Metabarcoding unsorted kick-samples facilitates macroinvertebrate-based biomonitoring with increased taxonomic resolution, while outperforming environmental DNA

Abstract

Many studies have highlighted the potential of DNA-based methods for the biomonitoring of freshwater macroinvertebrates, however only a few studies have investigated homogenisation of bulk samples that include debris to reduce sample-processing time. In order to explore the use of DNA-based methods in water quality assessment in South Africa, this study compares morphological and molecular-based identification of freshwater macroinvertebrates at the mixed higher taxon and mOTU level while investigating abundance and comparing mOTU recovery with historical species records. From seven sites across three rivers in South Africa, we collected a biomonitoring sample, an intensive-search comprehensive sample and an eDNA sample per site. The biomonitoring sample was picked and scored according to standard protocols and the leftover debris and comprehensive samples were homogenised including all debris. DNA-based methods recovered higher diversity than morphology, but did not always recover the same taxa, even at the family level. Regardless of the differences in taxon scores, most DNA-based methods except some eDNA samples, returned the same water quality assessment category as the standard morphology-based assessment. Homogenised comprehensive samples recovered more freshwater invertebrate diversity than all other methods. The eDNA samples recovered 2 to 10 times more mOTUs than any other method, however 90% of reads were non-target and as a result eDNA recovered the lowest target diversity. However, eDNA did find some target taxa that the other methods failed to detect. This study shows that unsorted samples recover the same water quality scores as a morphology-based assessment and much higher diversity scores than both picked and eDNA samples. As a result, there is potential to integrate DNA-based approaches into existing metrics quickly while providing much more information for the development of more refined metrics at the species or mOTU level with distributional data which can be used for conservation and biodiversity management.

Assessment of stream macroinvertebrate communities with eDNA is not congruent with tissue‐based metabarcoding

“…To evaluate which sampling and identification method had the strongest stream-level signal, we used a distance-based redundancy analysis (RDA) with stream as the constraining factor. We calculated Jaccard dissimilarity indices based on presence/absence data for our three identification methods (coarse level for morphology and OTU level for both bulk sample and eDNA metabarcoding) and performed three ordinations using the "capscale" function in the R package vegan (Oksanen et al, 2017). The sample sites were symbolized by stream, and samples from the same stream were joined by a line for visualization.…”

“…While sorting benthic macroinvertebrate samples does not require taxonomic expertise, it is still a time‐consuming process and can bottleneck freshwater monitoring research. In some cases, specimens can be ground without sorting (Pereira‐da‐Conceicoa et al., 2020), but it is not clear if that is a possibility for all stream conditions (e.g. samples with a lot of organic debris or sand).…”

“…(2018), more metazoan (and classical bioindicator taxa) OTUs were detected using bulk sample metabarcoding. Since environmental sampling contains all the microorganisms present in the water sample, it is possible that eDNA metabarcoding results in a higher amount of nonspecific amplification than bulk sample metabarcoding (Pereira‐da‐Conceicoa et al., 2020). While Macher et al.…”

“…Few studies have directly compared the community composition of aquatic macroinvertebrate samples collected using eDNA and kicknets and those that have focused on overall community dissimilarities between the two methods (Deiner et al., 2016; Macher et al., 2018; Pereira‐da‐Conceicoa et al., 2020). By pooling all samples and comparing them, it is not possible to observe variations in community composition that occur at the site or stream level.…”

Metabarcoding reveals different zooplankton communities in northern and southern areas of the North Sea

“…Such an approach, i.e. filtering out low abundant OTUs based on relative abundance, is commonly used in metabarcoding studies Pereira-da-Conceicoa et al, 2019;Theissinger et al, 2019). After this quality filtering step, the reads of the two technical replicates per sample were summed up to build the final dataset.…”