DNA metabarcoding is the taxonomic identification of organisms present in a bulk or environmental sample through the use of DNA amplification of standard regions of a genome (i.e. DNA barcodes) coupled with high-throughput sequencing (HTS) (Taberlet et al. 2012). DNA metabarcoding studies have been developed for diverse taxonomic groups (e.g. terrestrial arthropods: Elbrecht et al. 2019; freshwater macroinvertebrates: Bista et al. 2018; Giebner et al. 2020; meiofaunal organisms: Fais et al. 2020b; marine communities: Leray and Knowlton 2015; Aylagas et al. 2018), using a wide range of laboratory procedures (Andújar et al. 2018) and addressing questions about species richness, taxonomic composition, as well as biodiversity patterns (McGee et al. 2019; Piñol et al. 2019).

Metabarcoding allows for comparison across studies, however the harmonization and standardization of protocols is still far from being established (van der Loos and Nijland 2020; Duarte et al. 2021). While DNA-based approaches for assessing and monitoring marine macroinvertebrate species are constantly evolving (Andújar et al. 2018), the diversity of the adopted methodologies, including the use of different primer pairs or molecular markers, the lack of accurate and complete reference databases, and the continuous emergence of new sequencing innovations and bioinformatics pipelines, implies low standardization and comparability among studies (Cowart et al. 2015; Leray and Knowlton 2017), which remain important challenges that should be addressed.

Targeting marine species is especially challenging due to the broad taxonomic and phylogenetic composition of marine communities, and the choice of marker usually depends on the target taxa. However, the balance between the range of taxonomic coverage and the taxonomic discrimination ability should be considered in the choice of target genomic region and/or primer pairs, since it may affect the number of species and the taxonomic groups detected (van der Loos and Nijland 2020). Initially, a fundamental and critical decision in a DNA metabarcoding study is centered on which genomic region should be targeted. The 5’ end of the mitochondrial cytochrome oxidase I gene (COI) is the standard barcode for animal life and the backbone of the universal Barcode of Life species identification system (Hebert et al. 2003), being the recommend marker for community metabarcoding (Andújar et al. 2018) and by far the most well represented genomic region in public databases (Porter and Hajibabaei 2020). After COI, the nuclear small subunit rRNA gene (18S) is among the most widely used markers in marine biodiversity studies (e.g. Lejzerowicz et al. 2015; Wangensteen et al. 2018; Zhang et al. 2018; Fais et al. 2020a; Duarte et al. 2021).

PCR-based methodologies are highly influenced by amplification biases thereby encouraging the use of several primer pairs in different metabarcoding studies (Bista et al. 2018; Elbrecht et al. 2019; Hajibabaei et al. 2019; Porter and Hajibabaei 2020). Amplicon length, primer mismatches, GC content, and polymerase errors can also affect the ability to detect the species present in bulk or environmental samples, (Kebschull and Zador 2015; Piñol et al. 2015; Nichols et al. 2018; Derycke et al. 2020). Even broad-range primers (i.e. able to amplify a DNA fragment over a broad taxonomic scope) demonstrated more affinity for some species and consequently do not perfectly match the DNA of all species present in a bulk sample. Low species detection ability has been associated with the use of non-degenerate primers (Clarke et al. 2014; Elbrecht et al. 2017; Collins et al. 2019), however, degenerate primers seem to have a higher capability to detect species compared with nondegenerate ones. The Leray-Geller fragment (mlCOIintF/jgHCO2198 – Leray et al. 2013, Geller et al. 2013) is a degenerate primer pair widely used in DNA metabarcoding studies targeting different types of taxa (Leray and Knowlton 2015; Clarke et al. 2017; Ransome et al. 2017; Aylagas et al. 2018), mostly due to their design for marine organisms with a wide phylogenetic coverage and fair amplicon length (313 bp). Yet, the inclusion of inosine bases in the reverse primer jgHCO2198 may impair the performance of the most commonly used high-fidelity polymerases for generating amplicon libraries for HTS (Knittel and Picard 1993, Jungbluth et al. 2021). The combination of mlCOIintF with LoboR1 amplifies exactly the same fragment and with demonstrated success in the amplification of DNA barcodes of marine taxa (Haenel et al. 2017; Hollatz et al. 2017; Chang et al. 2020; Castro et al. 2021). Primers amplifying the nuclear genes (e.g. TAReuk454FWD1/TAReukREV3, Lejzerowicz et al. 2015) constitute alternatives to COI primers, mainly because of their slower rate of evolution which results in more conserved regions which may facilitate the design of primers. However, reference databases for 18S target regions are less populated than COI (Andújar et al. 2018) and COI-primers often outperform primers for rDNA loci on taxon recovery (Clarke et al. 2017; Elbrecht et al. 2017, 2019; Atienza et al. 2020) and species discrimination ability (Tang et al. 2012; Clarke et al. 2017). Choosing the appropriate molecular markers and/or primer pairs is a principal step to influence the quality of data through PCR amplification, to accurately and efficiently determine the taxonomic composition of the bulk sample and to assign a species level identification.

