Genetic distinctiveness and population size trends of a Genetic distinctiveness and population size trends of a peripheral metapopulation of the southern two-lined peripheral metapopulation of the southern two-lined salamander (salamander (Eurycea cirrigeraEurycea cirrigera) in Will County, Illinois) in Will County, Illinois

Maria BoyleMaria Boyle11, Eric L. Peters, Eric L. Peters11,,Timothy J. BellTimothy J. Bell11, and Olga Tsyusko, and Olga Tsyusko22

11Department of Biological Sciences, Chicago State UniversityDepartment of Biological Sciences, Chicago State University22Department of Plant and Soil Sciences, University of KentuckyDepartment of Plant and Soil Sciences, University of Kentucky

3 August 20103 August 2010

Southern two-linedSouthern two-linedsalamander,salamander,Eurycea cirrigeraEurycea cirrigera

Member of the FamilyMember of the FamilyPlethodontidae, part of a subgroup known as brook Plethodontidae, part of a subgroup known as brook salamanders that are lungless as adults. Some species are salamanders that are lungless as adults. Some species are troglodytic and/or neotenic.troglodytic and/or neotenic.

Forest-dependent, requiring cool water and rocky streams free Forest-dependent, requiring cool water and rocky streams free of predatory fishes. Live under rocks, logs and other debris in of predatory fishes. Live under rocks, logs and other debris in streams.streams.

Courtship occurs in fall. Eggs are laid in winter or spring.Courtship occurs in fall. Eggs are laid in winter or spring.

Larvae emerge in mid-late spring. In northern parts of U.S., Larvae emerge in mid-late spring. In northern parts of U.S., larvae metamorphose in their second year.larvae metamorphose in their second year.

WC

AC

Illinois localities (as of 1990)www.inhs.uiuc.edu/cbd/herpdist/species/

eu_cirrige.htmlMierzwa K. 1989. Bull. Chicago Herpetol.

Soc.24:61-9.

Redrawn from Conant et al.Redrawn from Conant et al.1991. 1991. A Field Guide to A Field Guide to ReptilesReptilesand Amphibians of Eastern and Amphibians of Eastern and Central North Americaand Central North America. . 33rdrd Ed. Ed.

KRSP

WC

Distribution of the Distribution of the ““Eurycea cirrigeraEurycea cirrigera Complex Complex””

KRSP

Image ©2005, DigitalGlobe, EarthsatImage ©2005, DigitalGlobe, Earthsat

Habitat Fragmentation at the KRSPHabitat Fragmentation at the KRSP

Survey Localities in the KRSPSurvey Localities in the KRSP

South Side of Kankakee River:South Side of Kankakee River:Sites 113-2 and 113-4Sites 113-2 and 113-4

North side of Kankakee River:North side of Kankakee River:Sites 102-2, 102-3, and 102-5Sites 102-2, 102-3, and 102-5

E. cirrigeraE. cirrigera Population Sampling Population SamplingSamples collected July-August 2003.Samples collected July-August 2003.• Transects measured and search time recorded for Transects measured and search time recorded for

comparison with VIS encounter rates.comparison with VIS encounter rates.

Sites were visited three times. Salamanders were hand-Sites were visited three times. Salamanders were hand-collected and marked by clipping toes with collected and marked by clipping toes with microdissection scissors:microdissection scissors:• Permit restriction: onePermit restriction: one

clip/foot, maximumclip/foot, maximumtwo clips/individual.two clips/individual.

• Recaptures marked on adjacent limb.Recaptures marked on adjacent limb.

• Toe and 1-2 mm tail clips retained for DNA analysis.Toe and 1-2 mm tail clips retained for DNA analysis.

Data AnalysesData AnalysesMark-recapture data were analyzed using Triple-catch Mark-recapture data were analyzed using Triple-catch (modified Jolly-Seber), Bailey’s, and Chapman’s (modified Jolly-Seber), Bailey’s, and Chapman’s methods.methods.

Genetic data were analyzed using GENPOP and Genetic data were analyzed using GENPOP and Mantel’s test calculatorMantel’s test calculator..

FFisis: describes inbreeding-like effects within : describes inbreeding-like effects within

subpopulationssubpopulations

FFstst: correlation measurement of the genetic difference : correlation measurement of the genetic difference

between pairs of populations.between pairs of populations.

Mantel correlation coefficient: calculates the Mantel correlation coefficient: calculates the correlation between correlation between FFstst and geographical distance.and geographical distance.

Probability tests conducted using Fisher’s Exact Test.Probability tests conducted using Fisher’s Exact Test.

