We present an in silico approach for the reconstruction of complete mitochondrial genomes of non-model organisms directly from next-generation sequencing (NGS) data—mitochondrial baiting and iterative mapping (MITObim). The method is straightforward even if only (i) distantly related mitochondrial genomes or (ii) mitochondrial barcode sequences are available as starting-reference sequences or seeds, respectively. We demonstrate the efficiency of the approach in case studies using real NGS data sets of the two monogenean ectoparasites species Gyrodactylus thymalli and Gyrodactylus derjavinoides including their respective teleost hosts European grayling (Thymallus thymallus) and Rainbow trout (Oncorhynchus mykiss). MITObim appeared superior to existing tools in terms of accuracy, runtime and memory requirements and fully automatically recovered mitochondrial genomes exceeding 99.5% accuracy from total genomic DNA derived NGS data sets in <24 h using a standard desktop computer. The approach overcomes the limitations of traditional strategies for obtaining mitochondrial genomes for species with little or no mitochondrial sequence information at hand and represents a fast and highly efficient in silico alternative to laborious conventional strategies relying on initial long-range PCR. We furthermore demonstrate the applicability of MITObim for metagenomic/pooled data sets using simulated data. MITObim is an easy to use tool even for biologists with modest bioinformatics experience. The software is made available as open source pipeline under the MIT license at https://github.com/chrishah/MITObim.

Ian J. 2016. Environmental DNA metabarcoding of lake fish communities reflects long-term data from established survey methods. Molecular Ecology, 25 (13). 3101-3119. 10.1111/mec.13660 Contact CEH NORA team at noraceh@ceh.ac.ukThe NERC and CEH trademarks and logos ('the Trademarks') are registered trademarks of NERC in the UK and other countries, and may not be used without the prior written consent of the Trademark owner. Accepted ArticleThis article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/mec.13660This article is protected by copyright. All rights reserved.

Environmental DNA (eDNA) analysis is a rapid, cost‐effective, non‐invasive biodiversity monitoring tool which utilises DNA left behind in the environment by organisms for species detection. The method is used as a species‐specific survey tool for rare or invasive species across a broad range of ecosystems. Recently, eDNA and “metabarcoding” have been combined to describe whole communities rather than focusing on single target species. However, whether metabarcoding is as sensitive as targeted approaches for rare species detection remains to be evaluated. The great crested newt Triturus cristatus is a flagship pond species of international conservation concern and the first UK species to be routinely monitored using eDNA. We evaluate whether eDNA metabarcoding has comparable sensitivity to targeted real‐time quantitative PCR (qPCR) for T. cristatus detection. Extracted eDNA samples (N = 532) were screened for T. cristatus by qPCR and analysed for all vertebrate species using high‐throughput sequencing technology. With qPCR and a detection threshold of 1 of 12 positive qPCR replicates, newts were detected in 50% of ponds. Detection decreased to 32% when the threshold was increased to 4 of 12 positive qPCR replicates. With metabarcoding, newts were detected in 34% of ponds without a detection threshold, and in 28% of ponds when a threshold (0.028%) was applied. Therefore, qPCR provided greater detection than metabarcoding but metabarcoding detection with no threshold was equivalent to qPCR with a stringent detection threshold. The proportion of T. cristatus sequences in each sample was positively associated with the number of positive qPCR replicates (qPCR score) suggesting eDNA metabarcoding may be indicative of eDNA concentration. eDNA metabarcoding holds enormous potential for holistic biodiversity assessment and routine freshwater monitoring. We advocate this community approach to freshwater monitoring to guide management and conservation, whereby entire communities can be initially surveyed to best inform use of funding and time for species‐specific surveys.

Environmental DNA offers great potential as a biodiversity monitoring tool. Previous work has demonstrated that eDNA metabarcoding provides reliable information for lake fish monitoring, but important questions remain about temporal and spatial repeatability, which is critical for understanding the ecology of eDNA and developing effective sampling strategies. Here, we carried out comprehensive spatial sampling of England's largest lake, Windermere, during summer and winter to (1) examine repeatability of the method, (2) compare eDNA results with contemporary gill-net survey data, (3) test the hypothesis of greater spatial structure of eDNA in summer compared to winter due to differences in water mixing between seasons, and (4) compare the effectiveness of shore and offshore sampling for species detection. We find broad consistency between the results from three sampling events in terms of species detection and abundance, with eDNA detecting more species than established methods and being significantly correlated with rank abundance determined by long-term data. As predicted, spatial structure was much greater in the summer, reflecting less mixing of eDNA than in the winter. For example Arctic charr, a deepwater species, was only detected in deep, midlake samples in the summer, while littoral or benthic species such as minnow and stickleback were more frequently detected in shore samples. By contrast in winter, the eDNA of these species was more uniformly distributed. This has important implications for design of sampling campaigns, for example, deep-water species could be missed and littoral/benthic species overrepresented by focusing exclusively on shoreline samples collected in the summer. K E Y W O R D S eDNA, fish, lakes, metabarcoding, monitoring | 27 LAWSON HANDLEY Et AL.