Presentation on theme: "Environmental DNA ‘eDNA’"— Presentation transcript:
1Environmental DNA ‘eDNA’ Karen Mock & Torrey RodgersUtah State University
2Noninvasive Genetic Sampling eDNA is a noninvasive technique
3Species Identification Not individual IDPresence/Absence DataAssay designed to ID a particular species or group of speciesRequires comparison with known reference sequencesOften a mitochondrial genome target sequencee.g. CTATACGGGTCNGCCCTATTAGTCATTTTATCAT CATATTTCCAAGGTATTCTATTCCCTTCACCCAA CCGACTAATTAATAACCGCCTAGTCTCACTACAC AATGACTAGTACAACTAAATCAAAACAAATACG GAAeDNA so far is just a species ID tool, not a tool for identifying individualsOften mtDNA gene is used because mtDNA has a high copy number and there is a large amount of public sequence data available for mt genes.
4Reference sequences are available, e. g Reference sequences are available, e.g. from the Barcode of Life project and GenBank
5eDNAis DNA that can be extracted from environmental samples without capturing or even seeing the target organism.DNA may be cellular or extracellularSubstrate may be water, snow, soil, vegetation…Target may be single species, a small number of species, or everything present (meta-barcoding).
6How it WorksField LabUses quantitative PCR (qPCR) to detect certain species; can be more sensitive and more specific than conventional PCR
7Taxon-specific PCR primers and probe to detect a particular DNA sequence in a mixture of DNA molecules from different sources.
10eDNA applications Species distribution especially for rare, endangered, or cryptic speciesEarly detection of invasive speciesRelative density
11Species distributions Especially useful for rare, endangered, or cryptic speciesSpeciesLocationCitationRocky Mountain tailed frogIdaho giant salamandersIdahoGoldberg et al. 2011Pilliod et al. 2013Eastern HellbenderIndiana, Missouri, Ohio, Kentucky, North CarolinaOlsen et al. 2012Santas et al. 2013Spear et al. 2015Bull TroutMontanaWilcox et al. 2013Brook TroutMassachusettsJane et al. 2014Great Crested NewtUnited KingdomBiggs et al. 2015Weather LoachDenmarkSigsgaard et al. 2015CetaceansFoote et al. 2014Spadefoot Toad, Great Crested Newt, Weather Loach, White-faced darter,Eurasian otterThroughout EuropeThomsen et al. 2002Chinook SalmonUpper Columbia River BasinLaramie et al. 2015Can be used in conjunction with occupancy modelling to calculate probability of detection (if sampling is repeated). Presents absence data can be used to model species distributions and habitat preferences
12Invasive Species Detection Early detection of invasive species before they become establishedeDNA may be able to detect invasive species prior to traditional methodsSpeciesLocationCitationBighead Carp (Asian carp)Great lakes regionJerde et al. 2011, 2013Mahon et al. 2013Burmese PythonFloridaPiaggio et al. 2014American Bull FrogFranceDejean et al. 2012New Zealand Mud SnailIdahoGoldberg et al. 2013Zebra MusselMichiganEgan et al. 2013BluegillJapanTakahara et al. 2013Red Swamp CrayfishTreguier et al. 2014Didymo (Rock Snot)New Zealand,Western USCary et al. 2007Mock, Rodgers, OlsenUnpublished
13Relative DensityThe quantity of eDNA (from qPCR) in a sample should correlate with species biomass and thus densityFig. 2 Combined scatter plots of eDNA shedding rate against biomass of fish in tanks.Klymus et al. 2015
14Relative Density eDNA vs. traditional kicknet surveys Pilliod et al. 2013
15Relative DensityIn fish, eDNA shedding rates may vary with diet (Klymus et al. 2015)More research needed to determine eDNA shedding rates, across species and environmental conditionsSuch factors may bias the link between biomass and relative densityMore empirical lab and field studies needed compare eDNA quantities and relative density
16Sampling Considerations eDNA persistence/degradationMovement of eDNA in lotic systemsCross contamination and false positives
17eDNA persistence/degradation In fresh water, eDNA degrades, and is no-longer detectable, in days or weeks (at longest 58 days, Strickler et al. 2015)Thus eDNA detection should be indicative of contemporary species presenceHow long eDNA persists in the environment after it is shedFig.1. Time-dependant changes in eDNA concentration afterfish removal . Maruyama et al 2014
18eDNA persistence/degradation eDNA degradation is influenced by temperature, UV and pHEnvironments favorable for microbe activity increase rate of eDNA degradation (Strickler et al. 2015)eDNA may persist much longer, (from months to hundreds of years) in aquatic sediments (Turner at al. 2015).
