Molecular Ecology Resources Starting a DNA barcode reference library for shallow water polychaetes from the southern European Atlantic coast Journal:For Molecular Review Ecology Resources Only Manuscript ID: Draft Manuscript Type: Resource Article Date Submitted by the Author: n/a Complete List of Authors: Lobo, Jorge; CBMA – Centre of Molecular and Environmental Biology, Departamento de Biologia, Universidade do Minho; MARE – Marine and Environmental Sciences Centre, Departamento de Ciências e Engenharia do Ambiente, Universidade Nova de Lisboa Teixeira, Marcos; CBMA – Centre of Molecular and Environmental Biology, Departamento de Biologia, Universidade do Minho Borges, Luisa; CBMA – Centre of Molecular and Environmental Biology, Departamento de Biologia, Universidade do Minho; Helmholtz-Zentrum Geesthacht, Centre for Material and Coastal Research, Ferreira, Maria; CBMA – Centre of Molecular and Environmental Biology, Departamento de Biologia, Universidade do Minho Hollatz, Claudia; CBMA – Centre of Molecular and Environmental Biology, Departamento de Biologia, Universidade do Minho Gomes, Pedro; CBMA – Centre of Molecular and Environmental Biology, Departamento de Biologia, Universidade do Minho Sousa, Ronaldo; CBMA – Centre of Molecular and Environmental Biology, Departamento de Biologia, Universidade do Minho; CIIMAR/CIMAR – Interdisciplinary Centre of Marine and Environmental Research, Universidade do Porto, Ravara, Ascensão; CESAM – Centre for Environmental and Marine Studies, Departamento de Biologia, Universidade de Aveiro, Costa, Maria; MARE – Marine and Environmental Sciences Centre, Departamento de Ciências e Engenharia do Ambiente, Universidade Nova de Lisboa Costa, Filipe; CBMA – Centre of Molecular and Environmental Biology, Departamento de Biologia, Universidade do Minho Annelida, Benthos, Cytochrome c oxidase subunit I (COI-5P), Estuaries, Keywords: Taxonomy Page 1 of 39 Molecular Ecology Resources 1 Starting a DNA barcode reference library for shallow water polychaetes from the southern European 2 Atlantic coast 3 Keywords : Annelida, benthos, cytochrome c oxidase subunit I (COI-5P), estuaries, taxonomy 4 5 JORGE LOBO 1,2,*, MARCOS A. L. TEIXEIRA 1, LUISA M. S. BORGES 1,3, MA RIA S. G. FERREIRA 1, 6 CLAUDIA HOLLATZ 1, PEDRO A. GOMES 1, RONALDO SOUSA 1,4, ASCENSÃO RAVARA 5, MARIA H. 7 COSTA 2, FILIPE O. COSTA 1 8 For Review Only 9 1CBMA – Centre of Molecular and Environmental Biology, Departamento de Biologia, Universidade do Minho, 10 Campus de Gualtar, 4710-057 Braga, Portugal, 2MARE – Marine and Environmental Sciences Centre, 11 Departamento de Ciências e Engenharia do Ambiente, Faculdade de Ciências e Tecnologia da Universidade 12 Nova de Lisboa, 2829-516 Monte de Caparica, Portugal, 3Helmholtz-Zentrum Geesthacht, Centre for Material 13 and Coastal Research, Max-Planck-Straße 1, 21502, Germany, 4CIIMAR/CIMAR – Interdisciplinary Centre of 14 Marine and Environmental Research, Universidade do Porto, Rua dos Bragas, 123, 4050-123, Porto, Portugal, 15 5CESAM – Centre for Environmental and Marine Studies, Departamento de Biologia, Universidade de Aveiro, 16 Campus de Santiago, 3810-193 Aveiro, Portugal. 17 18 * Correspondence: [email protected] 19 20 Abstract 21 22 Annelid polychaetes, as a group, have been seldom the focus of dedicated DNA barcoding studies, despite their 23 ecological relevance and often dominance, particularly in soft-bottom estuarine and coastal marine ecosystems. 24 Here we report the first assessment of the performance of DNA barcodes in the discrimination of shallow water 25 polychaete species from the southern European Atlantic coast, focusing on specimens collected in estuaries and 26 coastal ecosystems of Portugal. We analysed cytochrome oxidase I DNA barcodes (COI-5P) from 164 27 specimens, which were assigned to 51 morphospecies. To our dataset from Portugal, we added available 28 published sequences selected from the same species, genus or family, to inspect for taxonomic congruence 29 among studies and collection location. The final dataset comprised 79 morphospecies and 290 specimens, which 1 Molecular Ecology Resources Page 2 of 39 30 generated 99 Barcode index numbers (BINs) within Barcode of Life Datasystems (BOLD). Among these, 22 31 BINs were singletons, 47 other BINs were concordant, confirming the initial identification based on 32 morphological characters, and 30 were discordant, most of which consisted on multiple BINs found for the same 33 morphospecies. Some of the most prominent cases include Hediste diversicolor (O.F. Müller, 1776) (7), Eulalia 34 viridis (Linnaeus, 1767) (2) and Owenia fusiformi s (delle Chiaje, 1844) (5), all of them reported from Portugal 35 and frequently used in ecological studies as environmental quality indicators. However, our results showed 36 discordance between molecular lineages and morphospecies, or added additional relatively divergent lineages. 