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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)

1

2

3

5

4

6

7

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

0

20

40

60

80

100

Per Swab (n=28) Per Carcass (n=10)

% S

ucc

ess

Kill Wound Species ID Success (%)

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

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

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

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

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 %

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