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PRIMING A DNA BARCODE LIBRARY FOR MARINE GASTROPODA OF THE CONTINENTAL
PORTUGUESE COAST AND AZORES ISLANDS
L. M. S. Borges1,2, C. Hollatz1, J. Lobo1,3, A. M. Cunha1, A. P. Vilela1, G. Calado3,4,5, R. Coelho5, A. C. Costa6, M. S. G. Ferreira1., M. H. Costa3, F. O. Costa1
1CBMA- Centre of Molecular and Environmental Biology, Braga, Portugal
2Helmholtz-Zentrum Geesthacht, Centre for Material and Coastal Research, Geesthacht, Germany
3MARE-Marine and Environmental Sciences Centre, Lisbon New University, Monte de Caparica, Portugal
4Department of Life Science, School of Psychology , University Lusofona, Lisbon, Portugal
5IPM– Portuguese Institute of Malacology, Zoomarine, Albufeira, Portugal
6InBIO – Centre of Investigation of Biodiversity and Genetic Resources, University of the Azores, Ponta Delgada, Azores, Portugal
The Gastropoda are among the most diverse taxonomic groups of marine invertebrates and together with other
dominant classes, such as Malacostraca and Polychaeta, constitute major components of marine benthic communities.
As part of a wider effort to compile reference libraries for dominant benthic invertebrates of the NE Atlantic, we used
DNA barcodes to investigate the diversity of gastropods from continental Portugal and Azores Islands.
Marine gastropod specimens were collected between 2009 and 2015, at sites along the coasts of mainland Portugal and
two Azorean Islands, São Miguel and Terceira, Portugal (Fig.1). DNA barcodes were obtained using published protocols
for invertebrates (Costa et al., 2007) according to the methodology summarized in Fig. 2. Patterns of distance between
sequences were analysed in a Neighbour-Joining phenogram (K2P model). The Barcode Index Number (BIN) algorithm
was applied to produce Molecular Operational Taxonomic Units (MOTUs) and the ranking system proposed by Costa and
co-authors was applied to assess the taxonomic reliability of our DNA barcode records (e.g. Lobo et al., 2015).
Materials and Methods
The source or discordance was due to:
1) cases of species names that have not yet been updated in public repositories e.g. Osilinus lineatus that has not yet been
updated to Phorcus lineatus;
2) distinct species still identified as a single species e.g. Patella ulyssiponensis and Patella aspera identified interchangeably
as P. ulyssiponensis and P. aspera.
To this dataset, we added publicly available COI-5P sequences of the same or taxonomically close species, to inspect for DNA
barcode discriminatory ability and data congruence. In total, we examined 183 specimens belonging to 51 species.
The percentage of concordant BINs came up to over 80% after revision, which shows a good reliability of our DNA barcode
library. Nonetheless, the amount of discordant data highlights the need for continuous revision and DNA barcode-based
probation of the supposedly well-known gastropod fauna from the European Atlantic coasts.
Results and Discussion
Figure 3. Neighbour-joining phenogram based on mitochondrial cytochrome c oxidase (COI)
barcode nucleotide distance (K2P). Data was generated from circa 32 gastropod species and 105
specimens from Portuguese coast and Azores Islands. *Indicates species sequenced in this study.
Costa FO, deWaard JR, Boutillier J, Ratnasingham S, Dooh Hajibabaei M, Hebert PDN (2007). Biological identification through DNA barcodes : the case of the Crustacea. Can J Fish Aquat Sci. 64: 272-295
Costa, F. O. et al. (2012). A ranking system for reference libraries of DNA barcodes: application to marine fish species from Portugal. PLoS One 7, e35858
Cristescu, ME. (2014).From barcoding single individuals to metabarcoding biological communities: towards an integrative approach to the study of global biodiversity. Trends Ecol. Evol. 29, 566–571
Lobo, Jorge, et al. (2015). Starting a DNA barcode reference library for shallow water polychaetes from the southern European Atlantic coast. Mol. Ecol. Res. DOI: 10.1111/1755-0998.12441
Figure 2. Schematic summary of the methodology used for the DNA barcode library of gastropods of
DATA COLLECTION BARCODE OF LIFE DATA BASE
Corresponding author: email@example.com
Barcode COI-5P region
5 novel barcodes
AFTER BIN REVISION
27 concordant 7 discordant
22 concordant 12 discordant 1 singleton
Figure 1. Location of the sites where specimens were collected in the continental coast of
Portugal, and Terceira and São Miguel, Azores.
ACKNOWLEDGEMENTS: project FCOMP-01-0124-FEDER-015429 (FCT), UID/BIA/04050/2013 (FCT-IP), International Barcode of
Life (iBOL). Claudia Hollatz was supported by a CAPES Post-doctoral fellowship (Ministry of Education, Brazil), while Jorge
Lobo was supported by a PhD fellowship. (SFRH/BD/69750/2010) from FCT.