ProcedureProcedurePCR - candidate primers PCR - candidate primers

(all DNA samples)(all DNA samples)Run products on Run products on

electrophoretic gels with electrophoretic gels with standardstandard

View gels on Fluor-S View gels on Fluor-S imagerimager

Evaluate PCR products Evaluate PCR products andand

determine the lengths determine the lengths (bp)(bp)

of allelesof alleles

Coordinate input data Coordinate input data sets for sets for

use in GENEPOP and use in GENEPOP and MantelMantel’’ss

test calculatortest calculator

Statistical analyses:Statistical analyses:• Evaluate genetic difference Evaluate genetic difference

((FFstst) among the three state ) among the three state populationspopulations

• Correlate genetic difference Correlate genetic difference with geographical distancewith geographical distance

• Evaluate fine scale (<10 km) Evaluate fine scale (<10 km) differences at KRSP & WCdifferences at KRSP & WC

Collect, isolateCollect, isolateand quantify DNAand quantify DNA

Amplify DNA via PCRAmplify DNA via PCRInitially testing allInitially testing all

loci (primers)loci (primers)with randomly chosen with randomly chosen

samplessamples

0.00010.0001

0.00100.0010

0.01000.0100

0.10000.1000

1.00001.0000

102-2102-2 102-3102-3 102-5102-5

2009200920082008

2007200720062006

20052005200420042003200320022002200120012000200019991999199819981997199719961996

E. cirrigeraE. cirrigera Population Densities Population DensitiesA

du

lt E

ncou

nte

r R

ate

(In

div

idu

als

/m/o

bs/

min

)A

du

lt E

ncou

nte

r R

ate

(In

div

idu

als

/m/o

bs/

min

)

E. cirrigeraE. cirrigera Population Densities Population Densities

00

0.0010.001

0.0020.002

0.0030.003

0.0040.004

102-2102-2 102-3102-3 102-5102-5 113-2113-2 113-4113-4

LocationLocation

Mea

n (

SE

) C

aptu

re R

ate

Mea

n (

SE

) C

aptu

re R

ate

(No.

of

adu

lts/

m/m

in)

(No.

of

adu

lts/

m/m

in)

0.00010.0001

0.00100.0010

0.01000.0100

0.10000.1000

1.00001.0000

102-2102-2 102-3102-3 102-5102-5

2009200920082008

2007200720062006

20052005200420042003200320022002200120012000200019991999199819981997199719961996

E. cirrigeraE. cirrigera Population Densities Population DensitiesA

du

lt E

ncou

nte

r R

ate

(In

div

idu

als

/m/o

bs/

min

)A

du

lt E

ncou

nte

r R

ate

(In

div

idu

als

/m/o

bs/

min

)

113-2, -4113-2, -4

Population Density EstimatesPopulation Density Estimates

Site (transect Site (transect length)length)

Number of adults/mNumber of adults/m

Triple-Triple-CatchCatch Bailey’sBailey’s Chapman’sChapman’s

102-2 (70 m)102-2 (70 m) 0.400.40 0.310.31 0.500.50

102-3 (90 m)102-3 (90 m) 1.181.18 1.131.13 1.151.15

102-5 (100 m)102-5 (100 m) 0.580.58 1.101.10 1.161.16

113-4 (50 m)113-4 (50 m) 0.180.18 0.570.57 0.600.60

ResultsResultsSeven of 13 microsatellite loci developed for Seven of 13 microsatellite loci developed for Plethodon Plethodon cinereuscinereus were also found in were also found in E. cirrigeraE. cirrigera..• Five these seven loci were variable and were used in this Five these seven loci were variable and were used in this

study.study.• The total number of alleles detected ranged from 8 to 12.The total number of alleles detected ranged from 8 to 12.

Both KRSP and WC had a greater percentage of average Both KRSP and WC had a greater percentage of average heterozygosity across loci than WCheterozygosity across loci than WC• Percent of polymorphic loci was 40% for KRSP (Percent of polymorphic loci was 40% for KRSP (““isolatedisolated””

population), 13% for WC (peripheral population)population), 13% for WC (peripheral population)

Both KRSP and WC populations showed significant Both KRSP and WC populations showed significant departures from HWE. AC population did not.departures from HWE. AC population did not.

FFisis (describes inbreeding-like effects w/in subpopulations) (describes inbreeding-like effects w/in subpopulations)• FFisis values were consistently < 0.values were consistently < 0.• No evidence of a deficiency of heterozygotes in any of the No evidence of a deficiency of heterozygotes in any of the

subpopulations.subpopulations.

00

0.20.2

0.40.4

0.60.6

0.80.8

11

0.10.1 11 1010 100100 10001000

Distance between populations (km)Distance between populations (km)

FFstst/(

1 –

/(1

– FF

stst) )

yy = 0.186 + 0.166 log = 0.186 + 0.166 log xxFF1,341,34 = 17.51; = 17.51; PP = 0.0002 = 0.0002 rr22 = 0.34 = 0.34

Genetic Difference and Geographical DistanceGenetic Difference and Geographical Distance

Is the Kankakee River a barrier to gene flow?Is the Kankakee River a barrier to gene flow?

