ARTICLES

Chelonian Conservation and Biology, 2008, 7(1): 3–11� 2008 Chelonian Research Foundation

Migration, Gene Flow, and Genetic Diversity Within and Among Iowa Populations ofOrnate Box Turtles (Terrapene ornata ornata)

REBECCA J. RICHTSMEIER1, NEIL P. BERNSTEIN

2, JAMES W. DEMASTES1, AND

ROBERT W. BLACK3

1Department of Biology, University of Northern Iowa, Cedar Falls, Iowa 50614 USA[bjrichtsmeier@hotmail.com; jim.demastes@uni.edu];

2Department of Biology, Mount Mercy College, 1330 Elmhurst Drive NE, Cedar Rapids, Iowa 52402 USA[nbernstein@mtmercy.edu; correspondnig author];

3Department of Biology, Cornell College, Mount Vernon, Iowa 52302 USA [bblack@cornellcollege.edu]

ABSTRACT. – Like many fragmented reptile populations, the ornate box turtle (Terrapene ornataornata) is located in isolated, often small, populations in eastern Iowa. If populations are to remainviable, genetic diversity within these populations must be maintained, which is done mostefficiently by migration among populations. Population viability was accessed using 3microsatellite loci to determine population genetic structure in 2 locally dispersed subpopulationsof ornate box turtles. Although these subpopulations were determined to be 1 large populationwith the normal range of heterozygosity, further examination revealed evidence of geneticdivergence from a once larger population that is now geographically separated into fragments. Weconcluded that the central population studied was genetically healthy, and with propermanagement that continues to promote gene flow, the population should remain viable in thenear future.

KEY WORDS. – Reptilia; Testudines; Emydidae; population genetics; microsatellites; Iowa

The worldwide decline of amphibians is well

documented (e.g., Lannoo 2005). Although declines

among amphibians have received considerable media

attention, reptilian declines also appear significant due to

pollutants, invasive species, habitat loss, and increased

predation along edges of fragmented habitats (Gibbons et

al. 2000). Fragmented habitats, without regular gene flow

between populations, also reduce genetic diversity within

small, isolated populations, partially explained by meta-

population theory (Levins 1969).

Among reptiles, terrestrial turtles are limited in their

dispersal abilities, which limit gene flow. Therefore,

success of recolonization by terrestrial turtles after a

population crash may depend on patch and/or corridor

habitat quality, the distance separating subpopulations

from the main population, or migration barriers such as

rivers or roads (Lidicker and Koenig 1996). Turtle home

ranges are often fragmented by highways and housing

developments. In such fragmented conditions, marginal

populations generally show reduced numbers and are

expected to have lower average fitness (Weins 1997;

Rubin et al. 2001). All of these conditions potentially

apply to the ornate box turtle (Terrapene ornata ornata),

which is protected in Indiana, threatened in Iowa, and

endangered in Wisconsin (Swarth and Hagood 2005). All

of these states contain isolated populations on the margins

of their US range (Conant and Collins 1991).

In eastern Iowa, most ornate box turtle populations are

scattered and small. However, the Hawkeye Wildlife Area

(HWA) of Johnson County, Iowa has a relatively large

population (Fig. 1). Two intensely studied subpopulations

within the HWA (Mallard Pools and Greencastle) are

separated by less than 1 km with no apparent barriers to

immigration (Fig. 2). However, studies by Bernstein et al.

(2007) indicated that ornate box turtles have a high degree

of philopatry and that turtles do not travel between the 2

sites.

The HWA ornate box turtle population is one of the 2

largest in Iowa (over 600 turtles have been marked in the

75-ha area that contains the 2 subpopulations); the only

other population that exceeds 100 individuals in eastern

Figure 1. Map of Iowa indicating locations of the HawkeyeWildlife Area (HWA) and Muscatine.

Iowa exists near Muscatine, approximately 120 km away

(Fig. 1).

Herein, we analyze data from microsatellite loci to

compare levels of genetic diversity among these popula-

tions to determine if there is genetic evidence to support

the hypothesis of no migration between the HWA

subpopulations.

METHODS

Turtles were marked with a modified Cagle (1939)

system and located at HWA by walking through the sites

at various times of day during April and early May. As

described in Bernstein et al. (2007), HWA is largely

composed of sandy habitats, which are required by ornate

box turtles (Dodd 2001; Bernstein and Black 2005). Blood

samples were collected from the Mallard Pool subpopu-

lation (n ¼ 47) and from the Greencastle subpopulation

(n ¼ 41). In addition, 14 samples were collected from

Muscatine, Iowa, for comparison (Fig. 1).

DNA Extraction. — Whole blood was extracted from

the caudal vein in the tail using disposable 1-cc syringes.

