Environmental DNA (eDNA) metabarcoding can greatly enhance our understanding of global biodiversity and our ability to detect rare or cryptic species. However, sampling effort must be considered when interpreting results from these surveys. We explored how sampling effort influenced biodiversity patterns and nonindigenous species (NIS) detection in an eDNA metabarcoding survey of four commercial ports. Overall, we captured sequences from 18 metazoan phyla with minimal differences in taxonomic coverage between 18 S and COI primer sets. While community dissimilarity patterns were consistent across primers and sampling effort, richness patterns were not, suggesting that richness estimates are extremely sensitive to primer choice and sampling effort. The survey detected 64 potential NIS, with COI identifying more known NIS from port checklists but 18 S identifying more operational taxonomic units shared between three or more ports that represent un-recorded potential NIS. Overall, we conclude that eDNA metabarcoding surveys can reveal global similarity patterns among ports across a broad array of taxa and can also detect potential NIS in these key habitats. However, richness estimates and species assignments require caution. Based on results of this study, we make several recommendations for port eDNA sampling design and suggest several areas for future research.
MOTU Cleaning and Analyses Script
This script removes cleans the raw MOTU data and runs all of the analyses in the paper including within-sample metazoan MOTU accumulation curves, sample richness estimates, site metazoan MOTU accumulation curves by sample number or read count, dissimilarity NMDS ordination, dissimilarity by distance analysis for Adelaide sites, and metazoan MOTU richness estimates standardize to 20 samples across all sites. This script was written for R version 3.4.1, requires input files "Unfiltered_18S.csv", "Unfiltered_COI.csv", "SampleIDs_18S.csv", "SampleIDs_COI.csv", and "geo dists adelaide.csv", and requires packages "vegan", "reshape2", and 'iNEXT".
MOTU Cleaning and Analyses.R
Unfiltered 18S MOTU Dataset
This dataset contains all of the 18S MOTUs obtained after the bioinformatics pipeline described in the manuscript. For each MOTU, there is taxonomic assignment information from two algorithms – SAP and Geneious BLAST – and the number of reads per MOTU per sample. Field descriptions are:
"OTU_ID" = unique MOTU identifier
"SAP" = taxonomic assignment from the SAP algorithm
"MetazoanPhylum_SAP" = Metazoan phylum assigned by SAP
"Level1_SAP"= higher level SAP assignment (e.g., Metazoan, Fungi, etc.)
"Geneious Accession" = the NCBI accession of the best BLAST hit
"Geneious1"= higher level BLAST assignment (e.g., Metazoan, Fungi, etc.)
“MetazoanPhylum_Geneious”= metazoan phylum of best BLAST hit
“Geneious2”…”Geneious6”= progressively lower-level taxonomic assignment of best BLAST hit
“Geneious_coverage” = Percent coverage of best BLAST hit
“Geneious_identity” = Percent identity of best BLAST hit
“Geneious_eval” = E-value of best BLAST hit
“Level1” = final higher-level categorization of the MOTU
“MetazoanPhylum” = final Metazoan phylum of the MOTU
Other fields: See the SampleIDs_18S for more information
Unfiltered_18S.csv
Unfiltered_COI
This dataset contains all of the COI MOTUs obtained after the bioinformatics pipeline described in the manuscript. For each MOTU, there is taxonomic assignment information from two algorithms – SAP and Geneious BLAST – and the number of reads per MOTU per sample. Field descriptions are:
"OTU_ID" = unique MOTU identifier
"SAP" = taxonomic assignment from the SAP algorithm
"MetazoanPhylum_SAP" = Metazoan phylum assigned by SAP
"Level1_SAP"= higher level SAP assignment (e.g., Metazoan, Fungi, etc.)
"Geneious Accession" = the NCBI accession of the best BLAST hit
"Geneious1"= higher level BLAST assignment (e.g., Metazoan, Fungi, etc.)
“MetazoanPhylum_Geneious”= metazoan phylum of best BLAST hit
“Geneious2”…”Geneious6”= progressively lower-level taxonomic assignment of best BLAST hit
“Geneious_coverage” = Percent coverage of best BLAST hit
“Geneious_identity” = Percent identity of best BLAST hit
“Geneious_eval” = E-value of best BLAST hit
“Level1” = final higher-level categorization of the MOTU
“MetazoanPhylum” = final Metazoan phylum of the MOTU
Other fields: See the SampleIDs_18S for more information
SampleIDs_18S
Dictionary linking samples to sites and ports. Also provides the 18S sequencing ID and bioinformatics pipeline ID for each sample.
SampleIDs_COI
Dictionary linking samples to sites and ports. Also provides the COI sequencing ID and bioinformatics pipeline ID for each sample.
Adelaide Site Over-Water Distances
Over-water distances in kilometers between the seven Adelaide sites.
geo dists adelaide.csv