For DNA metabarcoding studies, multiple sets of primers amplifying different molecular markers have been used to target a broad range of taxonomic groups in different marine communities. However, the majority of studies used a single primer pair or single marker loci strategy (Duarte et al. 2021). In two studies which comprehensively review DNA metabarcoding studies over the last 10 years, the authors concluded that only a minor portion of the publications used more than one marker (25% in van der Loos and Nijland 2020; 24% in Duarte et al. 2021).

Although DNA metabarcoding studies aim to provide species level assignments (Taberlet et al. 2012), the existence of gaps in the reference sequence databases (Weigand et al. 2019; Leite et al. 2020; Mugnai et al. 2021), associated with the limited species level discrimination ability of some markers (e.g. 18S rRNA; von Ammon et al. 2018) in some taxonomic groups, reduces the discrimination level of taxonomic identifications. However, the trade-off between the range of taxonomic coverage (species successfully amplified) and the taxonomic discrimination ability should be considered to enable accurate identifications at lower taxonomic ranks (Porter and Hajibabaei 2020).

Considering the importance of choice of marker and primer to improve taxonomic coverage and discrimination of DNA metabarcoding, we investigated the impact of these factors on the composition and structure of marine macrozoobenthic communities. We selected two different primer pairs targeting COI and one targeting 18S rDNA V4 region, to compare their ability to detect macroinvertebrates at species level and to evaluate the benefits of the use of two molecular markers on species recovery success. We also conducted an assessment of the availability of reference sequences for all species detected in the study, in order to compare the representativeness of species, identifying the existence of gaps in both databases (BOLD for COI and SILVA for 18S rRNA gene) and attempting to infer the reasons for failed detections.

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). Although criteria of choice such as practical efficiency and cost-effectiveness would favor finding the “best” single primer pair that fits a particular study, our results indicated that, when the goal is to assess as comprehensively as possible the species composition of a marine macrozoobenthic sample, the ideal primer pair may be hard to find. Both biased primer affinity for the DNA templates and gaps in species representativeness in reference databases appear to concur for this difficulty.

Together, the three primer pairs used in this study enabled the detection of a fair number of macrozoobenthic species. Quantitatively, the 18S V4 region and the two COI primer pairs displayed similar ability to detect marine macroinvertebrate species. However, considering the high complementarity in the species recovered between these markers, the choice of the best performing primer was not obvious. Similar COI – 18S comparisons described in the literature report somewhat distinct and even contradictory results. For example, DNA metabarcoding studies using mock zooplankton communities demonstrated different taxonomic recovery ability between COI and 18S: whereas one of the studies detected similar patterns of species detection ability among markers (Clarke et al. 2017), the other reported higher detection efficiency with 18S V4 region than with COI, and with higher levels of overlapping between markers (Zhang et al. 2018). Contrary to these results, in a study using marine hard-bottom communities the authors showed that using COI (Leray-XT) they were able to recover four times more diversity than using 18S region (V7) (Wangensteen et al. 2018). However, a direct comparison with our study is limited given the employment of different primers, reference libraries and thresholds. The differences obtained for the 18S V4 region in assignment for some taxonomic groups could be related to more conserved sequences in priming regions (Tang et al. 2012; Brown et al. 2015). In addition, taxonomic discrimination efficiency of the targeted marker loci, possible mismatches between primer and DNA template or difficulties in primer affinity for specific taxa can be possible reasons for the obtained differences between primers and marker region in amplification efficiency.

The significant complementarity observed between the two molecular markers, with each single marker capturing at the very best approximately 66% of the detected species in a sample, revealed that, by using only one marker, a fair portion of the marine macroinvertebrate species may fail detection. Both markers detected different communities, raising high concerns for monitoring studies, since the biodiversity detected will be different and many species may be overlooked due to methodological steps only. For example, while isopods were only detected by COI, platyhelminthes and tunicates were exclusively detected by 18S V4. Although few metabarcoding studies compared the performance of molecular markers on species recovery (Dowle et al. 2015; Drummond et al. 2015), our results are generally consistent with previous findings in marine invertebrate communities (Wangensteen et al. 2018), where high levels of complementarity at species level between these markers were reported. Since our goal is to capture the widest possible diversity of macrozoobenthic species, compared to using a single molecular marker, the combination of a multi-locus strategy improves the number of retrieved species, which we recommend as the best practice to be used in marine macroinvertebrates assessments.

