Evaluation of two short overlapping rbcL markers for diatom metabarcoding of environmental samples: Effects on biomonitoring assessment and species resolution.

Two short diatom rbcL barcodes, 331 bp and 263 bp in length, have frequently been used in diatom metabarcoding studies. They overlap in a common 263-bp region but differ in the presence or absence of a 68-bp tail at the 5' end. Though the effectiveness of both has been demonstrated in separate biomonitoring and diversity studies, the impact of the 68-bp non-shared region has not been evaluated. Here we compare the two barcodes in terms of the values of a biotic index (IPS) and the ecological status classes derived from their application to an extensive metabarcoding dataset from United Kingdom rivers; this comprised 1703 samples and was produced using the 331-bp primers. In addition, we assess the effectiveness of each barcode for discrimination of genetic variants around and below the species level. The strong correlation found in IPS values between barcodes (Pearson's R = 0.98) indicates that the choice of the barcode does not have major implications for current WFD ecological assessments, although a very few sites (55: 3.23% of those analysed) were downgraded from an acceptable WFD class ("Good") to an unacceptable one ("Moderate"). Analyses of the taxonomic resolution of the two barcodes indicate that for many ASVs, the use of either marker - 263-bp and 331-bp - gives unambiguous assignations at species level though with differences in bootstrap confidence values. Such differences are caused by the stochasticity involved in the naïve Bayesian classifier used and by the fact that genetic distance, regarding closely related species, is increased when using the 331-bp barcode. However, in three cases, species differentiation fails with the shorter marker, leading to underestimates of species diversity. Finally, two ASVs from Nitzschia species evidenced that the use of the shorter marker can sometimes lead to false positives when the extent and nature of infraspecific variation are poorly known.

Assessment of marine benthic diatom communities: insights from a combined morphological-metabarcoding approach in Mediterranean shallow coastal waters.

We investigated the advantages and disadvantages of light microscope (LM)-based identifications and DNA metabarcoding, based on a 312-bp rbcL marker, for examining benthic diatom communities from Mediterranean shallow coastal environments. For this, we used biofilm samples collected from different substrata in the Ebro delta bays. We show that 1) Ebro delta bays harbour high-diversity diatom communities [LM identified 249 taxa] and 2) DNA metabarcoding effectively reflects this diversity at genus- but not species level, because of the incompleteness of the DNA reference library. Nevertheless, DNA metabarcoding offers new opportunities for detecting small, delicate and rare diatom species missed by LM and diatoms that lack silica frustules. The primers used, though designed for diatoms, successfully amplified rarely reported members of other stramenopile groups. Combining LM and DNA approaches offers stronger support for ecological studies of benthic microalgal communities in shallow coastal environments than using either approach on its own.

Assessment of a novel microalgae-cork based technology for removing antibiotics, pesticides and nitrates from groundwater.

Groundwater pollution has increased in recent years due to the intensification of agricultural and livestock activities. This results in a significant reduction in available freshwater resources. Here, we have studied the long term assessment of a green technology (1-4 L/day) based on a photobioreactor (PBR) containing immobilised microalgae-bacteria in polyurethane foam (PF) followed by a cork filter (CF) for removing nitrates, pesticides (atrazine and bromacil), and antibiotics (sulfamethoxazole and sulfacetamide) from groundwater. The prototype was moderately effective for removing nitrates (58%) at an HRT of 8 days, while its efficiency decreased at a HRT of 4 and 2 days (<20% removal). The combined use of PBR-CF enabled antibiotics and pesticides to be attenuated by up to 95% at an HRT of 8 days, but their attenuation decreased with shorter HRT, with pesticides being the compounds most affected (reducing from 97 to 98% at an HRT of 8 days to 23-45% at an HRT of 2 days). Pesticide transformation products were identified after the CF, supporting biodegradation as the main attenuation process. A gene-based metataxonomic assessment linked the attenuation of micropollutants to the presence of specific pesticide biodegradation species (e.g. genus Phenylobacterium, Sphingomonadaceae, and Caulobacteraceae). Therefore, the results highlighted the potential use of microalgae and cork to treat polluted groundwater.

DNA metabarcoding reveals differences in distribution patterns and ecological preferences among genetic variants within some key freshwater diatom species.

Our study evaluates differences in the distribution and ecology of genetic variants within several ecologically important diatom species that are also key for Water Framework Directive monitoring of European rivers: Fistulifera saprophila (FSAP), Achnanthidium minutissimum (ADMI), Nitzschia inconspicua (NINC) and Nitzschia soratensis (NSTS). We used DADA2 to infer amplicon sequence variants (ASVs) of a short rbcL barcode in 531 environmental samples from biomonitoring campaigns in Catalonia and France. ASVs within each species showed different distribution patterns. Threshold Indicator Taxa ANalysis revealed three ecological groupings of ASVs in both ADMI and FSAP. Two of these in each species were separated by opposite responses to calcium and conductivity. Boosted regression trees additionally showed that both variables greatly influenced the occurrence of these groupings. A third grouping in FSAP was characterized by a negative response to total organic carbon and hence was better represented in waters with higher ecological status than the other FSAP ASVs, contrasting with what is generally assumed for the species. In the two Nitzschia species, our analyses confirmed earlier studies: NINC preferred higher levels of calcium and conductivity. Our findings suggest that the broad ecological tolerance of some diatom species results from overlapping preferences among genetic variants, which individually show much more restricted preferences and distributions. This work shows the importance of studying the ecological preferences of genetic variants within species complexes, now possible with DNA metabarcoding. The results will help reveal and understand biogeographical distributions and facilitate the development of more accurate biological indexes for biomonitoring programmes.

Evaluation and sensitivity analysis of diatom DNA metabarcoding for WFD bioassessment of Mediterranean rivers.

Our study of 164 diatom samples from Catalonia (NE Spain) is the first to evaluate the applicability of DNA metabarcoding, based on high throughput sequencing (HTS) using a 312-bp rbcL marker, for biomonitoring Mediterranean rivers. For this, we compared the values of a biotic index (IPS) and the ecological status classes derived from them, between light microscope-based (LM) and HTS methods. Very good correspondence between methods gives encouraging results concerning the applicability of DNA metabarcoding for Catalan rivers for the EU Water Framework Directive (WFD). However, in 10 sites, the ecological status class was downgraded from "Good"/"High" obtained by LM to "Moderate"/"Poor"/"Bad" by HTS; these "critical" sites are especially important, because the WFD requires remedial action by water managers for any river with Moderate or lower status. We investigated the contribution of each species to the IPS using a "leave-one-out" sensitivity analysis, paying special attention to critical sites. Discrepancies in IPS between LM and HTS were mainly due to the misidentification and overlooking in LM of a few species, which were better recovered by HTS. This bias was particularly important in the case of Fistulifera saprophila, whose clear underrepresentation in LM was important for explaining 8 out of the 10 critical sites and probably reflected destruction of weakly-silicified frustules during sample preparation. Differences between species in the rbcL copy number per cell affected the relative abundance obtained by HTS for Achnanthidium minutissimum, Nitzschia inconspicua and Ulnaria ulna, which were also identified by the sensitivity analysis as important for the WFD. Only minor IPS discrepancies were attributed to the incompleteness of the reference library, as most of the abundant and influential species (to the IPS) were well represented there. Finally, we propose that leave-one-out analysis is a good method for identifying priority species for isolation and barcoding.