Summary Aqueous environmental DNA (eDNA) is an emerging efficient non‐invasive tool for species inventory studies. To maximize performance of downstream quantitative PCR (qPCR) and next‐generation sequencing (NGS) applications, quality and quantity of the starting material is crucial, calling for optimized capture, storage and extraction techniques of eDNA. Previous comparative studies for eDNA capture/storage have tested precipitation and ‘open’ filters. However, practical ‘enclosed’ filters which reduce unnecessary handling have not been included. Here, we fill this gap by comparing a filter capsule (Sterivex‐GP polyethersulfone, pore size 0·22 μm, hereafter called SX) with commonly used methods. Our experimental set‐up, covering altogether 41 treatments combining capture by precipitation or filtration with different preservation techniques and storage times, sampled one single lake (and a fish‐free control pond). We selected documented capture methods that have successfully targeted a wide range of fauna. The eDNA was extracted using an optimized protocol modified from the DNeasy® Blood & Tissue kit (Qiagen). We measured total eDNA concentrations and Cq‐values (cycles used for DNA quantification by qPCR) to target specific mtDNA cytochrome b (cyt b) sequences in two local keystone fish species. SX yielded higher amounts of total eDNA along with lower Cq‐values than polycarbonate track‐etched filters (PCTE), glass fibre filters (GF) or ethanol precipitation (EP). SX also generated lower Cq‐values than cellulose nitrate filters (CN) for one of the target species. DNA integrity of SX samples did not decrease significantly after 2 weeks of storage in contrast to GF and PCTE. Adding preservative before storage improved SX results. In conclusion, we recommend SX filters (originally designed for filtering micro‐organisms) as an efficient capture method for sampling macrobial eDNA. Ethanol or Longmire's buffer preservation of SX immediately after filtration is recommended. Preserved SX capsules may be stored at room temperature for at least 2 weeks without significant degradation. Reduced handling and less exposure to outside stress compared with other filters may contribute to better eDNA results. SX capsules are easily transported and enable eDNA sampling in remote and harsh field conditions as samples can be filtered/preserved on site.
DNA obtained from environmental samples such as sediments, ice or water (environmental DNA, eDNA), represents an important source of information on past and present biodiversity. It has revealed an ancient forest in Greenland, extended by several thousand years the survival dates for mainland woolly mammoth in Alaska, and pushed back the dates for spruce survival in Scandinavian ice-free refugia during the last glaciation. More recently, eDNA was used to uncover the past 50 000 years of vegetation history in the Arctic, revealing massive vegetation turnover at the Pleistocene/Holocene transition, with implications for the extinction of megafauna. Furthermore, eDNA can reflect the biodiversity of extant flora and fauna, both qualitatively and quantitatively, allowing detection of rare species. As such, trace studies of plant and vertebrate DNA in the environment have revolutionized our knowledge of biogeography. However, the approach remains marred by biases related to DNA behaviour in environmental settings, incomplete reference databases and false positive results due to contamination. We provide a review of the field.
Summary1. The keystone piscivore northern pike Esox lucius can structure fish communities, and models predicting pike-focused connectivity will be important for management of many waters. 2. We explored the ability of pike to colonize upstream locations and modelled presence-absence in lakes based on landscape features derived from maps. An upstream connectivity model (UC model) was generated using data from 87 lakes. We validated the UC model with retrospective whole-lake experiments involving introductions ( n = 49) and extirpations (by rotenone) of pike ( n = 96), as well as with the natural distribution of pike in lakes ( n = 1365) within 26 drainage basin networks in northern Sweden.3. The UC model predicted the incidence of pike in lakes with stream-connections with 95·4% accuracy, based mainly on a single variable, S V5max , that measures the minimum distance found between 5 m elevation intervals ( = maximum stream slope) along watercourses from nearest downstream source of potential immigrants. Recolonizations of pike in rotenone lakes generated a near-identical classification tree, as in the UC model. The classification accuracy of pike presence in the external validation procedure ranged from 88·7 to 98·7% between different drainage basins. Predictions of pike absence were not as accurate, due possibly to undetected introductions, but still lead to 86·6% overall accuracy of the external validation. Most lakes lacking pike, but misclassified as having pike based on low S V5max , were isolated from downstream sources of pike by subsurface streamflow through bouldery areas (SSB). 4. Synthesis and applications . The variable S V5max provide managers with a tool for revealing the location and severity of natural dispersal barriers to pike (and logically also barriers to other species with equivalent or less dispersal capacity). Because presented models only require map-based information, and have high predictive power, they may have the potential to be of fundamental use in predicting distribution of freshwater fish. These predictions may provide the means for prioritizing in risk assessment and control programmes to combat pike invasions, as well as contribute to determining a reference state of species incidence in specific lakes. Our results also point towards a possibility that, even where stream slope is low, long-term effective barriers may be designed that mimic natural SSB.
