DNA Metabarcoding Methods for the Study of Marine Benthic Meiofauna: A Review

Abstract

Meiofaunal animals, roughly between 0.045 and 1 mm in size, are ubiquitous and ecologically important inhabitants of benthic marine ecosystems. Their high species richness and rapid response to environmental change make them promising targets for ecological and biomonitoring studies. However, diversity patterns of benthic marine meiofauna remain poorly known due to challenges in species identification using classical morphological methods. DNA metabarcoding is a powerful tool to overcome this limitation. Here, we review DNA metabarcoding approaches used in studies on marine meiobenthos with the aim of facilitating researchers to make informed decisions for the implementation of DNA metabarcoding in meiofaunal biodiversity monitoring. We found that the applied methods vary greatly between researchers and studies, and concluded that further explicit comparisons of protocols are needed to apply DNA metabarcoding as a standard tool for assessing benthic meiofaunal community composition. Key aspects that require additional consideration include: (1) comparability of sample pre-treatment methods; (2) integration of different primers and molecular markers for both the mitochondrial cytochrome c oxidase subunit I (COI) and the nuclear 18S rRNA genes to maximize taxon recovery; (3) precise and standardized description of sampling methods to allow for comparison and replication; and (4) evaluation and testing of bioinformatic pipelines to enhance comparability between studies. By enhancing comparability between the various approaches currently used for the different aspects of the analyses, DNA metabarcoding will improve the long-term integrative potential for surveying and biomonitoring marine benthic meiofauna.

Comparing species detection success between molecular markers in DNA metabarcoding of coastal macroinvertebrates

“…DNA metabarcoding-based biomonitoring of aquatic communities would benefit from the establishment of standardized approaches (Blackman et al 2019;Duarte et al 2021;Gielings et al 2021), which would enable direct inter-study comparisons, broader implementation and greater scientific gains. Choice of marker and primer pairs to employ is a critical decision in DNA metabarcoding, that depends on the goals of the study, targeted communities and aimed rank of taxonomic assignment (van der Loos and Nijland 2020; Duarte et al 2021).…”

Mitochondrial cytochrome c oxidase subunit I (COI) metabarcoding of Foraminifera communities using taxon-specific primers

“…The availability of an easily obtainable molecular marker additionally to the commonly used 18S rRNA for metabarcoding of Foraminifera will be valuable for future studies, as it will open up new possibilities for studies on diversity and ecology of Foraminifera, and allows including multiple markers into molecular studies. This is becoming more common in metabarcoding studies ( Dupuis, Roe & Sperling, 2012 ; Gao et al, 2014 ; Fais et al, 2020 ; Eberle et al, 2020 ; Gielings et al, 2021 ) and could improve studies on phylogeny, species diversity and ecology of Foraminifera.…”

“…We extracted meiofauna from the larger sediment core (≈200 ml) by adding 500 ml of MgCl 2 solution to the sediment in a sterile bottle, which anaesthetises meiofauna and allows subsequent separation from the sediment by decantation ( Somerfield, Warwick & Moens, 2005 ). After 5 min, samples in MgCL 2 solution were carefully swirled and the supernatant containing the meiofauna decanted through a 1 mm and 41 um sieve cascade, as commonly done in beach meiofauna studies ( Haenel et al, 2017 ; Martínez et al, 2020 ; Castro et al, 2021 ; Gielings et al, 2021 ). The meiofauna fraction retained on the 41um sieve was rinsed into sterile 15 ml Falcon tubes and preserved with 10 ml 96% EtOH.…”

Environmental RNA as a Tool for Marine Community Biodiversity Assessments

“…To date, there is no one loci and associated primer pair that will effectively detect all eukaryotic organisms, and in particular metazoans, so it is beneficial to use multiple loci when looking for a variety of taxa 6 . Previous marine eDNA studies have demonstrated the preferred method of using two loci to achieve comprehensive metabarcoding for taxonomically diverse environments 5 , 30 , 40 . The current study further demonstrates the need to use at least two different loci for targeted PCR and sequencing to truly capture the wide diversity in rich systems, such as the top oxygenated layer of the marine benthos.…”

“…Zhang et al (2018) compared the utility of various primer pairs for different loci and found that using two markers allows for a more comprehensive overview of the species present in the community of interest, particularly when the markers are evolutionarily independent, such as 18S and COI 6 . Additionally, many metabarcoding studies on benthic communities use two primer pairs to maximize taxa detection 30 . Therefore, we selected the Uni18S (V4 region of 18S) and Leray COI primer pairs.…”