Do inferences about freshwater phytoplankton communities change when based on microscopy or high‐throughput sequencing data?

Abstract

Microscopy and high‐throughput sequencing (HTS) detect and quantify algae differently. It is not known if microscopy‐based abundance or biomass better compare to HTS data or how methodological differences affect ecological inferences about the phytoplankton communities studied. We investigated methodological (abundancemicroscopy vs. abundanceHTS, biomassmicroscopy vs. abundanceHTS), habitat (littoral, pelagic, deep hypolimnion), and year (2014 vs. 2017) differences for phytoplankton communities of Lake Tovel (Italy) using ANOVA. Specifically, we tested the hypothesis that depending on comparing abundancemicroscopy or biomassmicroscopy to abundanceHTS different effects would be indicated; we called this the metric effect. Furthermore, using samples from 2014 to 2017, we investigated environment–community relationships by a redundancy analysis based on abundancemicroscopy, biomassmicroscopy, and abundanceHTS, and compared the results. Approximately 9 times more operational taxonomic units were reported with HTS (n2014 = 819, n2017 = 891) than algal taxa with microscopy (n2014 = 90, n2017 = 109) in 2014 and 2017. While microscopically assessed algal taxa were evenly distributed among phyla, the vast majority of operational taxonomic units were attributed to Chrysophyta (2014 = 54%, 2017 = 62%) and Bacillariophyta (2014 = 19%, 2017 = 17%). A metric effect for method differences was generally observed comparing abundancemicroscopy to abundanceHTS with Chlorophyta, Cryptophyta, and Dinophyta showing higher % abundance with microscopy while richness and Chrysophyta showed higher values with HTS. Almost no metric effects were found in 2014, but they were common across phyla in 2017. Bacillariophyta and Eustigmatophyta showed the same habitat differences when comparing biomassmicroscopy to abundanceHTS. Dinophyta showed habitat differences only with microscopy, while Chyrsophyta showed habitat differences only with HTS; these results were probably related to technical bias and strengths of HTS, respectively. Habitat differences of phyla were reasonably related to their ecological niche and linked to factors such as temperature and feeding preferences; furthermore, phyla often showed a significant 2014‐versus‐2017 year effect. The year 2014 was very wet while 2017 had a dry winter, and we attributed the patterns found to allochthonous nutrient input by rain and decreased turbulence. Redundancy analyses based on phytoplankton communities assessed with microscopy and HTS, respectively, equally indicated the importance of hydrology, nutrients, and temperature for phytoplankton communities and discriminated the littoral from the deep hypolimnion. However, variance explained was higher with HTS, and the pelagic was similar to the deep hypolimnion with microscopy but to the littoral with HTS. Despite the different strengths of microscopy and HTS for biodiversity assessment, both datasets outlined similar large‐scale patterns linked to strong environmental control of phytoplankton communities as they related to habitat...

Metabarcoding vs Microscopy: Comparison of Methods To Monitor Phytoplankton Communities

“…Among genera, 36–55% were identified by both methods, while for species 9–27%, common identifications were found. These results are in agreement with previous reports on freshwater phytoplankton, where only a few common taxa were found by microscopy and metabarcoding operational taxonomic units (OTUs). ,, …”

“…The biodiversity of eukaryotic nano-and microphytoplankton showed higher values for metabarcoding than for microscopy (Figure 2). This is in agreement with some earlier studies, 16,27 but a recent meta-analysis of marine and freshwater environments showed rather similar alpha diversity of plankton and microphytobenthos for microscopy compared to DNA metabarcoding. 28 Furthermore, a previous study reported that traditional microscopy identified more taxa than metabarcoding for certain phyla, for example, Cryptophyta and Dinophyta.…”

“…13 In this study, we found two-fold more taxa with metabarcoding than with microscopy (Figure 2, left panel), which can be compared to earlier studies observing 9-to 22fold more taxa in metabarcoding. 14,27 The higher similarity between the methods in our study may partly be due to the focus on the same size range, i.e., nano and micro-sizes, but also by combining ASVs annotated to the same taxa. ASVs are often considered to host inter-specific and intra-specific diversity since a difference in only one nucleotide will lead to a unique ASV.…”

“…These results are in agreement with previous reports on freshwater phytoplankton, where only a few common taxa were found by microscopy and metabarcoding operational taxonomic units (OTUs). 16,27,28 Some species annotated by the metabarcoding in this study are not included in the HELCOM PEG list of species or the list of species in the European standard for Water quality -Guidance on the estimation of phytoplankton biovolume EN 16695. This mismatch can be due to intraspecific dissimilarities, due to evolutionary processes, such as genetic drift, gene duplication, or horizontal gene transfer, and those phenomena could in principle cause a misannotation by metabarcoding.…”

High-throughput sequencing and marker pigment analysis of freshwater phytoplankton: A direct comparison with microscopic count data in the tropical crater lakes of Western Uganda

“…Whereas this method can provide quantitative estimates of phytoplankton biomass and composition at high taxonomic resolution (down to morphospecies level), it is also time-consuming, requires expert taxonomic knowledge that is not generally available, and often fails to properly include the picophytoplankton (Paerl, 1978;Culverhouse et al, 2003). Two alternative approaches promising faster, cost-efficient and potentially more comprehensive assessment of phytoplankton community composition are high-throughput amplicon sequencing (HTS) of taxonomically diagnostic DNA sequences (Baird & Hajibabaei, 2012;Caron, 2013;Eiler et al, 2013;Abad et al, 2016;Keck et al, 2017;Pawlowski et al, 2018;Obertegger et al, 2020) and high-performance liquid chromatography (HPLC) of photosynthetic marker pigments (Jeffrey et al, 1997(Jeffrey et al, , 1999Descy et al, 2000Descy et al, , 2005Sarmento et al, 2006;Lauridsen et al, 2011). Both methods allow the simultaneous processing of multiple samples, and can detect small and fragile phytoplankton taxa that are often overlooked by microscopy.…”