In marine and estuarine benthic communities, the inventory and estimation of species richness are often hampered by the need for broad taxonomic expertise across several phyla. The use of DNA metabarcoding has emerged as a powerful tool for the fast assessment of species composition in a diversity of ecological communities. Here, we tested the amplification success of five primer sets targeting different COI-5P regions by 454 pyrosequencing to maximize the recovery of two simulated macrobenthic communities containing 21 species (SimCom1 and SimCom 2). Species identification was first performed against a compiled reference library of macrobenthic species. Reads with similarity results to reference sequences between 70% and 97% were then submitted to GenBank and BOLD to attempt the identification of concealed species in the bulk sample. The combination of at least three primer sets was able to recover more species than any primer set alone, achieving 85% of represented species in SimCom1 and 76% in SimCom2. Our approach was successful to detect low-frequency specimens, as well as concealed species, in the bulk sample, indicating the potential for the application of this approach on marine bioassessment and inventory, including the detection of "hidden" biodiversity that would hardly be possible based on morphology only.This work was supported by FEDER through POFCCOMPETE by national funds from 'Fundacao para a Ciencia e a Tecnologia (FCT)' in the scope of the grant FCOMP-01-0124-FEDER-015429 and also by the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER007569). This work was also funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalizacao (POCI). J.L. was supported by a PhD fellowship (SFRH/BD/69750/2010) from FCT and C.H. by a CAPES Post-doctoral fellowship, under Science Without Borders Program (Ministry of Education, Brazil)info:eu-repo/semantics/publishedVersio
