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1 No place left to hide Tracking animals via their DNA Arjen de Groot Animal Ecology, Alterra Wageningen UR Kennisnetwerk Milieu, 27 september2013

2 Contents 1. Making the invisible visible 2. Genetic approaches for population ecology The Dutch otter population 3. Species identification via DNA barcoding A wolf in the Netherlands? 4. (e)dna-based species inventories Early detection of invasive crayfish Screening for soil diversity 5. Conclusions

3 1. Making the invisible visible: why? Conventional animal ecology often involves visual and manual contact: Observations Capturing of animals But what to do in case the study species: Occurs in extremely low densities Is covered by water, soil or vegetation Hides in unreachable places Has a nocturnal and/or solitary life style

4 1. Making the invisible visible: why? Many smart tricks available: Camera traps Telemetry Track searching Extracting animals from their environment But this results in: High effort and costs Disturbance Often anecdotic / incomplete data

5 1. Making the invisible visible: how? Nowadays, DNA-based analyses provide excellent alternatives: Succesful extraction of small amounts of DNA from indirect evidence : Faeces, hairs, feathers, egg shells, etc., left in the field Environmental samples that contain traces of (e)dna Increasing amounts of data, reducing costs Simultanous information on many parameters But extraction of low quality and quantity DNA requires: Specialized protocols and laboratory facilities Proper method validation and replication to detect false information

6 2. Population ecology: the Dutch otters 1989: Eurasian otter (Lutra lutra) goes extinct in the Netherlands 2002: Start of reintroduction programme in Wieden/Weerribben In combination with intensive monitoring

7 Dental status

8 2. Population ecology: the Dutch otters DNA extraction Spraints (faeces) Genetic characterization: - Variation at 15 DNA fragments - Stepwise procedure (bad samples discarded) replicates per marker Traffic casualties Consensus profile per spraint Kuiters et al. (2011) Alterra rapport 2262; Koelewijn et al. (2010) Cons. genetics

9 2. Population ecology: population dynamics Koelewijn et al. 2010, Conservation Genetics

10 2. Population ecology: pedigree Female parent Male parent

11 Number of identified individuals 2. Population ecology: population expansion 84 Released New recruits Cumm. nr. of releases Koelewijn et al. 2010; De Groot et al /13

12 2. Population ecology: spatial structure De Groot et al. 2013

13 2. Population ecology: spatial structure Stefan Vriend, 2013

14 3. Species identification Identifying animal traces observed in the field: which animal did they come from? Does a hair or scat sample prove the presence of a new or uncommon species? Saliva analysis on carcasses: which predator was responsible for the kill? Holzwarth & Erlbruch, 1994; Uitgeverij Vries-Brouwers

15 3. Species identification: DNA barcoding Species identification based on the variation in the sequence of a standard DNA fragment (=DNA barcode) 1. Selecting a fragment that varies among, but not within species 2. Create a reference database with all potentially encountered species 3. Obtain the DNA barcode of the unknown sample 3.Bepaal de DNA barcode van het onbekende monster 4. Compare with reference set and find best match 1.Keuze van fragment met variatie tussen, maar niet binnen soorten 2.Maak een referentieset met DNA barcodes van alle mogelijk aan te treffen soorten Database Unknown specimen DNA extract DNA barcode

16 3. Species identification: the Dutch wolf 4 juli 2013: a young female wolf was found dead near Luttelgeest Pure wolf, no hybrid Related to eastern European populations Still not sure how she got here Photo: Joke van der Linde Source: ANP / Natuurmonumenten

17 4. (e)dna-based species inventories Demand for proper monitoring of specific aquatic species: Species conservation policies Early detection of invasive species to prevent damage to ecosystem and/or infrastructure Kaderrichtlijn Water (KRW) But low densities: conventional detection difficult New alternative: environmental DNA (edna) free DNA that animals leave behind in their environment Detection via a simple water sample Presence/absence assays for specific species

18 4. Environmental DNA: state of the art First results are promising: Ficetola et al. 2008: higher detection rates based on edna Methods are now available for several aquatic species More research needed: more species & better validation under varying circumstances Lithobates catesbeianus Lodge et al. (2012) Thompson et al. (2012)

19 4. Environmental DNA: crayfish 8 Example: Invasive crayfish in the Netherlands Damage to ecosystems: endemic European crayfish (Astacus astacus) riparian / aquatic vegetation Damage to infrastructure: burrowing Procambarus clarkii (Red swamp crayfish) Roessink et al. 2010, Alterra-report 2052

20 4. Environmental DNA: invasive crayfish Development of a detection method for Procambarus clarkii: 1. Marker development finding a discriminating barcode fragment 2. Optimizing qpcr (quantitative PCR) protocols 3. Testing sensitivity what is the detection limit? 4. Tests on other crustaceae (Astacus, Gammarus, etc) false positives? 5. Tests with field samples with known presence / absence Promising results: succesful detection in ditch systems Work in progress: Finetuning of method for P. clarkii Development of a more general assay for all invasive crayfish in NL Future: towards quantitative methods for population size estimation

21 3. (e)dna-based species inventories DNA metabarcoding = determining total biodiversity for multiple taxonomic groups via high-throughput DNA barcoding Goal: faster processing of high numbers of samples Currently under development (e.g. EcoFINDERS) Reference Requires large reference datasets Requires complex bioinformatics What about relative abundances? Soil sample DNA extraction Mass sequencing Bioinformatics Species composition

22 Conclusions DNA-based methods ready to be applied in ecological studies: New techniques allow the use of traces of DNA left behind in the environment no need for visual or manual contact no need for disturbance allows new types of questions to be answered DNA sample can simultanously yield many types of information Methods become cheaper and simpler But working with low quality samples requires: Proper validation of the methods Proper sample replication both in the field and in the lab De Groot & Laros, 2013, Vakblad NBL

23 Thanks to: Hugh Jansman Ivo Laros Jan Bovenschen Dennis Lammertsma Geert Groot Bruinderink Loek Kuiters Ivo Roessink Hans Peter Koelewijn Questions: Arjen de Groot: / Hugh Jansman: /

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