COI metabarcoding primer choice affects richness and recovery of indicator taxa in freshwater systems

Abstract

Mixed community or environmental DNA marker gene sequencing has become a commonly used technique for biodiversity analyses in freshwater systems. Many cytochrome c oxidase subunit I (COI) primer sets are now available for such work. The purpose of this study is to test whether COI primer choice affects the recovery of arthropod richness, beta diversity, and recovery of target assemblages in the benthos kick-net samples typically used in freshwater biomonitoring. We examine six commonly used COI primer sets on samples collected from six freshwater sites. Biodiversity analyses show that richness is sensitive to primer choice and the combined use of multiple COI amplicons recovers higher richness. Thus, to recover maximum richness, multiple primer sets should be used with COI metabarcoding. In ordination analyses based on community dissimilarity, samples consistently cluster by site regardless of amplicon choice or PCR replicate. Thus, for broadscale community analyses, overall beta diversity patterns are robust to COI marker choice. Recovery of traditional freshwater bioindicator assemblages such as Ephemeroptera, Trichoptera, Plectoptera, and Chironomidae as well as Arthropoda site indicators were differentially detected by each amplicon tested. This work will help future biodiversity and biomonitoring studies develop not just standardized, but optimized workflows that either maximize taxon-detection or the selection of amplicons for water quality or Arthropoda site indicators.

Fig 1. Map of primers and amplicons tested in this study.

The reference sequence shown in black is Drosophila yakuba, cytochrome c oxidase region 1470–3009 bp (1540 nt). Secondary structure is shown for reference, comprised of six alpha helices in the standard DNA barcode region shown here.

Fig 2. ESV richness continues to increase as COI amplicons are added but species—Order richness reaches a plateau.

For the primer comparison experiment that used the soil DNA extraction kit, we pooled the results from the six sites and show the top COI amplicon combinations that detected the greatest richness. We report the recovered richness when up to six amplicons are combined at the 1) ESV, 2) species, 3) genus, 4) family, and 5) order ranks. ESV = exact sequence variant; A = BR5; B = F230R; C = ml-jg; D = BF1R2; E = BF2R2; F = fwh1.

Fig 3. Each amplicon differentially recovers site and water quality indicators.

In the top panel, the number of site indicator taxa from across the Arthropoda are shown. In the bottom panel, the number of typical water quality indicator taxa from the EPTC are shown. This analysis was based on normalized data. ESV = exact sequence variant; EPTC = Ephemeroptera, Plecoptera, Trichoptera, Chironomidae.

Fig 4. Site indicator taxa chosen based on metabarcode sequencing are comprised of Coleoptera, Diptera, Ephemeroptera, and Trichoptera.

Presence is indicated by a dark square, absence by a white square. The total number of Arthropoda site indicator taxa detected by each amplicon is shown in the bottom row.

Fig 5. Samples cluster mainly by site despite differences in amplicons and replicates.

Results are based normalized data. COI amplicons are labelled directly in the plot. Amplicons shown twice represent the two PCR replicates. Sites are grouped by color according to the legend.