Representativeness of Environmental DNA Metabarcoding signal in River Fish Biodiversity Assessment Pont D., Civade R., Valentini A., T. Dejean et P. Taberlet
Ability of edna Metabarcoding * to describe fish species communities in river? Sensus Taberlet et al. 2012. : Multispecific approach using environmental samples DNA released in water Species occurrence Comparison of classical eletrofishing and edna metabarcoding approach Spatial representativeness of edna metabarcoding samples Species relative abundance edna metabarcoding: a semi-quantitative method? Civade Raphaël. 2016. A new indicator for biodiversity : the environmental DNA (edna). Application to fish communities. (Co-Direction D. Pont & P. Taberlet). PhD. Paris. Civade R., T. Dejean, A. Valentini, N. Roset, J-C. Raymond, A. Bonin, P. Taberlet & D. Pont (2016). Spatial representativeness of environmental DNA metabarcoding signal for fish biodiversity assessment in a natural freshwater system. Plos One, 30, 1:19.
edna metabarcoding pipeline for aquatic studies (Valentini et al., 2016,Molecular Biology) Sampling - In situ tangential filtration (capsule 1µm porosity) - Disposable sterile tubing for each sample 1 filtration = 36 mn ~ 45 litres Several filtrations / site Avoid potential contaminations DNA extraction Controlled atmosphere Separated room Disposable equipment DNA amplification Universal primers 12S rrna region List of taxa Nb of reads / taxon Species sequences reference database Taxonomic assignation of sequences 50 PCR cycles 12 PCR replicates / sample 3Negative controls DNA sequencing Illumina MiSeq sequencer OBITools package
Comparison edna versus classical electrofishing Tier River - 0.2 m3/s Max. depth: 0.3 m River width: 6.5 m Complete electrofishing (3 pass with stop nets) Ardèche River - 65 m3/s Max. depth: 4 m River width: 74 m Partial electrofishing (along the shoreline) 1990-2014 : 13-17 electrofishing sessions / site 2014 : edna sampling ( 11-12 filtrations /site) Rhône River by-passed section -100 m3/s Max. depth: 10m River width: 95 m
2014: edna sampling versus one simultaneous electrofishing session 35 common species only edna only electrofishing 30 + 10 sp + 16 sp 25 20 15 + 4 sp 10 5 0 Rhône Ardèche Tier S1+S2 With edna sampling richness is increased by 45% to 96% Additional species: true presence or false positive?
edna sampling (2014) versus all previous electrofishing sessions 35 30 25 1 sp 3 sp 5 sp 20 2 sp 15 10 5 0 Rhône Ardèche Tier S1+S2 common species only edna only electrofishing 85 to 90 % of the total number of species are caught in common by the 2methods when considering all the previous electrofishing sessions (13-17) against only one edna session
Spatial representativeness of environmental DNA metabarcoding samples Is edna method able to describe spatial pattern of fish communities in rivers? What represents an edna sample in space? - comparable to a classical eletrofishing fished area? - few kilometers upstream? - upstream catchment?
Test on a lake-river longitudinal gradient Number of filtrations Lake: 3 Impoundment: 2 Tier S0: 3 Tier S1: 6 Tier S2: 6 edna water samples Lentic habitat Hydropower plant Impoundment Im2 Im1 Aiguebelette Lake Slow flowing section S2 S0 Dam S1 Tier River (residual flow) Fast Flowing section
Correspondance Analysis C. lavaretus E. lucius C. carpio T. tinca S. erythrophtalmus A. alburnus Lake & Impoundment S. fluviatilis A. Brama P. fluviatilis S. cephalus R. rutilus L. gibbosus S0 Leuciscus sp. Gobio sp. S1 S. trutta S2 P. Phoxinus B. barbatula O. mykiss 1st Axis (49.5%) B. barbus A. bipunctatus Cottus sp. Lampetra spp edna samples (2014) 2014 Others years Electrofishing Gillnets fishing Carassius sp. Micropterus sp. Salvelinus spp Lake Im1 Im2 S0 S1 S2 Tier river 0.2 1.3 dam 1.7 3.6 6.9 km Distance to the lake
Coregonus lavaretus European whitefish Im1 Im2 S0 S1 S2 Lake Tier river 0.2 1.3 dam 1.7 3.6 6.9 km Distance to the lake Presence / Absence Detection distance of typical lake species downstream in the river of around 2 km Discrimination between two fish assemblages from sites only distant 3 k downstream
edna metabarcoding: a semi-quantitative method?
Number of reads (edna) vs Relative species abondance in electrofishing samples Comparison on 2 rivers edna vs electrofishing (boating) Rhône River: R Pearson = 0.68 Ardèche River : R Pearson = 0.87 Species edna electrofishing Alburnus alburnus 19.57% 21.99% Squalius cephalus 7.53% 25.99% Rutilus rutilus 10.56% 13.38% Gobio sp. 6.55% 12.63% Barbus barbus 10.38% 1.38% Lepomis gibbosus 4.84% 5.53% Perca fluviatilis 6.17% 2.31% Blicca bjoerkna 2.73% 4.23% Gymnocephalus cernuus 4.80% 0.50% Silurus glanis 4.81% 0.37% Cyprinidae 3.58% 0.92% Rhodeus sericeus 2.56% 1.91% Cottus 3.71% 0.28% Abramis brama 3.16% 0.15% Alburnoides bipunctatus 1.66% 0.74% Ameiurus (melas) 0.11% 1.99% Barbatula 0.36% 1.37% Esox lucius 1.44% 0.26% Sander lucioperca 1.27% 0.38% Leuciscus 0.64% 0.63% Tinca tinca 0.46% 0.75% Salaria fluviatilis 0.71% 0.09% Cyprinus carpio 0.60% 0.17% Squalius laietanus 0.75% 0.00% Pseudorasbora parva 0.17% 0.54% Phoxinus phoxinus 0.16% 0.35% Scardinius erythrophthalmus 0.11% 0.37% Salmo trutta 0.18% 0.19% Anguilla anguilla 0.06% 0.28% Gasterosteus aculeatus 0.09% 0.12% > 10% DNAqua.net EU Cost Action 2 to Essen 10% March 6-9 Rhône River: % reads (edna) vs % individuals caught 0.5 to 2% 0.1 to 0.5% < 0.1%
Conclusion Efficiency of edna metabarcoding to describe fish diversity in rivers Spatial representativeness of edna within a large range of streams and rivers (water velocity, temperature, conductivity, ph) Increase the species reference database (in progress) Perspectives Short term: Application for biodiversity studies rare-patrimonial species, invasives species, global warming impact Mid term: Bio-indication (species relative abundance)