The blood was placed in lysis buffer (Seutin et al. 1991;

Pearse et al. 2001) and stored at 208C. As a backup, blood

was absorbed onto filter-paper tabs, placed into 1.0-mL

cryogenic storage tubes, and stored at �708C. DNA was

Figure 2. Aerial photo of Hawkeye Wildlife Area showing locations of Mallard Pools and Greencastle subpopulations. Note farm fieldsand houses south of Mallard Pools. (Photo from Iowa Geological Survey, Iowa Department of Natural Resources, Iowa City, IA.)

4 CHELONIAN CONSERVATION AND BIOLOGY, Volume 7, Number 1 – 2008

extracted using standard phenol-chloroform techniques

(Hillis et al. 1990). When necessary, DNA was extracted

from frozen filter tabs using Chelex 100 (Sigma, St. Louis,

MO) following Walsh et al. (1991).

Microsatellite Analysis. — If microsatellites are

flanked by highly conservative sequences, primers may

work on multiple, closely related species (Primmer et al.

1996). Therefore, polymerase chain reaction (PCR)

primers reported by Pearse et al. (2001), who successfully

amplified microsatellites from the confamilial painted

turtle (Chrysemys picta), were chosen for this study.

Likewise, PCR conditions also followed Pearse et al.

(2001). The 3 loci examined were designated CP2, CP3,

and CP10. PCR amplifications were carried out using a

Primus thermal cycler (MWG-Biotech, High Point, NC).

Both positive and negative controls were used in each

PCR batch.

PCR products were size-fractionated at the Iowa State

University Sequencing and Synthesis Facility. The pro-

gram Genographer 1.6 (Benham 2001) was utilized for

electromorph size determination and genotype scoring of

individuals. All resulting chromatograms were also

examined by eye to ensure accuracy of scoring (e.g.,

determine presence of stutter peaks, etc.) and multiple

reference individuals were included in different runs to

standardize size determinations between batches and to

confirm reproducibility of results.

Because the nature of the tandem repeats was not

reported by Pearse et al. (2001) one individual was directly

sequenced (using the same PCR primers) for each locus

using standard cycle-sequencing procedures at the DNA

Sequencing and Synthesis Facility at Iowa State Univer-

sity. This sequencing confirmed that the microsatellites

were dinucleotide repeats with a motif of CA. We did

regenotype individuals with genotypes that fell outside the

normal range.

Statistical Analysis. — Three different population

combinations were analyzed for each statistical test. In the

first group, Mallard Pools, Greencastle, and Muscatine

were analyzed as separate populations. The second group

combined Mallard Pools with Greencastle to form the

Hawkeye population, and this was compared with

Muscatine. The third group compared Mallard Pools with

Greencastle as 2 separate populations, leaving out

Muscatine.

With the exception of calculations for RST, an FST

analog based on allele size, all analyses were performed

using Genepop version 3.3 (Raymond and Rousset 1995).

Expected and observed heterozygosities and homozygos-

ities were compared per locus and per population; an

excess of homozygosity indicated either an inbred

population or the existence of a null allele.

The Markov chain method in Genepop version 3.3

(Raymond and Rousset 1995) was used to test for

deviation from Hardy-Weinberg equilibrium (Guo and

Thompson 1992). The dememorization number was set to

1000, the number of batches was set to 500, and the

number of iterations per batch was set to 1000 to achieve a

standard error (SE) , 0.01. The result of this test was the

fixation index (Fis), a measure of heterozygote deficiency

or excess for codominant diploid alleles where Fis ¼ 0

indicates the meeting of Hardy-Weinberg expectations and

Fis ¼ 1 signals no heterozygotes.

Genotypic linkage disequilibrium, whether one locus

was independent from another, was also tested. Genepop

accomplishes this using contingency tables for all loci and

estimating the p value by the Markov chain method

(Raymond and Rousset 1995). The dememorization number

was set to 1000, the number of batches was set to 1000, and

the number of iterations per batch was set to 1000.

The genotypic distribution across populations was

also calculated using Genepop version 3.3 (Raymond and

Rousset 1995). For each locus, a contingency table was

created, and the p value was estimated for each population

or pair of populations by using the Markov chain method

(Raymond and Rousset 1995). The dememorization

number was 1000, the number of batches was 500, and

the number of iterations per batch was 1000. FST, a

measure of population differentiation, was also generated

as defined by Weir and Cockerham (1984).

RST was generated by FSTAT (Goudet 1995) and

SPAGeDI version 1.0 (Hardy and Vekemans 2002).

SPAGeDI uses spatial coordinates to analyze pairwise

comparisons. Latitude and longitude were taken directly

from locations of turtles plotted in ArcView version 3.2

(ESRI, Redlands, CA) and were converted to decimal by

the Universal Transverse Mercator conversion program

from the US Geological Survey.

RESULTS

All 3 loci were polymorphic, with 7 alleles at locus

CP3, 3 at CP2, and 10 at CP10 (Table 1). There were 2

unique alleles at Mallard Pools, allele 145 at CP3 and

allele 225 at CP10, and Muscatine did not show alleles

208, 215, 225, and 230 from CP10 (Table 1).

