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Non-Invasive Genetic Sampling of Wildlife
Lisette Waits
Dept Fish and Wildlife
Laboratory for Ecological and Conservation Genetics
Laboratory for Ecological And Conservation Genetics
Est. 1998
Overview
• Sources
• Uses
• Examples
–Bears
–Wolves
–Snow leopards
• Tiger Genetics
1992
Saliva
Benefits
Detect/monitor rare species
J.
Witha
m
Individual ID/Population estimation
Diversity, gene flow, mating system
Demographic Data Sex Ratio, Survivorship, Population Turnover
Identify Predators
Detecting diet items or pathogens in scat
Two Types of DNA
Nucleus
Mitochondrion
Animal Cell
MtDNA
nDNA
Overview
• Sources
• Uses
• Examples
–Bears
–Wolves
–Caribou predators
–Snow leopards
• Tiger Genetics
Monitoring The Brown Bear in the Italian Alps through
Non-Invasive Genetic Sampling
Marta De Barba, Lisette Waits, Piero Genovesi, Ettore Randi
Western Trentino
Translocation Plan 1999-2002
10 bears from Slovenia
3 males, 7 females
Fitted with radiocollars
In 2003, bears no
longer radiocollared
3 Sampling approaches (DeBarba et al. 2010 J. Appl Ecol)
Systematic Collection
1. Baited Hair Trapping Grid
2. Transects for Sign Survey
Opportunistic
3. Patrols/Damage sites
Grid
Traps
fixed
active May-Aug
active Aug-Oct
PNAB
Opportunistic Collection
• Trails during patrol
• Damage site evaluations
• Year round collection
• Throughout the bear range
Laboratory Genetic Methods
• DNA extraction (Qiagen kit, room designated to low quantity/quality DNA)
• Species ID – mtDNA fragment analysis
• Individual ID at 10 usat loci – 3 – 7 replicates/sample
• Sex ID (Ennis and
Gallagher,1994)
• Parentage Analyses (Exclusion Test)
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NGS monitoring 2002 – 2008 DeBarba et al 2010 Mol Ecol 19:3938
• 26,000 km2 found 2781 samples
• 9 founders to > 27 individuals
• lambda = 1.17–1.19
• Stage specific mortality rates
• Sex ratio
• Change in genetic diversity
• Effective population size
• Generation time
• Reconstruct pedigree
No
immigrants
Evaluating the potential of noninvasive genetic sampling for
long-term monitoring of gray wolves (Canis lupus) in Idaho
Jennifer Stenglein, Curt Mack, Dave Ausband, Mike Mitchell, Pete Zager,
Steve Nadeau and Lisette Waits
NPS Photo NPS Photo by Jim Peaco NPS Photo
NPS Photo by Bill Campbell
Objective
Evaluate the potential of
noninvasive genetic
sampling (NGS) for long-
term monitoring of gray
wolves in Idaho
www.firstpeople.us
Stenglein et al 2010
JWM 74:1050
Study Area
• 11,335 km2 area of central Idaho
• Good radio-telemetry data
• Low density = 1-3 packs
• High density = 4-7 packs
Compare field-based
surveys with NGS surveys
Research Questions
INDIVIDUAL WOLVES
WOLF PACKS
STUDY AREA
• Did the sample come from a wolf?
• Which wolf?
• Detection?
• Pack count?
• Mating system?
• Minimum count?
• Population count?
• Density?
