BackgroundThis study reports progress in assembling a DNA barcode reference library for Ephemeroptera, Plecoptera, and Trichoptera ("EPTs") from a Canadian subarctic site, which is the focus of a comprehensive biodiversity inventory using DNA barcoding. These three groups of aquatic insects exhibit a moderate level of species diversity, making them ideal for testing the feasibility of DNA barcoding for routine biotic surveys. We explore the correlation between the morphological species delineations, DNA barcode-based haplotype clusters delimited by a sequence threshold (2%), and a threshold-free approach to biodiversity quantification--phylogenetic diversity.ResultsA DNA barcode reference library is built for 112 EPT species for the focal region, consisting of 2277 COI sequences. Close correspondence was found between EPT morphospecies and haplotype clusters as designated using a standard threshold value. Similarly, the shapes of taxon accumulation curves based upon haplotype clusters were very similar to those generated using phylogenetic diversity accumulation curves, but were much more computationally efficient.ConclusionThe results of this study will facilitate other lines of research on northern EPTs and also bode well for rapidly conducting initial biodiversity assessments in unknown EPT faunas.

DNA barcoding of aquatic macroinvertebrates holds much promise as a tool for taxonomic research and for providing the reliable identifications needed for water quality assessment programs. A prerequisite for identification using barcodes is a reliable reference library. We gathered 4165 sequences from the barcode region of the mitochondrial cytochrome c oxidase subunit I gene representing 264 nominal and 90 provisional species of mayflies (Insecta: Ephemeroptera) from Canada, Mexico, and the United States. No species shared barcode sequences and all can be identified with barcodes with the possible exception of some Caenis. Minimum interspecific distances ranged from 0.3–24.7% (mean: 12.5%), while the average intraspecific divergence was 1.97%. The latter value was inflated by the presence of very high divergences in some taxa. In fact, nearly 20% of the species included two or three haplotype clusters showing greater than 5.0% sequence divergence and some values are as high as 26.7%. Many of the species with high divergences are polyphyletic and likely represent species complexes. Indeed, many of these polyphyletic species have numerous synonyms and individuals in some barcode clusters show morphological attributes characteristic of the synonymized species. In light of our findings, it is imperative that type or topotype specimens be sequenced to correctly associate barcode clusters with morphological species concepts and to determine the status of currently synonymized species.

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DNA barcoding is an effective approach for species identification and for discovery of new and/or cryptic species. Sanger sequencing technology is the method of choice for obtaining standard 650 bp cytochrome c oxidase subunit I (COI) barcodes. However, DNA degradation/fragmentation makes it difficult to obtain a full-length barcode from old specimens. Mini-barcodes of 130 bp from the standard barcode region have been shown to be effective for accurate identification in many animal groups and may be readily obtained from museum samples. Here we demonstrate the application of an alternative sequencing technology, the four-enzymes single-specimen pyrosequencing, in rapid, cost-effective mini-barcode analysis. We were able to generate sequences of up to 100 bp from mini-barcode fragments of COI in 135 fresh and 50 old Lepidoptera specimens (ranging from 53–97 year-old). The sequences obtained using pyrosequencing were of high quality and we were able to robustly match all the tested pyro-sequenced samples to their respective Sanger-sequenced standard barcode sequences, where available. Simplicity of the protocol and instrumentation coupled with higher speed and lower cost per sequence than Sanger sequencing makes this approach potentially useful in efforts to link standard barcode sequences from unidentified specimens to known museum specimens with only short DNA fragments.

This work is intended as a general and concise overview of Ephemeroptera biology, diversity, and services provided to humans and other parts of our global array of freshwater and terrestrial ecosystems. The Ephemeroptera, or mayflies, are a small but diverse order of amphinotic insects associated with liquid freshwater worldwide. They are nearly cosmopolitan, except for Antarctica and some very remote islands. The existence of the subimago stage is unique among extant insects. Though the winged stages do not have functional mouthparts or digestive systems, the larval, or nymphal, stages have a variety of feeding approaches—including, but not limited to, collector-gatherers, filterers, scrapers, and active predators—with each supported by a diversity of morphological and behavioral adaptations. Mayflies provide direct and indirect services to humans and other parts of both freshwater and terrestrial ecosystems. In terms of cultural services, they have provided inspiration to musicians, poets, and other writers, as well as being the namesakes of various water- and aircraft. They are commemorated by festivals worldwide. Mayflies are especially important to fishing. Mayflies contribute to the provisioning services of ecosystems in that they are utilized as food by human cultures worldwide (having one of the highest protein contents of any edible insect), as laboratory organisms, and as a potential source of antitumor molecules. They provide regulatory services through their cleaning of freshwater. They provide many essential supporting services for ecosystems such as bioturbation, bioirrigation, decomposition, nutrition for many kinds of non-human animals, nutrient cycling and spiraling in freshwaters, nutrient cycling between aquatic and terrestrial systems, habitat for other organisms, and serving as indicators of ecosystem health. About 20% of mayfly species worldwide might have a threatened conservation status due to influences from pollution, invasive alien species, habitat loss and degradation, and climate change. Even mitigation of negative influences has benefits and tradeoffs, as, in several cases, sustainable energy production negatively impacts mayflies.