Environmental Barcoding of Aquatic Invertebrates More about EBAI By Tove Eivindsen Freshwater ecosystems hold almost 6% of all described species supporting at least 100 000 species out of approximately 2 million. The European Commission’s Water Framework Directive (WFD, Directive 2000/60/EC) sets ambitious goals and state that all water bodies are to be classified, and included in a management plan. The EBAI project will develop and test new ways of monitoring freshwater invertebrates. Sampling localities The background knowledge of the faunal community composition is important if different sampling methods are to be appropriately compared. Therefore, the EBAI project does its fieldwork at two well-investigated localities, one stream and one lake ecosystem. Atna watershed at Skranglehaugan and Lake Jonsvatn near Trondheim The Atna River originates in the Rondane National Park in Central Norway. The river is one of the best documented Nordic freshwater ecosystems and since the initiation of FORSKREF in the 1980’s it has been a reference river system with yearly sampling of insect larvae. In addition, Torbjørn Ekrem and Elisabeth Stur performed an extensive weekly sampling with Malaise traps in the area in 2008. This material has been stored refrigerated since then and can be used to fill the gaps in the barcode library of aquatic insects. Lake Jonsvatn near Trondheim is moderately large lake (14 km2) with a multitude of different habitats and it is the main source for drinking water for the city of Trondheim. Substantial information is thus available with regards to the water quality and also to the bottom fauna in the lake. The NTNU University Museum has been sampling planktonic crustaceans in the lake quantitatively six times a year since 1983. Monitoring So far, monitoring of freshwater invertebrates has followed the practice where bottom of streams and lakes have been sampled using kick sampling and benthos sampling, respectively. From these samples, invertebrates are sorted based on morphological characters and identified to species, genus or family level. The sorting and identification are time consuming and the success of the identification relies on the taxonomic expertise of the investigator. While species level identification clearly adds necessary information and increases the explanatory potential for freshwater assessments, the majority of the aquatic insects are larval stages of Diptera that are challenging to identify to species level. Therefore, developing methodology that increases efficiency and power in species-level identifications in freshwater assessments is important. By expanding the taxonomic coverage in traditional monitoring programs one can increase the explanatory power without increasing the sample effort. This is the one of the main objectives of the EBAI project. DNA barcoding The expansion of taxonomic coverage in monitoring is achieved using DNA to identify the species. An approach called DNA barcoding employs sequence diversity in short segments of standardized gene regions to create a digital system for species recognition. For this purpose, The 648 bp segment of the mitochondrial cytochrome c oxidase subunit1 gene (COI) is used. The COI sequence of a given individual is permanent from birth to death, and thus, morphologically inseparable immature stages of the aquatic invertebrates can be identified to species level using DNA barcoding. DNA barcoding requires a high quality reference library to be effective. The reference library is a collection of barcodes from individuals that have been taxonomically identified to species level by an expert taxonomist. The newly collected barcodes are just strings of letters denoting the bases in the DNA strand (A, C, G, T). These need to be compared to the barcodes in the reference library in order to be identified to which species the barcodes belong to. A high quality DNA barcode library of Norwegian species is currently being developed as part of the national research infrastructure Norwegian Barcode of Life Network (NorBOL). DNA barcoding of environmental samples, so called environmental barcoding or metabarcoding, presents a new way to monitor biodiversity. In environmental barcoding, DNA is extracted directly from unsorted environmental samples. Thus, the extracts contain DNA from all organisms present in the samples and can be used to identify species and at least partially quantify the species composition in a particular environment. However, although this approach has been explored on various types of environmental samples, the effectiveness and accuracy of environmental barcoding on samples of aquatic invertebrates needs further testing in order to establish solid reliable and cost-effective protocols for freshwater monitoring. This is the main objective of the EBAI project.