19Movement of eDNA in lotic systems In lotic systems, is eDNA detection indicative of local presence, or presence upstream instead?Controlled experiment with caged trouteDNA still detectable at 239m from cage siteLow flow 4-7 L/seDNA might become undetectable down stream due to dilution, settling of eDNA and cells, degradation over time as moving downstream. Likely dependent on flowHigh flows >10 L/sJane et al. 2014
20Movement of eDNA in lotic systems In two invertebrate species, eDNA was detectable up to nearly 12 kilometersfrom the source
21Cross contaminationIt is very important to avoid cross contamination in the field and lab to avoid false positivesCareful handling of samples (changing gloves between samples)Bleach decontamination of collection equipment between sitesInclusion of field negative controls to monitor for contamination
22Novel eDNA applications Moving eDNA beyond aquatic environments to terrestrial environmentseDNA from browsed vegetationeDNA from drinking watereDNA from snow trackseDNA from invertebrate “samplers”eDNA from salt licks?Any other ideas?So far most eDNA research has been focused on aquatics (for vertebrates at least)
23Used eDNA from saliva on browsed twigs to identify ungulate species (moose, roe deer, fallow deer and red deer)Found the DNA could be amplified up to 12 weeks laterCould be used to study browsing habits of different species without direct observationCould be used to detect rare ungulates
24eDNA from drinking water eDNA sampling of watering holes could be used to detect species that drank thereWe were able to recover and sequence coyote eDNA from drinking water at the Coyote research facilityCould be especially useful to survey desert speciese.g. kit foxes in S. Utah
25eDNA from Snow Tracks eDNA extracted from snow tracks of Polar bear
26Used mammalian DNA in blood collected from leeches 2012Used mammalian DNA in blood collected from leeches21 of 25 leeches collected in Vietnam contained mammal DNA from 6 different speciesThis included the recently discovered (in 1997) and extremely rare Truong Son munjtacThis technique could have great promise for surveying mammals in areas where leaches are common such as SE Asia
27Used DNA from carrion flies, flies which feed on dead animals and wounds on live animals and scat Of 115 flies collected in Africa and Madagascar, 46 contained mammal DNA from 20 different speciesIncluded small and large mammals, canopy living mammals and bats
28Carrion flies exist world-wide, and are easy to trap This tool could greatly improve upon surveys of mammal diversity
29Advantages of eDNADoes not require sighting or handling of target speciesDoes not require taxonomic expertise or animal handling skillsGenerally has higher detection probabilities and greater sensitivity than traditional sampling techniquesIs often (but not always) more economical than traditional sampling techniquesOnce an assay is established, samples are very inexpensive to run (e.g. ~$20 per sample for multiples of 48 samples)Multiple assays (different species queries) can be run on the same samples
30Limitations of eDNA No detection of individuals Primer/probe designs are species-specific and require considerable up-front investmentPilot work is necessary to understand variance in detectability for different species and systems
31Recent PublicationsThe journal Biological Conservation has an upcoming special issue dedicated to eDNA:
32Labs USU Molecular Ecology Lab assay development, pilot testing service lab billinglinkage to graduate/ undergrad projectsKaren MockPisces Molecularprivate company in Boulder, COexcellent choice for high throughput work for established assays