00

0.20.2

0.40.4

0.60.6

0.80.8

0.10.1 11 1010

WCWCKRSPKRSP

Distance between populations (km)Distance between populations (km)

FFstst/(

1 –

/(1

– FF

stst) )

yy = 0.158 + 0.350 log = 0.158 + 0.350 log xxFF1,111,11 = 5.919; = 5.919; PP = 0.0332 = 0.0332 rr22 = 0.35 = 0.35

Genetic Difference and Geographical DistanceGenetic Difference and Geographical Distance

yy = 0.094 + 0.195 log = 0.094 + 0.195 log xxFF1,31,3 = 75.58; = 75.58; PP = 0.003 = 0.003 rr22 = 0.96 = 0.96

FFstst/(

1 –

/(1

– FF

stst) )

00

0.10.1

0.20.2

0.30.3

0.40.4

0.50.5

0.60.6

0.70.7

11 1010 100100 10001000

Mean Distance (km)Mean Distance (km)

WC-1 + WC-2 & WC-3WC-1 + WC-2 & WC-3

KRSP N & SKRSP N & S

KRSP & WCKRSP & WC

KRSP KRSP & AC& AC

WC & ACWC & AC

WCWC

ACAC

KRSPKRSP

Genetic Difference and Geographical DistanceGenetic Difference and Geographical Distance

ResultsResultsFFstst values were mostly very significantly greater than zero values were mostly very significantly greater than zero

((PP < 0.0001); indicating that, to varying extents, the state < 0.0001); indicating that, to varying extents, the state populations were genetically different from one another.populations were genetically different from one another.• In pair-wise comparisons among the statewide populations, In pair-wise comparisons among the statewide populations,

94.7% were significantly different.94.7% were significantly different.

• Within the KRSP, 80% of theWithin the KRSP, 80% of thepair-wise comparisons werepair-wise comparisons weresignificantly different. Overall,significantly different. Overall,greater differences were seengreater differences were seenamong sampling sites on oppositeamong sampling sites on oppositesides of the river compared withsides of the river compared withsites on the same side of the river.sites on the same side of the river.

• Two population pairs located in close proximity (102-2 & 102-3 Two population pairs located in close proximity (102-2 & 102-3 and 102-3 and 113-2) without obvious physical barriers were not and 102-3 and 113-2) without obvious physical barriers were not significantly different.significantly different.

Population Genetic Studies of Amphibians Population Genetic Studies of Amphibians Using Using FFstst as a Measure of Genetic Difference as a Measure of Genetic Difference

SpeciesSpecies DistanceDistance FFstst No. of lociNo. of loci

Ambystoma maculatumAmbystoma maculatum(spotted salamander)(spotted salamander)11 10–650 km 10–650 km 0.08-0.140.08-0.14 1515

Rana luteiventrisRana luteiventris(Columbia spotted frog)(Columbia spotted frog)22 Montana/IdahoMontana/Idaho 0.008-0.5080.008-0.508 66

Rana sylvaticaRana sylvatica(wood frog)(wood frog)33 0.05–5.5 km0.05–5.5 km 0-0.0590-0.059 66

Litoria aureaLitoria aurea(bell frog)(bell frog)44 1–1000 km          1–1000 km           0.034-0.4540.034-0.454 44

Eurycea cirrigeraEurycea cirrigera(southern two-lined salamander)(southern two-lined salamander) 1–1032 km1–1032 km 0-0.5000-0.500 55

11Julian et al. (2003)     Julian et al. (2003)     33Newman and Squire (2001)Newman and Squire (2001)

22Funk et al. (2005)       Funk et al. (2005)       44Burns et al. (2004)Burns et al. (2004)

AcknowledgementsAcknowledgements

• Michelle Boyle, Angela Boyle and Tim Thompson (collection assistance)Michelle Boyle, Angela Boyle and Tim Thompson (collection assistance)• IL & IN Departments of Natural Resources (permitting)IL & IN Departments of Natural Resources (permitting)• David Mauger – Will County Forest Preserve District (KRSP habitat David Mauger – Will County Forest Preserve District (KRSP habitat

information and collection assistance)information and collection assistance)• Paul Cabe and Lisa Connors – Washington and Lee University (primers)Paul Cabe and Lisa Connors – Washington and Lee University (primers)• Ache Gana, Kevin Swier, and Mark Erhart – Chicago State University Ache Gana, Kevin Swier, and Mark Erhart – Chicago State University

(electrophoresis assistance)(electrophoresis assistance)• Tony Mills – Savannah River Ecology Lab (collection assistance)Tony Mills – Savannah River Ecology Lab (collection assistance)• Steve Harper – Savannah River Ecology Lab (GIS assistance)Steve Harper – Savannah River Ecology Lab (GIS assistance)

webs.csu.edu/~epeters/KRSP.ppswebs.csu.edu/~epeters/KRSP.ppse-peters@csu.edue-peters@csu.edu