant from FCT co-financed by ESF (SFRH/BD/131527/2017) and from the DNAqua-Net STSM grant “Rich and hidden biodiversity not yet barcoded in the Canary archipelago (Spain) as an opportunity to enrich the DNA barcode reference library for European polychaetes,” under the EU Cost action CA15219–Developing new genetic tools for bio-assessment of aquatic ecosystems in Europe. Pedro E. Vieira was supported by national funds through the Portuguese Foundation for Science and Technology (FCT, I.P.) in the scope of the project (early detection and monitoring of non-indigenous species in coastal ecosystems based on high-throughput sequencing tools, PTDC/BIA-BMA/29754/2017). Ascensão Ravara was supported by national funds, through FCT, I.P., in the scope of the framework contract foreseen in the numbers 4, 5, and 6 of the article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 19. Arne Nygren was supported by the Norwegian Taxonomy Initiative [https://www.biodiversity.no/Pages/135523] (Cryptic polychaete species in Norwegian waters, knr 49–13, pnr 70184228), the Swedish Taxonomy Initiative [https://www.artdatabanken.se/en/the-swedish-taxonomy-initiative/] (Polychaete species complexes in Swedish waters, dnr 140/07 1.4 and 166/08 1.4), and Kungliga Fysiografiska sällskapet Nilsson-Ehle donationerna [https://www.fysiografen.se/sv/]

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

The crown pearl: a draft genome assembly of the European freshwater pearl mussel Margaritifera margaritifera (Linnaeus, 1758)

Since historical times, the inherent human fascination with pearls turned the freshwater pearl mussel Margaritifera margaritifera (Linnaeus, 1758) into a highly valuable cultural and economic resource. Although pearl harvesting in M. margaritifera is nowadays residual, other human threats have aggravated the species conservation status, especially in Europe. This mussel presents a myriad of rare biological features, e.g. high longevity coupled with low senescence and Doubly Uniparental Inheritance of mitochondrial DNA, for which the underlying molecular mechanisms are poorly known. Here, the first draft genome assembly of M. margaritifera was produced using a combination of Illumina Paired-end and Mate-pair approaches. The genome assembly was 2.4 Gb long, possessing 105,185 scaffolds and a scaffold N50 length of 288,726 bp. The ab initio gene prediction allowed the identification of 35,119 protein-coding genes. This genome represents an essential resource for studying this species' unique biological and evolutionary features and ultimately will help to develop new tools to promote its conservation.A.G.-d.-S. was funded by the Portuguese Foundation for Science and Technology (FCT) under the grants SFRH/BD/137935/2018, EF (CEECIND/00627/2017) and MLL (2020.03608.CEECIND). This research was developed under ConBiomics: the missing approach for the Conservation of freshwater Bivalves Project No. NORTE-01-0145-FEDER- 030286, co-financed by COMPETE 2020, Portugal 2020 and the European Union through the ERDF, and by FCT through national funds. Additional strategic funding was provided by FCT UIDB/04423/2020 and UIDP/04423/2020. Authors’ interaction and writing of the article was promoted and facilitated by the COST Action CA18239: CONFREMU—Conservation of freshwater mussels: a pan- European approach.info:eu-repo/semantics/publishedVersio

ATLANTIC-PRIMATES: a dataset of communities and occurrences of primates in the Atlantic Forests of South America

Primates play an important role in ecosystem functioning and offer critical insights into human evolution, biology, behavior, and emerging infectious diseases. There are 26 primate species in the Atlantic Forests of South America, 19 of them endemic. We compiled a dataset of 5,472 georeferenced locations of 26 native and 1 introduced primate species, as hybrids in the genera Callithrix and Alouatta. The dataset includes 700 primate communities, 8,121 single species occurrences and 714 estimates of primate population sizes, covering most natural forest types of the tropical and subtropical Atlantic Forest of Brazil, Paraguay and Argentina and some other biomes. On average, primate communities of the Atlantic Forest harbor 2 ± 1 species (range = 1–6). However, about 40% of primate communities contain only one species. Alouatta guariba (N = 2,188 records) and Sapajus nigritus (N = 1,127) were the species with the most records. Callicebus barbarabrownae (N = 35), Leontopithecus caissara (N = 38), and Sapajus libidinosus (N = 41) were the species with the least records. Recorded primate densities varied from 0.004 individuals/km 2 (Alouatta guariba at Fragmento do Bugre, Paraná, Brazil) to 400 individuals/km 2 (Alouatta caraya in Santiago, Rio Grande do Sul, Brazil). Our dataset reflects disparity between the numerous primate census conducted in the Atlantic Forest, in contrast to the scarcity of estimates of population sizes and densities. With these data, researchers can develop different macroecological and regional level studies, focusing on communities, populations, species co-occurrence and distribution patterns. Moreover, the data can also be used to assess the consequences of fragmentation, defaunation, and disease outbreaks on different ecological processes, such as trophic cascades, species invasion or extinction, and community dynamics. There are no copyright restrictions. Please cite this Data Paper when the data are used in publications. We also request that researchers and teachers inform us of how they are using the data. © 2018 by the The Authors. Ecology © 2018 The Ecological Society of Americ

Lançamento de uma biblioteca de referência de DNA barcodes para poliquetas de estuários de Portugal e do mar profundo da Peninsula Ibérica

