Molecular biodiversity and advanced monitoring tools for macrobenthic communities of Portuguese coastal ecosystems

Abstract

Benthic communities (BCs) are components of paramount importance for aquatic ecosystems function and therefore central elements in aquatic biodiversity monitoring programmes (e.g. European Union's Water Framework Directive – WFD). Current methodologies (e.g. morphological identification) are very time-consuming, technically difficult and frequently species-level data cannot be obtained. Molecular approaches such as DNA barcodes can greatly facilitate and speed up monitoring of BCs. DNA barcodes are also proficient in unravelling latent biodiversity and clarifying taxonomic ambiguities. The emergence of high throughput sequencing (HTS) technologies, combined with DNA barcodes, have the potential to overtake many of the limitations of the morphology-based method. In this thesis we use DNA barcodes to investigate the biodiversity of macrobenthic organisms of Portuguese coastal ecosystems and develop a high-throughput DNA metabarcoding approach for macrobenthic communities. To this purpose, a) we optimized the primer design in order to enhance the amplification success of DNA barcodes, b) compiled DNA barcodes for dominant groups and species and c) tested primer success in DNA metabarcoding of mock communities, and compared the effectiveness of morphology-based and DNA metabarcoding approaches in the inventory of wild estuarine macrobenthic communities. DNA barcodes were used to contribute to the enrichment of the global reference library with prominent species of the estuarine and marine benthic communities of Portugal, focusing on annelids, crustaceans and mollusks. This thesis contributed to generate DNA barcodes for macrobenthic morphospecies, which include 51 polychaetes, 43 amphipods and 34 gastropods. Evidence of hidden or cryptic species based on high intra-specific distances was found 6 polychaete, 7 amphipod and 3 gastropod morphospecies, corresponding to the detection of a possible total of 30 hidden species. To assess the ability of the HTS to recover DNA barcodes from all macrobenthic species present in a bulk community DNA extract, 3 phylogenetically diverse communities were assembled. Over 80% of the species of the tested communities were recovered. Subsequently, the efficiency of morphology-based identifications compared to metabarcoding-based approaches in the assessment of the species composition of macrobenthos was determined in four distinct sites of the Sado estuary, Portugal. The results indicate that the species richness would be underestimated if only morphological methods were used, since the metabarcoding approach was able to provide unambiguous species-level assignments for a much greater number of species, which are susceptible to scrutiny and verification in the future. We conclude that the DNA metabarcoding approach can perform equally or better than morphology based inventories, and that high-throughput biodiversity assessment of macrobenthic communities is attainable through this approach. Nevertheless, further refinement is still required to improve its performance and potential, as for example the improvement of the recovery rates through additional optimization of primers, or by testing alternative primer combinations. Finally, since the reference libraries are still incipient for marine invertebrates, the continuing completion will be decisive to fully materialize the potential of metabarcoding.