37 The potential inaccuraciesFor in environmental Review assessments, where Onlyunderpinning polychaete species diversity is 38 poorly resolved or clarified, demands additional and extensive investigation of the DNA barcode diversity in this 39 group, in parallel with alpha taxonomy efforts. 2 Page 3 of 39 Molecular Ecology Resources 40 Introduction 41 42 The estuarine and coastal intertidal areas have a large number of benthic invertebrates, where the polychaetes are 43 one of the most representative classes and, therefore, important indicators of environmental quality in these 44 ecosystems (Sousa et al. 2006, 2008). In addition, they are also an important trophic link in the food chain being 45 an important prey for many species with conservational importance (e.g. fish, birds and mammals), are 46 responsible for important ecosystem functions that include nutrient cycling and also being important ecosystem 47 engineers due bioturbationFor and bioirrigation Review activities (Kristensen Only et al . 1985; Volkenborn et al. 2007). Some 48 polychaete species are also used as baits and are, therefore, economically important for fisheries in several 49 European countries (Gambi et al. 1994; Olive 1994; Gillet & Torresanib 2003). However, polychaetes as a 50 whole are poorly studied in comparison to other taxa of similar ecological importance (Quijón & Snelgrove 51 2005). 52 Concerns with the adequate management of marine ecosystems gained more momentum with the 53 implementation of the Directive 2000/60 / EC of the European Parliament and Council, 23 October 2000. This 54 directive establishes a framework for Community action in the field of water policy, which requires the 55 preparation of management plans for watersheds for each river basin, including estuaries, in order to achieve 56 good ecological and chemical status, and also contributing to mitigate the effects of floods. Studies focusing on 57 polychaetes could contribute to this goal. However, species identification of this functional group based on 58 morphological characters is of great difficulty (Coull 1999). These difficulties are patent in the polychaete 59 records available in the BOLD database, where among the 10430 published records only 6055 were identified to 60 the species level (accessed in 8 October 2014). 61 An accurate identification based on morphological approaches can be extremely difficult and sometimes an 62 impossible task (Rouse & Pleijel 2001. The difficulties often linked to a reliable species discrimination include, a 63 shortage of taxonomists, the use of incomplete identification keys and the collection of degraded or bodily 64 injured specimens caused by sampling techniques (Knowlton 1993). In addition, taxonomic ambiguities and 65 uncertainties are frequently generated by the presence of complex life stages and cryptic or hidden species 66 (Knowlton 1993; Jarman & Elliott 2000; Bickford et al. 2006; Nygren 2014). 67 Ecological and biogeographic studies rely upon the ability to distinguish between morphologically similar 68 species, as well as knowledge of their evolutionary relationships. In order to respond to this limitation, several 3 Molecular Ecology Resources Page 4 of 39 69 studies have examined variation in the mitochondrial DNA sequences of the cytochrome oxidase I gene and 70 confirmed DNA barcodes (Hebert et al . 2003a) as a reliable approach to discriminate species of polychaetes (e.g. 71 Glover et al . 2005; Bleidorn et al . 2006; Rice et al . 2008; Olson et al . 2009; Pleijel et al. 2009; Barroso et al. 72 2010; Nygren & Pleijel 2011). Some studies revealed interesting findings in polychaete species that were 73 presumed to be cosmopolitan, but which were in fact species complexes comprising several cryptic species, as 74 for example Eurythoe complanata (Pallas, 1766) (Barroso et al. 2010) and the Eumida sanguinea (Örsted, 1843) 75 complex comprising between up to ten additional putative species (Nygren & Pleijel 2011). DNA barcoding 76 differs from the approachesFor and conventional Review taxonomic identification Only tools by allowing direct comparison of 77 specimens with a global reference library, allowing cryptic detection as well as the identification of species from 78 fragments, at any stage of the life cycle; thus creating a universal master key in a format that reduces ambiguity 79 (Costa & Carvalho 2007). In some cases, with the help of this molecular tool, the morphological, ecological and 80 behavioral differences that were once overlooked can now be detected after further examination of divergent 81 taxa (Hebert et al. 2004; Smith et al . 2006). Nevertheless, most existing