Preliminary Population Genetic Analysis of Spotted Turtles, Clemmys guttata, in Western New York.
Christine M. Binner1 Oceanprincess167@aol.com,
Amy McMillan1, Faculty Advisor, McMillam@buffalostate.edu; Kenneth Roblee2, Edward Standora1
1 Biology Department, State University of New York College at Buffalo, 2 New York State Department Of Environmental Conservation
Abstract
The spotted turtle (Clemmys guttata), a species of special concern in New York, is known to exist in fragmented populations in eastern North America. Historically, populations of Clemmys guttata were easily found due to large numbers but
these populations are declining in size. Western New York populations are particularly small and isolated. A recent reintroduction program was initiated to replace spotted turtles in appropriate habitat. This study uses population genetic
methods to explore the genetic diversity and relationships of spotted turtle populations in western New York. Genetic diversity is often used to determine population health; low genetic diversity may indicate inbreeding and isolation from
other populations. Two groups of turtles were sampled; a natural population from Niagara County (n = 10) and a reintroduced population in Erie County (n = 8). Ten polymorphic microsatellite loci were amplified and results analyzed with
GENEPOP. Our results suggest that within population genetic diversity in these groups was low and homozygosity was generally higher than expected. Significant population differentiation was detected between these populations (allelic
differentiation test, P < 0.05). Our long-term goals include using genetic methods to determine origins of the western NY spotted turtles and providing a baseline with which to understand the impacts of any reintroduction program in this area.
Materials and Methods
Background and Objectives
 Spotted turtle samples were collected from Oswego, Niagara, and Schenectady
Counties (NY), and Franklin County (PA). Blood samples were taken from the
hind leg with a small-bore syringe. Samples were stored in a phosphate buffered
saline solution and refrigerated until DNA was extracted.
Spotted turtles, Clemmys guttata, are found in the eastern portion of the United States and around
the Great Lakes. Populations of these turtles have been in steady decline throughout their range.
Loss of habitat, pollution causing a decline in water quality, and collecting for the pet trade are the
main reasons for recent declines in these turtles. C. guttata is Federally listed as endangered in
Ohio and Illinois and threatened in Maine and Vermont. They are protected in Massachusetts and
considered of special concern in Indiana and New York.
 DNA was extracted from blood samples using Qiagen DNeasy tissue kit.
The New York Department of Environmental Conservation has been interested in the distribution,
health, and size of C. guttata populations, particularly in western New York where their decline has
been dramatic. Recently, spotted turtles were reintroduced into Erie County and radio tracking
indicates these turtles were doing well in Fall of 2004. This study was conducted in order to better
understand the population dynamics of western New York C. guttata.
Spotted turtle distribution map
(http://www.dec.state.ny.us/website/dfw
mr/wildlife/endspec/sptufs.html).
The New York State Reptile and Amphibian Atlas
shows Spotted Turtle captures from 1990-1998
(http://www.dec.state.ny.us/website/dfwmr/wildlife/her
p/turtles.html). Circles indicate the counties
represented by spotted turtle samples in this study.
Oswego County turtles were reintroduced into Erie
County.
 Gels were visualized with a Kodak Digital Science Electrophoresis Documentation
System. Alleles were sized and scored by relative position of DNA ladder and of
bands within and between gels. Duplicates were run to maintain consistency of
calls between gels.
Locus
Niagara
(FIS)
Oswego
(FIS)
Schenectady Franklin
(FIS)
(FIS)
10
8
4
6
A18
5
3
4
4
A18
0.404
1
-0.333
0.100
A19
3
2
2
3
A19
-0.113
-0.091
-0.500
0.000
D16
7
4
4
4
D16
0.040
-0.448
0.100
-0.333
D28
6
5
5
5
D28
-0.059
0.623
0.478
0.478
Table 2: FIS values for each
locus by site. Positive FIS
values indicate a higher
number of homozygous
individuals than expected. NA
= no amplification. The final
row is the probability of
Hardy-Weinberg equilibrium.
