DNA metabarcoding of macrobenthic communities in the North Sea is most comparable with morphology based analyses when using the Leray primer set
There is considerable support for the use of the mitochondrial COI gene to assess metazoan diversity from bulk samples (Andujar et al., 2018; Leray & Knowlton, 2015; Lobo et al., 2017), yet finding the best primer set for a specific target group and study requires empirical data rather than relying on general rule of thumbs (Alberdi et al., 2018). In a study comparing the performance of five primer sets on mock communities of marine macrobenthos using the 454 sequencing platform, primer set E was amongst the best primer sets in terms of detecting many species (Hollatz et al., 2017). The amplification of the 3’ region of the Folmer region has resulted in better detection of marine macroinvertebrates than the full length Folmer fragment (Aylagas, Borja, et al., 2016; Hollatz et al., 2017; Lobo et al., 2017). In silico testing showed that the primer sets in the 3’ region generally detected almost twice the amount of species compared to the primer set in the 5’ region (Table 1), which was confirmed by our wet lab results: primer sets amplifying the 3’ region outperformed primer set D in recovering macrobenthic species. Our data clearly show that primer sets used by Leray et al. (2013) (Primer set A) and Lobo et al. (2013) (Primer set E) outcompete the other three primer sets in the number of high quality and non-chimeric reads (ESM Fig 1). Both primer sets amplify exactly the same fragment of the COI gene, but the reverse primer of primer set A has a higher degeneracy than the reverse primer of primer set E (Table 1). A higher degeneracy leads to a better performance of COI primers for insect communities (Marquina, Andersson, & Ronquist, 2019). Based on the wetlab results, primer set A outperformed primer set E at various levels: 1/ twice the amount of sequences received taxonomic assignment (22.6 vs 10.9 %); 2/ the best correspondence with morphological species numbers in the different locations was obtained (ESM Fig 4) and 3/ the highest number of morphologically identified species was detected with primer set A (Fig 3). Finally, the Leray primer set was able to differentiate the four macrobenthic communities. Consequently, for monitoring studies targeting marine macrobenthic communities the use of the Leray primer set is recommended.
Metabarcode studies typically detect a higher number of species compared to morphology based analyses (Elbrecht, Vamos, et al., 2017; Lobo et al., 2017), which has been explained by the fact that specimens are not lost during the sorting process and also small pieces of animals – which are morphologically not identified- are included in the bulk DNA and eDNA. In addition, metabarcode studies have reported the detection of species that were lacking in the morphological analyses from the same samples (Aylagas, Borja, et al., 2016). This was also the case for our study, but in contrast to what we expected, less species were detected using DNA metabarcoding (Fig 3): for the four samples that were processed both morphologically and with DNA metabarcoding, 57 species were morphologically identified, while 52 and 45 species were identified with the bulk DNA and eDNA, respectively. This may be partly explained by the reference database and algorithm used to assign taxonomy: 51 and 76 species for the bulk and eDNA, respectively, were identified when using the MIDORI reference database, while 60 and 103 species for the bulk and eDNA, respectively, were identified when using Blast and the NCBI database. The latter procedure was also used by Lobo et al. (2017) and Elbrecht, Vamos, et al. (2017).