1. Species detection using Environmental DNA from water samples 2012.10.25.

2. Biodiversity studies Species distribution Biogeography Conservation biology Ecology Problem: - Difficulty to detect a species, particular time periods or developmental stages Solution: - Detect the presence of a species using the DNA in the environment especially specific primers Position

3. Benefits Extraction of DNA from environmental samples: - Allows characterization of their micro-organisms - Provide information on extinct communities of macro organisms (eg: old sediments, permafrost and ice cores) - Unexplored potential about highly concentrated organisms in present-day Novel approach: based on the persistence of DNA in the environment Purpose: - To detect the presence of a species in fresh water, - To examine shether DNA fragments can be used for a reliable assessment of current species presence

4. American bullfrog( ): Rana catesbeiana (=Lithobates catesbeianus) 1. Controlled environments2. Natural field conditions invasive amphibian (high-quality census data) =>Reliable field validation American bullfrog: - Native to western North America - Introduced into ecosystems around the glove - One of the worlds most harmful invasive species

5. Materials and methods Controlled conditions - Tadpole in Aquarium with 3L of water - Natural alpine spring at 1000m sealevel, - 80km from the nearest bullfrog record. - 0, 1, 5, 10 tadpoles / aquarium Each density: 6 replicates, After 24 hrs, collected 15ml water sample from each aquarium

6. Low density: 3 ponds 1-2 adults, no reproduction High density: 3 ponds Dozens of adults, thousands of tadpoles Natural populations: Ponds: 1000-10000m 2 No detection: 3 ponds Never been detected, 30km from the nearest bullfrog record Immediately after collection * 1.5 ml sodium acetate 3M + 33ml absolute ethanol To recover precipitated DNA/cellular remains -> Centrifuge/ discard supernatant

7. PCR primers 5-TGCCAACGGAGCATCATTC-3 and 5- ATAAAGGTAGGAGCCGTAGT-3 :amplify a 79 bp segment of mitochondrial cyt-b, which is monomorphic in all 397 individuals analysed by population genetic studies covering the whole native and European range of the species ( Ficetola et al. 2008). -> Primers : -Specificity confirm-> Genbank -Try amplifying DNA of all other frog species living in France (genus Rana) 2 ind. from different sites per each species. -Each water sample: 3-5 amplification using the multi-tube approach PCR product of one pond was sequenced using the 454 pyrosequencing. Generalized mixed models: assuming a binomial error to compare the amplification rates among ponds with different bullfrog densities, fitted using lme4 in R

8. Results All 18 aquarium water samples: using selective primers PCR was successful. (0.3,1.7,3.3 tadpoles per L) - All PCR products were sequenced and corresponded perfectly to the published bullfrog cyt-b sequence. -674 fragments from one PCR product were sequenced using 454 pyrosequencing technology. -False negative: approximately 1.5% Using a multi-tube approach and ancient DNA precautions, which are suitable for analysing DNA that is degraded and/or at low concentrations

9. 22% 89% Average amplification success 0.37+/- 0.1 0.79+/- 0.08 0 - To ensure that the positive amplification is not due to artefacts -To ensure that the negative amplification is not due to chance -Generalized linear mixed model: significant Differences in amplification rates among ponds with differing densities of target species were significant (Average amplification success)

10. The way to use environmental DNA and Strengths - To ascertain species presence: discriminating between absence and presence even low density -To allow the reliable detection of secretive organisms in wetlands w/o direct observation -Answer to many situation where traditional census techniques give low-quality results -To quantify secretive harmful, invasive or threatened species -Assessment of distribution of rare threatened species(target of conservation plans) Disucssion

11. Several precautions Influence factor: the amount of DNA in environmental samples : volume of water, size and density of the organism and volume of secretions Duration time: difficult to evaluate how long DNA fragments persist in water (short DNA fragments can persist a long time under dry cold conditions… ) eg: 10000 year old dry cave sediments amplification(Willerslev et al. 2003) 400bp may persist up to 1 week at 18 in lake water(Matsui et al. 2001) New avenues for the study of biodiversity: DNA barcodes for identifying species from degraded DNA will be more applicable to more and more plant and animal species Massive sequencing techniques: To analyse PCR products generated with universal primers working on degraded substrates -> To make possible the assessment of the current biodiversity of macro-organisms from environmental samples