Allelic Differentiation (P)
Locus
FST
Niagara vs
Oswego
All four
sites
A18
-0.092
0.615
0.467
A19
0.125
0.041
0.000
D16
-0.004
0.572
0.465
NA
D28
0.004
0.209
0.748
0.002
0.378
0.371
D90
2
3
3
2
D114
5
3
1
3
D114
0.143
0.459
NA
0.500
D90
D121
5
5
4
4
D121
-0.077
-0.256
-0.200
-0.263
D114
-0.029
0.803
0.406
D79
4
3
5
4
D79
-0.108
0.100
0.143
0.200
D121
-0.032
0.782
0.772
D88
3
3
4
2
D99
0.789
0.188
0.000
NA
D79
0.107
0.042
0.025
A32
3
2
2
2
A32
0.697
1
NA
1
D88
-0.065
0.501
0.114
Avg #
alleles
4.3
3.3
3.4
3.3
H-W
Probability
0.012
0.045
0.990
0.275
A32
0.153
0.004
0.042
Combined
0.020
0.031
0.158
Polyacrylamide gel of locus
D121 showing genotypes of
individual turtles.
Future Research Objectives
N
D90
homozygote
100 bp
 Data were analyzed with GENEPOP® (Raymond and Rousset, 1995)
Despite the small sample size in this study, these sites showed a relatively large number of alleles (Table 1). Hardy Weinberg expectations were not met in Niagara and Oswego county
turtles. This was partially driven by the relatively high number of homozygous individuals found (Table 2). The high level of homozygosity may suggest these turtles exist in relatively
small, inbred populations. The high homozygosity in the Oswego site, however, is probably because these turtles came from a breeder, who may have a small breeding stock. FST, a
measure of population differentiation (Wright 1978), was relatively low overall but three loci indicated moderate genetic differentiation (A19, A32 and D79; Table 3). A statistical test for
population differentiation suggests Niagara and Oswego County turtles represent different populations, again driven by the same three loci (Table 3). Population differentiation was not
calculated for all sites because of the small sample number.
Niagara Oswego Schenectady Franklin
heterozygote
 PCR product was electrophoresed on 8% native polyacrylamide gels, post staining
was done with ethidium bromide or SYBR® Gold soak.
Results and Conclusions
Table 1. Sample size (N) and
number of alleles for each
locus by population. Final
row is average number of
alleles over all 10 loci.
10 bp ladder
 20 µl PCR reactions: 10 μM each forward and reverse primer, 0.25 mM each
dNTP, 1.5 mM MgCl2, 1 U Taq DNA polymerase, and 50 ng template DNA in a
 Thermal regime: 2 min at 94°C; 34 cycles of 45 sec denaturing at 94°C, 45 sec
annealing at 58°C, 1:30 min extension at 72°C; 5 min final extension at 72°C; hold
at 4°C
1. Determine the genetic diversity of spotted turtles in western New York. Genetic diversity is often
an indicator of population health. Low genetic diversity suggests small population size,
inbreeding, or low migration between populations.
3. Determine relatedness between a natural population (Niagara County) of spotted turtles and a
captively bred and reintroduced population (originating from Oswego County).
Sampling a spotted turtle.
buffer containing 20 mM Tris-HCl and 50 mM KCl (pH 8.0).
The specific objectives of this study were to:
2. Determine genetic relatedness of spotted turtle populations in western New York and other areas.
The distribution map of spotted turtles (below) shows that the western NY population is relatively
isolated from other areas.
 Nineteen microsatellite primer pairs developed for bog turtles (Glyptemys
muhlenbergii; King and Julian 2004) were tested for amplification and
polymorphism using polyacrylamide gels. Ten were chosen for this study.
-0.296
-0.125
Table 3: Niagara vs. Oswego
County FST and allelic
differentiation test Niagara vs.
Oswego sites and all four sites.
Locus names are after King and
Julian, 2004.
1. Collect more spotted turtle
samples from known
populations and conduct genetic
analysis on them to provide
larger sample size for more
accurate genetic analysis of
populations.
2. Obtain samples from western
Pennsylvania and more from
eastern New York to determine
the origin of western New York
C. guttata populations.
ACKNOWLEDGEMENTS
We would like to thank the USGS Leetown Science Center (Dr. Timothy King’s lab) for microsatellite markers.
We would like to thank Travis Morse, Susan Standora, and Brandon Wojcik for help in the field and laboratory.
Schenectady County samples were provided by Karl Parker, NYDEC. Franklin County samples were provided
by Tim Maret at Shippensburg University. Funding for this project was provided by the State University of New
York College at Buffalo and the NY State Department of Environmental Conservation.
Literature Cited
King, T.L. and S.E. Julian (2004) Conservation of microsatellite DNA flanking sequence across 13 Emydid
genera assayed with novel bog turtle (Glyptemys muhlenbergii) loci. Conservation Genetics 5: 719-725
Raymond, M. and F. Rousset (1995) GENEPOP Version 3.4. http://wbiomed.curtin.edu.au/genepop
Wright, S. (1978) Evolution and the genetics of populations 4: