The importance of standardization for biodiversity comparisons: A case study using autonomous reef monitoring structures (ARMS) and metabarcoding to measure cryptic diversity on Mo’orea coral reefs, French Polynesia

Abstract

The advancement of metabarcoding techniques, declining costs of high-throughput sequencing and development of systematic sampling devices, such as autonomous reef monitoring structures (ARMS), have provided the means to gather a vast amount of diversity data from cryptic marine communities. However, such increased capability could also lead to analytical challenges if the methods used to examine these communities across local and global scales are not standardized. Here we compare and assess the underlying biases of four ARMS field processing methods, preservation media, and current bioinformatic pipelines in evaluating diversity from cytochrome c oxidase I metabarcoding data. Illustrating the ability of ARMS-based metabarcoding to capture a wide spectrum of biodiversity, 3,372 OTUs and twenty-eight phyla, including 17 of 33 marine metazoan phyla, were detected from 3 ARMS (2.607 m2 area) collected on coral reefs in Mo’orea, French Polynesia. Significant differences were found between processing and preservation methods, demonstrating the need to standardize methods for biodiversity comparisons. We recommend the use of a standardized protocol (NOAA method) combined with DMSO preservation of tissues for sessile macroorganisms because it gave a more accurate representation of the underlying communities, is cost effective and removes chemical restrictions associated with sample transportation. We found that sequences identified at ≥ 97% similarity increased more than 7-fold (5.1% to 38.6%) using a geographically local barcode inventory, highlighting the importance of local species inventories. Phylogenetic approaches that assign higher taxonomic ranks accrued phylum identification errors (9.7%) due to sparse taxonomic coverage of the understudied cryptic coral reef community in public databases. However, a ≥ 85% sequence identity cut-off provided more accurate results (0.7% errors) and enabled phylum level identifications of 86.3% of the sequence reads. With over 1600 ARMS deployed, standardizing methods and improving databases are imperative to provide unprecedented global baseline assessments of understudied cryptic marine species in a rapidly changing world.

Anacapa Toolkit: An environmental DNA toolkit for processing multilocus metabarcode datasets

“…However, the lack of accurate and easily customizable bioinformatic pipelines is limiting the broader application of eDNA approaches. The Anacapa Toolkit provides enhanced functionality for eDNA projects and can be used for other common applications such as gut content analysis (Leray et al, ), autonomous reef monitoring structures (Ransome et al, ), and microbiomes (Bokulich et al, ). Importantly, the Anacapa Toolkit is modular and its parameters are easily modifiable, making it easily adapted to user specific needs in several important ways.…”

Reconstructing hyperdiverse food webs: Gut content metabarcoding as a tool to disentangle trophic interactions on coral reefs

“…DNA metabarcoding often results in many sequences with unknown identities, which creates a biased perception of prey items that neglects rare and cryptic fauna and flora (Leray & Knowlton, ). In our case, we were fortunate to have the Mo'orea BIOCODE database, which contains DNA barcodes for the majority of invertebrate and vertebrate organisms around Mo'orea (Leray & Knowlton, , ; Meyer, ; Plaisance et al, ; Ransome et al, ). Consequently, we identified the vast majority of our sequences to the level of phylum, with many taxonomic identifications to the genus or species level.…”

“…However, the efficacy of metazoan metabarcoding is heavily reliant on the existence of local genetic inventories that include cryptic species like meiofauna (Leray, Yang, et al, 2013;Ransome et al, 2017). Mo'orea, French Polynesia, provides a unique opportunity for fish dietary analysis due to the existence of an extensive DNA-barcode library created through the Mo'orea BIOCODE Project, a comprehensive inventory that contains barcodes of the majority of terrestrial and marine metazoans on the island (Leray, Boehm, Mills, & Meyer, 2012;Meyer, 2016).…”

Anacapa Toolkit: an environmental DNA toolkit for processing multilocus metabarcode datasets

“…However, the lack of accurate and easily customizable bioinformatic pipelines is limiting the broader application of eDNA approaches. The Anacapa Toolkit provides enhanced functionality for eDNA projects and can be used for other common applications such as gut content analysis (Leray et al, 2013), autonomous reef monitoring structures (Ransome et al, 2017), and microbiomes (Bokulich et al, 2018). Importantly, the Anacapa Toolkit is modular and its parameters are easily modifiable, making it easily adapted to user specific needs in several important ways.…”