DNA metabarcoding approach as a complementary technique for assessment of Portuguese rivers using diatoms

• Autor(a) principal: Mortágua, Andreia Filipa Henriques
• Data de Publicação: 2018
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/25173
• topic: Diatoms; DNA metabarcoding; Environmental DNA; Biomonitoring; Portuguese rivers; Ecological assessment of water; Freshwater systems

The Water Framework Directive (WFD) is the main political instrument for management of the waterbodies in Europe and includes the bioassessment of rivers and streams based on diatoms, through the calculation of an autoecological index, the Indice de Polluosensibilité Spécifique (IPS) officially adopted for Portugal. This index requires a high level of taxonomic expertise for morphological identification of individuals. Advances in genomics, such as the DNA metabarcoding combined with high-throughput sequencing (HTS) techniques offer a promising alternative to classical methods, limiting expertise requirement and therefore reducing time and costs. The aim of this study was to test the potential of DNA metabarcoding of diatoms in the bioassessment of Portuguese rivers by comparing the IPS classifications obtained with morphological and molecular approaches. A total of 88 samples from rivers in central Portugal were collected in the spring of 2017 following WFD standards. The morphological approach comprised taxonomic identification of at least 400 valves, under the light microscope. The molecular approach included eDNA extraction followed by DNA sequencing (Illumina MiSeq) using a 312bp rbcL DNA barcode. Sequences were analysed with Mothur software, producing Operational Taxonomic Units (OTUs) that were taxonomically assigned to the R-Syst::diatom reference library. It was also tested the effect of a correction factor (CF) for biovolume applied on molecular data. Inventories of diatom communities revealed a total number of 306, 125 and 111 species identified with morphology, molecular method without and with the CF, respectively. The total percentage of successfully assigned OTUs to the reference library was 32%, with an average of 47.5% per sample, while the average percentage of unassigned reads from the converted OTUs to taxa list was 52.5% and varied between 2 and 95%, among all samples. When comparing species’ abundances, the results showed statistical differences in the number of species between molecular and morphological inventories although the application of the CF approximated both approaches. The source of these differences may lay on the incompleteness of reference libraries, which currently represents the major difficulty in taxonomic assignment of eDNA sequencings. Regarding IPS values, the results indicated a good correlation between morphological and molecular methods, especially when applying the CF. NMDS and PCO diagrams based on species abundances revealed a gradient of quality classifications in all 3 methodologies. These support the hypothesis that DNA metabarcoding may be a valid approach for ecological quality assessment. Yet, there is still work to be done on this new methodology in order to be able to make a smooth transition between the traditional and this new approach without misspend accumulated knowledge from the last decades on water quality assessment