We determine if lacustrine salmonids show large-scale patterns of coexistence with the keystone predator northern pike (Esox lucius) and test an approach to predict fish communities using coexistence rules set in the context of three hierarchical filters that a species must pass to be present. The mutually exclusive species distribution patterns that we detected among 1029 lakes were repeatedly verified from results of whole-lake interventions with rotenone and introductions. Essentially, pike did not coexist with self-sustaining salmonid populations in lakes. High connectivity to pike (derived from maps) largely predicts the absence of lacustrine salmonids. Our analysis strongly suggests that pike prevented selfsustaining populations of brown trout (Salmo trutta), Arctic char (Salvelinus alpinus), and brook trout (Salvelinus fontinalis) in lakes. High connectivity to pike resulted in nonsalmonid lake fish communities, most often including both European perch (Perca fluviatilis) and roach (Rutilus rutilus). Our analysis suggests that if pike were not present in many boreal lakes where they now dwell, salmonid fish assemblages would prevail, a sharp contrast from the present pike-driven homogenized state with mainly nonsalmonid fish communities.Résumé : Nous déterminons si les salmonidés lacustres possèdent des patrons de coexistence à grande échelle avec le brochet du nord (Esox lucius), leur prédateur le plus important, et nous testons une méthode pour prédire les communautés de poissons à l'aide de règles de coexistence qui comprennent trois filtres hiérarchiques qu'une espèce doit traverser pour être présente. Les patrons de répartition des espèces mutuellement exclusives que nous avons détectés dans les 1029 lacs se vérifient à répétition lors d'interventions à la roténone ou d'introductions à l'échelle des lacs entiers. En somme, les brochets ne se retrouvent pas avec des populations autosuffisantes de salmonidés dans les lacs. Une forte connectivité avec le brochet (déduite à partir de cartes géographiques) permet la plupart du temps de prédire l'absence de salmonidés lacustres. Notre analyse indique clairement que le brochet empêche l'établissement de populations autosuffisantes de truites brunes (Salmo trutta), d'ombles chevaliers (Salvelinus alpinus) et d'ombles de fontaine (Salvelinus fontinalis) dans les lacs. Une connectivité au brochet produit des communautés de poissons lacustres sans salmonidés, mais contenant souvent à la fois la perche (Perca fluviatilis) et le gardon (Rutilus rutilus). Notre analyse laisse croire que si le brochet était absent de plusieurs lacs boréaux où il habite présentement, il y aurait prédominance de peuplements de poissons salmonidés -en contraste marqué avec l'état d'homogénéité actuel des communautés de poissons non salmonidés, causé par le brochet.[Traduit par la Rédaction]
This study of 193 boreal lakes of northern Sweden suggests a long-term detrimental impact of introduced brook trout (Salvelinus fontinalis) on brown trout (Salmo trutta) populations. Thirteen of 65 (20%) brown trout populations in lakes exposed to brook trout went extinct, whereas the extinction rate in unexposed lakes was significantly lower (2%). We verified other studies that indicate that altitude strongly affects the distribution of the two species; brown trout populations in our higher altitude lakes were more sensitive to impact from brook trout. In 28 lakes above 285 m, 12 trout populations exposed to brook trout went extinct, while only one population became extinct in 37 lakes below 285 m. No effects of other environmental factors were detected (e.g., water chemistry, stocking of rainbow trout (Oncorhynchus mykiss), fish species community assembly, migration barriers, or lake morphometry on brown trout extinction). The time lag between the first record of brook trout introduction and subsequent extinction of brown trout was two decades on average (maximum 70 years). Even though further stocking of brook trout has been stopped, our analysis suggest that existing sympatric populations may continue to pose an extinction threat to brown trout.
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Sound environmental management decisions - in accordance with the EU WFD for aquatic ecosystems – mainly depend on reliable species presence- and distribution- data. Here we present a workflow from sampling strategies to results and decision making using eDNA metabarcoding analyses for fish, amphibians, and mussels from habitat to landscape scales with focus on sampling strategies for "big data" in marine and freshwater ecosystems in Sweden. The project LifeDNAquatic highlights a solid eDNA pipeline and comparison of methods, which cover field planning and the entire pipeline generating data for Species Distribution Models (SDMs). Intense sampling over a large river catchment highlights previoulsy unanswered questionsand and provides insights to a priori settings for sampling strategies to retrieve "big data". The results provide novel insights to DNA distribution in the environment, seasonal and spatial changes in eDNA composition, and validation of data.
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