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Applied eDNA metabarcoding is increasingly being considered as a tool to inform management decisions, regulations, or policy development. Because these downstream considerations are coming to the forefront of eDNA applications, optimizing workflow elements is essential to increasing standardization, efficiency, and competency of metabarcoding results. Reference DNA sequences are critical workflow elements that currently lack consistent approaches to generating, curating, or publishing. We present a complete mitochondrial genome and nuclear ribosomal DNA cistron reference DNA sequence library for 92% of the freshwater fish species of British Columbia, Canada. This resource is published as the Novel Applied eDNA Metabarcoding Reference Sequences (NAMERS) repository (https://namers.ca), a user-friendly and interactive website for specialists and non-specialists alike to explore and generate custom reference libraries for taxa and genes of interest. We demonstrate the power of NAMERS for optimization of applied eDNA metabarcoding study design by analyzing the number of primer mismatches and species resolution power of existing metabarcoding markers. NAMERS demonstrates that high quality curated genomic information is within a reasonable reach to meet the increasing demand for actionable eDNA metabarcoding applications. The framework used here incorporating the pillars of accuracy, completeness and accessibility can be applied for new iterations of other reference sequence databases to bring DNA-based monitoring into a new era.

Abstract Background Whole genomes are commonly assembled into a collection of scaffolds and often lack annotations of autosomes, sex chromosomes, and organelle genomes (i.e., mitochondrial and chloroplast). As these chromosome types differ in effective population size and can have highly disparate evolutionary histories, it is imperative to take this information into account when analysing genomic variation. Here we assessed the accuracy of four methods for identifying the homogametic sex chromosome in a small population using two whole genome sequences (WGS) and 133 RAD sequences of white-tailed eagles (Haliaeetus albicilla): i) difference in read depth per scaffold in a male and a female, ii) heterozygosity per scaffold in a male and a female, iii) mapping to the reference genome of a related species (chicken) with annotated sex chromosomes, and iv) analysis of SNP-loadings from a principal components analysis (PCA), based on the low-depth RADseq data. Results The best performing approach was the reference mapping (method iii), which identified 98.12% of the expected homogametic sex chromosome (Z). Read depth per scaffold (method i) identified 86.41% of the homogametic sex chromosome with few false positives. SNP-loading scores (method iv) identified 78.6% of the Z-chromosome and had a false positive discovery rate of more than 10%. Heterozygosity per scaffold (method ii) did not provide clear results due to a lack of diversity in both the Z and autosomal chromosomes, and potential interference from the heterogametic sex chromosome (W). The evaluation of these methods also revealed 10 Mb of putative PAR and gametologous regions. Conclusion Identification of the homogametic sex chromosome in a small population is best accomplished by reference mapping or examining differences in read depth between sexes.

Abstract Environmental DNA (eDNA) methods are providing tools for detecting invasive species in aquatic environments. Targeted qPCR assays applied to eDNA samples promise to overcome limitations of traditional methods, especially for early detection. The European green crab (Carcinus maenas) is considered one of the most successful invasive species globally due to the large range it has invaded and negative impacts on native species, marine habitats, and shellfish industries. We developed, laboratory‐validated, and field‐tested a specific qPCR assay for the detection of green crab from eDNA samples. We also show that the assay can detect green crab in bulk DNA extracted from plankton samples. Assay design, optimization, sensitivity, and specificity testing generally followed the validation pathway recommended by the World Organization for Animal Health for assays used to manage global aquatic animal health and infectious disease. Assay specificity was verified in silico and in vitro by laboratory testing 26 nontarget species, none of which showed potential for amplification. Assay sensitivity was appropriately high, with the limit of detection approaching two gene copies/μl. The assay was field‐tested on eDNA samples collected from filtered seawater at five sites on the Pacific coast of Canada known to harbor green crab based on historical monitoring data; green crab DNA was amplified from all sites. We also present early pilot field testing of the assay done on bulk DNA extracted from plankton samples from four sites from Australia, two sites with and two sites without reported records of green crab presence. Green crab was detected at both sites with known green crab records. Significant inhibition was recorded for some plankton samples but not for eDNA samples. This is the first qPCR assay for detection of European green crab, providing researchers and managers with a valuable new tool to aid early detection and ongoing monitoring.

The analysis of phylogeographic patterns has often been based on mitochondrial DNA variation, but recent analyses dealing with nuclear DNA have in some instances revealed mito-nuclear discordances and complex evolutionary histories. These enigmatic scenarios, which may involve stochastic lineage sorting, ancestral hybridization, past dispersal and secondary contacts, are increasingly scrutinized with a new generation of genomic tools such as RADseq, which also poses additional analytical challenges. Here, we revisited the previously inconclusive phylogeographic history, showing the mito-nuclear discordance of an endemic groundwater amphipod from Iceland, Crangonyx islandicus, which is the only metazoan known to have survived the Pleistocene beneath the glaciers. Previous studies based on three DNA markers documented a mitochondrial scenario with the main divergence occurring between populations in northern Iceland and an ITS scenario with the main divergence between the south and north. We used double digest restriction-site-associated DNA sequencing (ddRADseq) to clarify this mito-nuclear discordance by applying several statistical methods while estimating the sensitivity to different analytical approaches (data-type, differentiation indices and base call uncertainty). A majority of nuclear markers and methods support the ITS divergence. Nevertheless, a more complex scenario emerges, possibly involving introgression led by male-biased dispersal among northern locations or mitochondrial capture, which may have been further strengthened by natural selection.

As a part of a PhD project we are investigating the genomics of the current Icelandic white-tailed eagle population, in comparison with genomic data from other larger and outbred populations, and to historic samples from before the bottleneck in Iceland. We are examining the population structure, potential effects of small population size, signs of inbreeding and genomic selection. We present here the results of an analyzes of a bottleneck using the Whitetailed Eagles as a case study.