Regarding the two primer pairs from the COI barcode region, although we observed a similar number of species globally detected by each primer (84 vs 63 species), they diverge qualitatively in the species detected (41% of the species exclusively detected with mlCOIintF/LoboR and 21% in LCO1490/Ill_C_R), hence this should be the main criteria to consider in order to maximize the scope of species detection. The efficiency of different COI-primers in macroinvertebrates assessment has been already compared in previous studies (Hollatz et al. 2017; Lobo et al. 2017; Ip et al. 2019; Derycke et al. 2020), however not always reaching the same conclusions. Different methodologies adopted in different studies may explain the discrepancies in the results (e.g.: PCR thermal cycling regimes, sequencing depths, informatics platforms, clustering and threshold values, reference libraries). Our results emphasize that using a single primer pair for the COI region will result in a fair amount of undetected diversity of marine zoobenthic taxa. Therefore, we suggest the simultaneous employment of at least two primer pairs to improve the efficiency of the taxonomic diversity recovery.

The three primer pairs used in this study were able to detect marine macroinvertebrate species in every sampling time, all of them consistently pointing to a higher species diversity after seven months of deployment of the substrates. These results highlight the benefit of the application of a multiple primer pair and multi-locus strategy for ecological assessments of marine species, since if we had only used one primer pair or marker we would have failed to detect important macrobenthic taxa, and the taxonomic composition of the community could emerge substantially different. Temporal and seasonal changes in a community could affect the potential of DNA-based species monitoring, especially when methodological bias originated by amplification procedures (choice of marker loci and primer pairs) could influence ecological interpretations (Clarke et al. 2017).

We performed an assessment of the availability of representative sequences for all species detected in this study in each of the reference databases employed, namely BOLD and SILVA, respectively for COI and 18S V4 markers. This enabled us to verify if the detection of a species by one marker, and not by the other, could be attributed to gaps in the library of the latter or, if no gap was found, it could be ascribed to faulty amplification. A sizable but minor proportion of gaps was recorded for both markers (18% for COI and 28% for 18S V4). The incompleteness, and possible inaccuracies of databases may explain some of the species detected exclusively by one marker, as for example, the flatworm Vorticeros auriculatum, a species detected by 18S V4 that does not have representatives in BOLD. On other hand, some of the detected species with reference sequences in both databases were only detected by one marker (e.g. the tunicate Asterocarpa humilis, undetected with COI despite having representative sequences in BOLD). Considering the complete 18S rRNA gene, the target region we selected should not be the main reason for failed detections, since V4 is reported to have high amplification success (Brown et al. 2015; Lejzerowicz et al. 2015; Zhang et al. 2018) and demonstrated to have a better performance on taxonomic assignments than other 18S regions (Fais et al. 2020b). However, since these primers failed to detect some species, this was probably due to faulty amplification or reduced resolution of taxonomic assignments. Indeed, although COI-based monitoring approaches may claim the advantage of having a verified and dedicated database for a large variety of taxa (Porter and Hajibabaei 2020), several studies already reported the existence of significant gaps in reference libraries particularly for dominant marine macrobenthic taxa (Weigand et al. 2019: 20% to 30% of completion of databases for dominant groups, i.e. Annelida, Mollusca and Arthropoda), including for the region that comprised the geographic area of the current study (Leite et al. 2020: 49% for Mollusca, 53% for Crustacea and 16% for Polychaeta). Hence, although the availability of sequences or reference libraries does not appear to have been the main factor affecting species detection, it revealed that more investment should be allocated to obtain reliable reference sequences to enhance species assignment accuracy, in order to recover the taxonomic composition of a target community as complete as possible.

Globally, these results highlight the influence of marker and primer pair complementarity on the ability to record marine macrozoobenthic species through metabarcoding. For future high-throughput assessments using DNA metabarcoding approaches, we recommend combining molecular markers and, if possible, multiple primer pairs, to increase the power of species detections and the accuracy of biodiversity assessments, thereby yielding more comprehensive and reliable results for marine macroinvertebrate monitoring.

Conceptualization: B.R.L., J.S.T. and F.O.C.; Methodology: B.R.L and P.E.V.; Formal analysis: B.R.L. and P.E.V.; Data curation: B.R.L and P.E.V.; Writing – original draft: B.R.L. and F.O.C.; Writing – review and editing: B.R.L., P.E.V., J.S.T. and F.O.C.; Visualization: B.R.L. and P.E.V. Supervision: J.S.T. and F.O.C. All authors have read and agreed to the published version of the manuscript.