Diatoms recording using metabarcoding

Abstract

Freshwater monitoring is crucial to preserve the ecological services these ecosystems provide. Diatoms are known to be reliable indicators of water quality, hence the historical analysis of their community for routine biomonitoring. This thesis aims to optimise the current diatom biomonitoring method based on the most recent metabarcoding tools, in order to assist freshwater environment surveillance. Light Microscopy (LM) coupled with morphological identification is the traditional approach for diatom surveys. The more recent alternative, metabarcoding, combines barcoding to identify species using DNA variations from short conservative sequences (barcodes) and High Throughput Sequencing (HTS), that allows the analysis of thousands of sequences simultaneously. Aspects of the methodology, including primers, were tested in a variety of environments, including rivers and mesocosms, enabling the optimisation of the whole process and confirmation of the reliability of the short barcode located in the rbcL gene. This experimentation showed the interchangeability of LM and metabarcoding approaches for most routine diatom biomonitoring surveys. Both Illumina and MinION HTS platforms were compared to the LM method and judged to be a success but no benefit was found using a longer rbcL barcode region with MinION. Bioinformatic pipelines were created for each sequencing technology, based on new bioinformatic tools and particularly the denoising/polishing algorithms which generated equal or better results than the current QIIME1 bioinformatic pipeline. As an appropriate reference library is crucial for taxonomic assignment of sequences, the current UK reference library was compared with and updated from the European reference library, diat.barcode. In addition, non-diatom phytoplankton taxa were added to the reference library, improving the species assignment. The evolutionary history of the barcode region, the rbcL gene, was investigated using a phylogenetic approach. This demonstrated the link between rbcL evolution and diatom morphology, and its suitability as a ‘barcode’ were discussed. This project succeeded in improving diatom biomonitoring via HTS, and further demonstrated the reliability of this approach.