Dissertação de mestrado em EcologiaOs invertebrados bentónicos, como os poliquetas, são importantes indicadores de qualidade ambiental sendo também um elo relevante nas cadeias tróficas. A taxonomia, com base nos caracteres morfológicos desta classe é difícil, demorada, e muitas vezes requer um conhecimento especializado que nem sempre está disponível. Este trabalho pretende contribuir para a implementação de uma biblioteca de referência de DNA barcodes (citocromo oxidase I (COI)) de poliquetas de modo a melhorar e facilitar futuras identificações taxonómicas, podendo mais tarde, em conjunto com bibliotecas de outros organismos bentónicos, ser usada em estudos de monitorização, utilizando a sequenciação de segunda geração. Com recurso a DNA barcodes não publicados e gerados por equipas de projectos de investigação LusoMarBoL e BestBarcode, foram compiladas bibliotecas de poliquetas de duas comunidades distintas, nomeadamente de estuários e zonas costeiras de Portugal e do mar profundo da Península Ibérica. Em conjunto com os 26 DNA barcodes de poliquetas de estuários obtidos no âmbito deste estudo, a primeira biblioteca totalizou 31 espécies e 71 sequências, enquanto a segunda englobou 37 espécies e 66 DNA barcodes. Aos alinhamentos das sequências destas duas bibliotecas foram adicionadas sequências homólogas da mesma família ou género disponíveis em bases de dados públicas, por forma a permitir a sua análise conjunta. Os valores médios das divergências intra-específicas e congenéricas do conjunto dos DNA barcodes dos poliquetas de estuários foi de 0,52% (0 a 2,82%) e 20,53% (12,03 a 27,58%) respectivamente, enquanto para os poliquetas do mar profundo foram de 0,61% (0 a 2,63%) e 20,10% (7,36 a 31,77%) respectivamente. Estes valores estão dentro da gama de variação reportada em outros estudos de DNA barcodes de poliquetas. Nas árvores construídas pelo método Neighbour-joining, 79% e 73% das espécies agruparam-se em clados monofiléticos de baixa divergência, no caso dos poliquetas de estuário e do mar profundo, respectivamente. Paralelamente foram detectadas várias incongruências taxonómicas relevantes, bem como divergências intra-específicas relativamente elevadas, evidenciando a necessidade de uma revisão taxonómica em vários taxa.Marine benthic invertebrates, such as the polychaetes, are important environmental indicators and an important link in the marine trophic chains. The taxonomy based on morphological characteristics for this class is difficult, time consuming and often requires specialized knowledge that is not always available. This work aims to contribute to the implementation of a reference library of DNA barcodes (cytochrome c oxidase (COI)) of polychaetes in order to improve and facilitate future taxonomic identifications, and latter to be used in monitoring studies using the next generation sequencing., together with other benthic organisms libraries. Using the unpublished DNA barcodes, generated by the research teams from the projects LusoMarBoL and BestBarcode, libraries for two distinct polychaete communities were compiled, including estuaries and coastal areas of Portugal and the deep sea of the Iberian Peninsula. Together with the 26 DNA barcodes from estuarine polychaetes obtained in this study, the first library had 31 species and 71 sequences, while the second comprised 37 species and 66 DNA barcodes. Homologous sequences of the same family or genus searched in public databases were added to the sequence alignments of these two libraries, in order to enable their joint analysis. The average values of intraspecific and congeneric divergences for the estuarine polychaetes were 0,52% (0 to 2,82%) and 20,53% (12,03 to 27,58%) respectively, whereas for deep sea polychaetes were 0,61% (0 to 2,63%) and 20,10% (7,36 to 31,77%) respectively. These values are within the range of variation reported in other studies examining DNA barcodes of polychaetes. In the Neighbor-joining trees, 79% and 73% of the species grouped in monophyletic taxonomic congruent clades with low divergence, in the case of estuarine and deep sea polychaetes respectively. Concurrently, were also detected several relevant taxonomic ambiguities, as well as considerable genetic divergence, indicating the need for a taxonomic revision in several of the studied taxa.Fundação para a Ciência e a Tecnologia (FCT) - Projetos FCOMP-01-0124-FEDER-007381 e PEst-C/BIA/UI4050/2011.FEDER através do Programa Operacional de Factores de Competitividade - COMPETE.A sequenciação no BIO (Biodiversity Institute of Ontario) foi financiado pelo iBOL (International Barcode of Life Project) através do Canadian Centre for DNA Barcoding, a partir do Ontario Genomics Institute (2008-OGI-ICI-03), Genome Canada, Ontario Ministry of Research and Innovation e pelo Natural Sciences and Engineering Research Council of Canada

Molecular and morphometric analyses identify new lineages within a large Eumida (Annelida) species complex

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)

Macrozoobenthos monitoring in Portuguese transitional waters in the scope of the water framework directive using morphology and DNA metabarcoding

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. In the current study, we addressed this research gap by building upon the biomonitoring network program of the Portuguese Environmental Agency (APA). The ecological condition of 20 sites from four major transitional ecosystems in the west coast of Portugal was assessed, namely Minho, Lima, Vouga and Mondego estuaries, by using metabarcoding and the morphology-based approach for characterizing macrozoobenthic communities. 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.This work was funded by the project “River2Ocean – Socio-ecological and biotechnological solutions for the conservation and valorization of aquatic biodiversity in the Minho Region” (NORTE-01-0145-FEDER-000068), co-financed by the European Regional Development Fund (ERDF), through Programa Operacional Regional do Norte (NORTE 2020), by the “Contrato-Programa” UIDB/04050/2020 funded by national funds through the FCT I.P. (Foundation for Science and Technology) and the project MESCLA – “Melhorar e Complementar os Critérios de Classificação do Estado das Massas de Água de Transição e Costeiras” (POSEUR-03-2013-FC-000001). Financial support granted by the FCT to SD (CEECIND/00667/2017), BRL (PD/BD/127994/2016), MALT (SFRH/BD/131527/2017), and PEV (through the project NIS-DNA: PTDC/BIA-BMA/29754/2017) is also acknowledged. The authors also want to thank to Àngel Borja for helping in the decision of the ecological categories of species that are not yet contemplated in the AMBI list