Developing a standard operating procedure for species identifications of dragonfly larvae using DNA barcoding: A contribution of the Dragonfly Mercury Project

Nelson, Sarah; Little, Megan; James, Karen; Marion, Michael; Greig, Hamish

doi:10.36967/nrr-2288457

Identification of larval dragonflies can be challenging even for experts, especially when larvae are in their earlier life stages (Tennessen 2019). DNA barcoding provides a promising new approach to species identification, where the DNA sequences of difficult-to-identify larval dragonflies are matched to a public, global DNA database based on adult dragonflies that are more often studied and collected, and more easily identified with formal taxonomy. The outcome is a species identification inferred solely from its DNA. Here we used DNA barcoding to identify species of individual dragonfly larvae that were collected as part of the Dragonfly Mercury Project (DMP), a citizen engagement initiative that enables long-term monitoring and research documenting mercury contamination in national parks and other protected lands nationwide. The DMP is currently managed by the National Park Service (NPS) and US Geological Survey (USGS), with collaborative engagement from the Appalachian Mountain Club (AMC) and Dartmouth College. Over the past 10 years, the project has assessed mercury concentrations in dragonfly larvae in more than 500 waterbodies across more than 100 U.S. national parks and other federal, state, and local protected areas. Citizen scientists collect dragonfly larvae from freshwaters in park units around the U.S., under supervision of park or partner staff, who may or may not have scientific backgrounds. Our interpretation of mercury concentrations determined in the DMP is hindered by the potentially confounding influence of dragonfly species identity. Specifically, there are important differences among species in three key aspects that influence mercury bioaccumulation: 1) diet, which directly controls the magnification of mercury with increasing trophic level; 2) the length of aquatic life stage, which influences the lifetime accumulation of mercury; and 3) habitat preference. Traditionally, identification to the genus or species level relies on a skilled, expert taxonomist who uses dichotomous keys and other relevant literature to visually identify specimens. In this project, we identified dragonfly larvae to the finest taxonomic level possible (genus or species) in 2013–2014, with visually-based identification by a taxonomist in 2013 and DNA barcoding in 2014. We developed DNA barcoding methodology and produced an SOP (Standard Operating Procedure) to allow the process to be replicated in other studies. Here we present results of these analyses and the detailed SOP for use in future work. In brief: ● We identified 61 unique dragonfly species, representing the six major dragonfly families in North America, from a sample set of 401 individual dragonfly larvae (Figure E-1), collected from 84 distinct freshwater sites. ● Visual identification resulted in 100% identification to genus, and further 59% identification to species. For DNA barcoding, of the 54% of samples that produced a DNA sequence, 87% were identified to species. Thus, both methods produced similar overall percentages of species identities; visual identification was more likely to produce identification to at least the genus level for all samples, but DNA barcoding had a greater success rate moving from genus- to species-level identification when DNA sequences were produced. ● The Standard Operating Procedure (Little et al. 2021) can be used and refined as the field of DNA barcoding progresses and associated DNA databases are expanded, to identify new specimens. This method was promising as 97–100% of the DNA-based species identities were corroborated by visual identifications.

Developing a standard operating procedure for species identifications of dragonfly larvae using DNA barcoding: A contribution of the Dragonfly Mercury Project

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