Detection of Macrobenthos Species With Metabarcoding Is Consistent in Bulk DNA but Dependent on Body Size and Sclerotization in eDNA From the Ethanol Preservative

Abstract

DNA metabarcoding is a promising method to increase cost and time efficiency of marine monitoring. While substantial evidence exists that bulk DNA samples adequately reflect diversity patterns of marine macrobenthos, the potential of eDNA in the ethanol preservative of benthic samples for biodiversity monitoring remains largely unexplored. We investigated species detection in bulk DNA and eDNA from the ethanol preservative in samples from four distinct macrobenthic communities in the North Sea. Bulk DNA and eDNA were extracted with different extraction kits and five COI primer sets were tested. Despite the availability of a nearly complete reference database, at most 22% of the amplicon sequence variants (ASVs) were assigned taxonomy at the phylum level. However, the unassigned ASVs represented only a small fraction of the total reads (13%). The Leray primer set outperformed the four other primer sets in the number of non-chimeric reads and species detected, and in the recovery of beta diversity patterns. Community composition differed significantly between bulk DNA and eDNA samples, but both sample types were able to differentiate the four communities. The probability of detecting a species in the eDNA from the ethanol preservative was significantly lower than for bulk DNA for macrobenthos species having small to medium body size and for species having chitine or CaCO3 in their cuticula. Detection in the bulk DNA samples was not affected by the investigated morphological traits, indicating that monitoring of macrobenthos species will be most robust when using bulk DNA as template for metabarcoding.

Towards harmonization of DNA metabarcoding for monitoring marine macrobenthos: the effect of technical replicates and pooled DNA extractions on species detection

“…Ayla gas et al ( 2018) noted a high number of non-metazoan taxonomic assignments, but in this study, even when these unassigned ASVs were matched against the NCBI data base using Blastn, only an additional 8% of the unique unassigned ASVs gave a reliable taxonomic species name. Similar observations were observed in another me tabarcoding study for macrobenthos using various primer sets (Derycke et al 2021), where the unassigned ASVs were explained as unknown diversity, genetic noise like PCR or sequencing errors, nuclear pseudogenes or aspe cific amplification of other genomic regions.…”

“…For the DNA metabarcode dataset, species detected in all six rep licates and uniquely in one replicate were listed. For each species, the corresponding size class was given based on a previous study (Derycke et al 2021). If the species was also found in the morphologically identified sample, the abundance (counts) was given.…”

“…A second possibility why these species are not found in all three PCR replicates could be the primer efficiency. However, the used primer, the Leray primers set, has been shown to amplify most macrobenthic species of the North Sea (Derycke et al 2021). When looking at the taxonomy of species present in only one replicate, some classes are highly present, like Polychaeta (43% of the species that are present in only one replicate).…”

“…PCR is a stochastic process, because a molecule will be either replicated or not based on whether the primers were able to sufficiently bind during each PCR cycle. The impact of primer choice on species detection is well known (El brecht and Leese 2017; Lobo et al 2017;Braukmann et al 2019;Derycke et al 2021), but the number of PCR replicates can also affect the detected diversity (Dopheide et al 2018). PCR replicates, where the same DNA extract is amplified in separate PCR reactions, can decrease the stochasticity of this process (Leray and Knowlton 2015).…”

Comparing DNA metabarcoding with morphology in the assessment of macrozoobenthos in Portuguese transitional waters in the scope of the Water Framework Directive monitoring

“…However, despite the demonstrated utility of metabarcoding to reliably generate assessments of aquatic environmental status, in particular based on benthic invertebrates (Aylagas et al 2014, 2018, Cowart et al 2015, Lobo et al 2017b, Derycke et al 2021, Duarte et al 2021, Van den Bulcke et al 2021), the adoption of metabarcoding in biomonitoring still face several challenges, in particular in coastal and transitional ecosystems. These ecosystems hold very different features from freshwaters, and are highly diverse, thereby requiring a tailored tool in order to overcome existing technological shortcomings that can prevent the detection of all taxa within a sample (e.g., Leite et al 2021, Wangensteen et al 2018a,b).…”