non-invasive genetic sampling of wildlifentgp.org.np/docs/workshops/stakeholders-workshop/... ·...
TRANSCRIPT
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Non-Invasive Genetic Sampling of Wildlife
Lisette Waits
Dept Fish and Wildlife
Laboratory for Ecological and Conservation Genetics
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Laboratory for Ecological And Conservation Genetics
Est. 1998
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Overview
• Sources
• Uses
• Examples
–Bears
–Wolves
–Snow leopards
• Tiger Genetics
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1992
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Saliva
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Benefits
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Detect/monitor rare species
J.
Witha
m
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Individual ID/Population estimation
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Diversity, gene flow, mating system
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Demographic Data Sex Ratio, Survivorship, Population Turnover
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Identify Predators
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Detecting diet items or pathogens in scat
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Two Types of DNA
Nucleus
Mitochondrion
Animal Cell
MtDNA
nDNA
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Overview
• Sources
• Uses
• Examples
–Bears
–Wolves
–Caribou predators
–Snow leopards
• Tiger Genetics
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Monitoring The Brown Bear in the Italian Alps through
Non-Invasive Genetic Sampling
Marta De Barba, Lisette Waits, Piero Genovesi, Ettore Randi
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Western Trentino
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Translocation Plan 1999-2002
10 bears from Slovenia
3 males, 7 females
Fitted with radiocollars
In 2003, bears no
longer radiocollared
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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
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Opportunistic Collection
• Trails during patrol
• Damage site evaluations
• Year round collection
• Throughout the bear range
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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)
1
2
3
5
4
6
7
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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
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No
immigrants
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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
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NPS Photo NPS Photo by Jim Peaco NPS Photo
NPS Photo by Bill Campbell
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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
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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
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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?
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Surveyed 488 predicted rendezvous sites
Collected ~1500 fecal and hair samples
Species ID success – 90%
Individual ID success – 50%
98 wolves detected
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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
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Molecular Determination of Caribou Calf Predators
By Matthew A. Mumma,
Lisette P. Waits, and Colleen E. Soulliere
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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
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Predator Species
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Predator Species
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Changing Predator Guild
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Predator Species Determination
• Kill site assessment performed to determine predator species
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Unable to Identify the Predator Species at 26% of Kills
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Methods • Collared/monitored
caribou calves
• Mortality Location
– Field assessment
Genetic Assessment
32 carcasses, 139 swabs
– Collection method
• Ethanol soaked swab
• Stored w/ silica desiccant
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Results
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0
20
40
60
80
100
Per Swab (n=28) Per Carcass (n=10)
% S
ucc
ess
Kill Wound Species ID Success (%)
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0
20
40
60
80
100
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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0
10
20
30
40
50
60
70
80
90
100
Per Swab (n=139) Per Carcass (n=32)
% S
ucc
ess
% Species ID Success
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0
10
20
30
40
50
60
70
80
90
100
Per Swab (n=47) Per Carcass (n=30)
% S
ucc
ess
% Individual ID Success
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0
10
20
30
40
50
60
70
80
90
100
Field Molecular
Sp
ecie
s (%
) Kills / Species (%)
Unknown
Bald Eagle
Red Fox
Lynx
Black Bear
Coyote
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0
10
20
30
40
50
60
70
80
90
100
Field Molecular
Sp
ecie
s (%
) Kills / Species (%)
Unknown
Bald Eagle
Red Fox
Lynx
Black Bear
Coyote25%
62.5 %
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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
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Non-invasive Monitoring of Snow Leopards McCarthy et al 2008 JWM 72:1826
• 3 study areas – 2 Kyrgyzstan and 1 China
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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
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Tiger Genetics
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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
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Systematics Luo et al 2004
• 4000 bp of mtDNA, 30 nDNA usat loci, MHC loci
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Systematics Luo et al 2004
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Species ID • Multiple mtDNA species specific primers designed
in India (Bhagavatula and Singh 2006, Mukherjee et al 2007)
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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
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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
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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?
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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?
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MtDNA diversity/Connectivity Sharma et al. 2010
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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
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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