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

Abstract

DNA-based biodiversity analysis has gained major attention due to the use of high throughput sequencing technology in approaches such as mixed community or environmental DNA metabarcoding. Many cytochrome c oxidase subunit I (COI) primer sets are now available for such work. The purpose of this study is to look at how COI primer choice affects the recovery of arthropod richness, beta diversity, and recovery of site indicator taxa in benthos kick-net samples typically used in freshwater biomonitoring. We examine 6 commonly used COI primer sets, on samples collected from 6 freshwater sites. Richness is sensitive to primer choice and the combined use of additional multiple COI amplicons recovers higher richness. Thus, to recover maximum richness, multiple primer sets should be used with COI metabarcoding. 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. Additionally, the recovery of traditional freshwater bioindicator assemblages such as Ephemeroptera, Trichoptera, Plectoptera, and Diptera may not fully capture the diversity of broadscale arthropod site indicators that can be recovered from COI metabarcoding. Based on these results, studies that use different COI amplicons may not be directly comparable. This work will help future biodiversity and biomonitoring studies develop not just standardized, but optimized workflows that maximize taxon-detection or order taxa along gradients.

Variations in terrestrial arthropod DNA metabarcoding methods recovers robust beta diversity but variable richness and site indicators based on exact sequence variants

“…Though the overall percentage of retained raw arthropod reads is low, it is consistent with previous work from bulk samples that are known to comprise a phylogenetically diverse mixture of taxa that are detected even when using primers originally developed to target arthropods. 15,16 Since only a proportion of arthropod ESVs could be identified with confidence, we present our results at the ESV rank wherever possible (Fig S3). Rarefaction curves show that we saturated the sequencing of our arthropod COI PCR products (Fig S4).…”

“…Our results are consistent with a previous study that showed how COI metabarcoding may actually recover a greater taxonomic diversity of site indicators in addition to the usual expected bioindicators. 15 Future studies should attempt to pair soil arthropod metabarcoding with local DNA barcoding to improve taxonomic assignment rates. Despite this, the use of soil arthropod metarbarcodes as site bioindicators was successful and samples from two similar jack pine stands with different origins were distinguished from each other based on beta diversity and the presence of site indicators.…”

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“…Marquina et al 2018). These results were also recently confirmed by (Hajibabaei et al 2019a), which 411 showed that the use of multiple primer sets did not substantially improve taxa detection. Conclusions 415 Our study demonstrates that the fwh2, BF1/2/3 + BR2 and mlCOIintF based primer sets all 416 perform well when metabarcoding terrestrial arthropod samples.…”

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“…Marquina et al 2018). These results were also recently confirmed by (Hajibabaei et al 2019a), which 411 showed that the use of multiple primer sets did not substantially improve taxa detection. Conclusions 415 Our study demonstrates that the fwh2, BF1/2/3 + BR2 and mlCOIintF based primer sets all 416 perform well when metabarcoding terrestrial arthropod samples.…”