Surveyed 488 predicted rendezvous sites
Collected ~1500 fecal and hair samples
Species ID success – 90%
Individual ID success – 50%
98 wolves detected
Summary
INDIVIDUAL WOLVES
WOLF PACKS
STUDY AREA
• Identify samples from wolves
• Individual ID
• Detect packs
• Pack structure/pedigrees
• Obtain accurate pack counts
• Get a min. count
• Estimate population size
• Compare densities
WE CAN…
NGS is valuable new tool for monitoring
Expanded and applied 2009 - 2011
Molecular Determination of Caribou Calf Predators
By Matthew A. Mumma,
Lisette P. Waits, and Colleen E. Soulliere
0
10,000
20,000
30,000
40,000
50,000
60,000
70,000
80,000
90,000
100,000
1995 2000 2005
Car
ibo
u A
bu
nd
ance
Newfoundland Caribou
Population
Population~95,000
Population~34,000
199
7
200
2
200
7
Predator Species
Predator Species
Changing Predator Guild
Predator Species Determination
• Kill site assessment performed to determine predator species
Unable to Identify the Predator Species at 26% of Kills
Methods • Collared/monitored
caribou calves
• Mortality Location
– Field assessment
Genetic Assessment
32 carcasses, 139 swabs
– Collection method
• Ethanol soaked swab
• Stored w/ silica desiccant
Results
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20
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Per Swab (n=28) Per Carcass (n=10)
% S
ucc
ess
Kill Wound Species ID Success (%)
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20
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Per Swab (n=28) Per Carcass (n=10)
% S
ucc
ess
Kill Wound Species ID Success (%)
30% Black Bear
70% Coyote
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10
20
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90
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Per Swab (n=139) Per Carcass (n=32)
% S
ucc
ess
% Species ID Success
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10
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Per Swab (n=47) Per Carcass (n=30)
% S
ucc
ess
% Individual ID Success
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Field Molecular
Sp
ecie
s (%
) Kills / Species (%)
Unknown
Bald Eagle
Red Fox
Lynx
Black Bear
Coyote
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Field Molecular
Sp
ecie
s (%
) Kills / Species (%)
Unknown
Bald Eagle
Red Fox
Lynx
Black Bear
Coyote25%
62.5 %
Conclusions • Valuable technique – need multiple swabs
• Effective and complementary to field methods
• Coyotes and black bears are the primary predators
• Many different individuals
• Limited scavenging
Non-invasive Monitoring of Snow Leopards McCarthy et al 2008 JWM 72:1826
• 3 study areas – 2 Kyrgyzstan and 1 China
Non-invasive Monitoring of Snow Leopards McCarthy et al 2008 JWM 72:1826
• Camera trapping (20 -24 stations – 7 months, 1000 – 1200 trap nights)
• 1 (S), 6 (J), 13 (T) leopard
• 1 (S), 5 (J), 4 (T) individuals
• Fecal DNA sampling
• 15 (S), 19 (J), 19 (T) leopard
• 3 (S), 5 (J), 9 (T) individuals
Tiger Genetics
Non-invasive Monitoring of Tigers Mondol et al 2009 Biol Cons 142:2350
• Bandipur National Park 671 km2
• Camera trapping and NGS of scat
• 1246 camera nights – 35 photos, 29 individuals
• NGS - 58 tiger and 4 leopard scat detected
• NGS - 38 successful for individual ID
• Camera count – 29 NGS count - 26
• Camera estimate and NGS estimate the same:
66 ± 13 tigers
Systematics Luo et al 2004
• 4000 bp of mtDNA, 30 nDNA usat loci, MHC loci
Systematics Luo et al 2004
Species ID • Multiple mtDNA species specific primers designed
in India (Bhagavatula and Singh 2006, Mukherjee et al 2007)
Individual ID
• nDNA microsatellite loci needed
• 14 microsatellite loci from Bengal tigers (Sharma et al 2008)
• 30 others from domestic cat and other (sub) species
• Only need 5 – 10 to identify individuals
• Example
Locus 1 Locus 2 Locus 3 Locus 4 Locus 5
Sample1 76/78 100/102 92/96 120/120 84/88
Sample2 76/78 100/102 92/96 120/120 84/88
Sample3 72/76 100/102 90/92 120/124 90/90
Sample4 72/76 100/102 90/92 120/124 90/90
Sex ID
• 2 primer sets that amplify X and Y chromosomes (Pilgrim et al 2005)
• Zinc-finger – 161 and 164 bp male, 164 female
• Amelogein – 194 and 216 male, 216 female
• Also Felid specific Y primer
Genetic Diversity
• Important for individual fitness
• Important for population viability
• Problems with inbreeding depression
• What are the levels of genetic diversity in each study area in Nepal?
• How do they compare to each other? How do they compare to other tiger populations?
Gene Flow/Connectivity
• Movement of individuals/genes
• Important for maintaining genetic variation
• Is there dispersal/gene flow among tigers in different protected areas in Nepal?
• Is there dispersal/gene flow among tigers in Nepal and tigers in India?
• Do tigers in Nepal represent a ESU or MU?
MtDNA diversity/Connectivity Sharma et al. 2010
Forensics
• Database of genotypes from tigers from each protected area
– Poached individual – Check database – individual or closely related individual
• Species and individual ID of livestock kills or damages
Summary
• NGS methods have much to contribute to the ecology, conservation and monitoring of tigers
• First study of tiger genetics in Nepal
• Establish a baseline for the future
• Need for international collaboration and standardization of methods