A next-generation sequencing approach to river biomonitoring using benthic diatoms

Abstract

Diatoms are main bioindicators used to assess the ecological quality of rivers, but their identification is difficult and time-consuming. Next Generation Sequencing (NGS) can be used to study communities of microorganisms, so we carried out a test of the reliability of 454 pyrosequencing for estimating diatom inventories in environmental samples. We used small subunit ribosomal deoxyribonucleic acid (SSU rDNA), ribulose-1, 5-bisphosphate carboxylase (rbcL), and cytochrome oxidase I (COI) markers and examined reference libraries to define thresholds between the intra-and interspecific and intra-and intergeneric genetic distances. Based on tests of 1 mock community, we used a threshold of 99% identity for SSU rDNA and rbcL sequences to study freshwater diatoms at the species level. We applied 454 pyrosequencing to 4 contrasting environmental samples (with one in duplicate), assigned taxon names to environmental sequences, and compared the qualitative and quantitative molecular inventories to those obtained by microscopy. Species richness detected by microscopy was always higher than that detected by pyrosequencing. Some morphologically detected taxa may have been persistent frustules from dead cells. Some taxa detected by molecular analysis were not detected by morphology and vice versa. The main source of divergence appears to be inadequate taxonomic coverage in DNA reference libraries. Only a small percentage of species (but almost all genera) in morphological inventories were included in DNA reference libraries. DNA reference libraries contained a smaller percentage of species from tropical (27.1-38.1%) than from temperate samples (53.7-77.8%). Agreement between morphological and molecular inventories was better for species with relative abundance >1% than for rare species. The rbcL marker appeared to provide more reproducible results (94.9% species similarity between the 2 duplicates) and was very useful for molecular identification, but procedural standardization is needed. The water-quality ranking assigned to a site via the Pollution Sensitivity diatom index was the same whether calculated with molecular or morphological data. Pyrosequencing is a promising approach for detecting all species, even rare ones, once reference libraries have been developed.

Molecular evidence of a Didymosphenia geminata (Bacillariophyceae) invasion in Chilean freshwater systems

“…Molecular identification is useful for more accurate species identification and for assessing the biodiversity of environmental samples (Kermarrec et al, 2013(Kermarrec et al, , 2014Leliaert et al, 2014;Zimmermann et al, 2015). The molecular markers most commonly employed for species delimitations in diatoms are: the plastid gene rbcL, the mitochondrial gene cythocrome oxidase I (COI), the small ribosomal subunit (SSU), the large ribosomal (LSU), and the internal transcribed spacer (ITS) of the nuclear rDNA (Amato et al, 2007;Evans et al, 2007;Hamsher et al, 2011;MacGillivary and Kaczmarska, 2011;Zimmermann et al, 2011;Kermarrec et al, 2013).…”

“…Most recently advances in nextgeneration sequencing (NGS) technologies offer the possibility to use molecular barcoding for fast and reliable diversity surveys. With this technology, a new approach for analyzing environmental samples has been developed and is called (e-DNA) barcoding or metabarcoding (Kermarrec et al, 2014). In this context, metabarcoding can provide a valuable information to study biodiversity, food web dynamics, environmental and invasive species monitoring, and for reconstructing changes in biodiversity through time.…”

Sedimentary DNA versus morphology in the analysis of diatom-environment relationships

“…After using the OBITools pipeline the final results were entered into a spreadsheet and were further filtered manually. First, all sequences with less \96 % match to entries in the diatom reference library were removed to guarantee good taxonomic assignation, which we based on the average of intra-genus variation in the rbcL gene (Kermarrec et al 2014). Second, we subtracted the diatom sequence counts found in the extraction blanks or NTCs from the relevant samples belonging to each extraction and/or PCR batch.…”

The potential of High-Throughput Sequencing (HTS) of natural samples as a source of primary taxonomic information for reference libraries of diatom barcodes

“…Such sequence length has been shown to be long enough for applied topics such as the one addressed here: diatom species identification for ecological assessment (e.g. Kermarrec et al 2014;Zimmermann et al 2015;Visco et al 2015). It will be straightforward to expand such methodology to wider monitoring networks, if taxonomic experts are available who can follow the recommendations we propose here.…”