Expected and Observed Heterozygosities. — No

significant differences in heterozygosity were indicated

for CP2 and CP3 (Table 2). However, Fis values indicated

heterozygotic deficiency for locus CP10 for the popula-

tions at Mallard Pools (Fis ¼ 0.34) and Greencastle

(Fis ¼ 0.42). This signaled a heterozygotic deficiency,

likely the result of 1 or more null alleles at CP10.

Therefore, all subsequent tests were performed on the full

data set and also on the 2 loci, CP2 and CP3, alone, to

alleviate potential effects of a null allele.

Hardy-Weinberg Exact Tests. — CP2 and CP3 did not

deviate significantly from Hardy-Weinberg equilibrium

(Table 3). When 3 loci were examined, the highest and

lowest p values were found in Muscatine ( pCP2 ¼ 0.1114;

pCP3 ¼ 0.9403). However, the genotype frequencies for

CP10 deviated significantly from Hardy-Weinberg with

the Mallard Pools and Greencastle populations (Table 3).

Subsequently, fixation indices were low for all loci except

RICHTSMEIER ET AL. — Migration, Gene Flow, and Genetic Diversity in Terrapene ornata 5

at CP10 for Mallard Pools and Greencastle and CP2 for

Muscatine.

Genotypic Linkage Disequilibrium. — Two cases of

significant linkage were indicated by the analysis. In both

cases, the linkage involved the loci CP10 and CP2 and

occurred within the Greencastle and Mallard Pool

populations ( p ¼ 0.02 in both cases) (Table 4). Between

CP2 and CP3, the genotype frequencies did not deviate

significantly, indicating that the loci were not linked.

Because of the likelihood of the presence of a null allele

and possible linkage, CP10 was dropped from all further

analyses.

Genotype Differentiation. — Muscatine and the

pooled HWA populations showed significant differentia-

tion for CP2 (Table 5). Populations differed significantly

at CP2 between Muscatine and Mallard Pools and also for

both loci analyzed for comparisons of Muscatine and

Greencastle (Table 6).

FST and RST. — Analysis of the 2 loci indicated no

significant differences between the populations using

either FST or RST (Table 7). FST values ranged from

�0.0108 to 0.0686, and traditional RST values were also

low, ranging from�0.016 to 0.055 (Table 7). The addition

of spatial data using SPAGeDI resulted in similarly low

RST values (�0.157 to �0.0081) (Table 7).

Identical Alleles. — After comparing all sites for

identical alleles, 1 pair of turtles was found within

Muscatine, 1 pair within Mallard Pools, and 2 pairs within

Greencastle.

DISCUSSION

Both FST and RST values indicate that the 2 putative

populations at HWA share a high degree of gene flow

(where 1.0 ¼ fixation and 0.0 ¼ panmixia) and were

essentially 1 panmictic population. In their study of

Blanding’s turtles (Emydoidea blandingii), Mockford et

al. (2007) reported FST values ranging from 0.0 to 0.465

and RST values ranging from �0.016 to 0.507. Thus, our

genetic information does not support the lack of evidence

for movement between the Mallard Pools and Greenscastle

subpopulations from a decade of mark–recapture and

radiotracking data (Bernstein et al. 2007). Similarly, the

FST and RST analyses indicate that the HWA and

Muscatine populations were not differentiated. However,

the results of the genotypic distribution test, the more

sensitive test, indicated that the Muscatine population was

relatively isolated, given the significant subdivision at

Table 1. Allele summary for CP3, CP2, and CP10 within the 3study populations of Terrapene ornata ornata showing poly-morphisms, 2 unique alleles at Mallard Pools, and 4 alleles absentfrom Muscatine.a

Allele size(base pairs) Muscatine Mallard Pools Greencastle Totals

CP3 202138 3 23 21 47140 1 4 1 6145 0 1 0 1153 2 7 4 13155 14 23 21 58157 6 18 11 35159 2 18 22 42

CP2 200170 2 12 10 24174 25 63 50 138178 1 19 18 38

CP10 200194 10 4 6 20196 9 39 31 79198 1 22 10 33200 3 3 6 12208 0 4 1 5210 3 3 5 11215 0 11 15 26219 2 5 3 10225 0 2 0 2230 0 1 1 2

a CP2 and CP3 are missing 1 pair of alleles.

Table 2. Expected and observed heterozygosity for each microsatellite locus within the 3 study populations of Terrapene ornata ornatawith no significant differences.

Expected heterozygosity Observed heterozygosity Sample size Fixation index

CP3Muscatine 9.89 11 28 �0.08Hawkeye 68.83 66 172 0.06Mallard Pools 37.98 39 94 �0.03Greencastle 30.99 27 78 �0.05

CP2Muscatine 2.85 2 28 0.01Hawkeye 43.75 42 172 0.02Mallard Pools 23.45 22 94 �0.01Greencastle 20.52 20 78 0.08

CP10Muscatine 10.74 10 28 0.00Hawkeye 66.21 30 172 0.37Mallard Pools 35.65 16 94 0.34Greencastle 30.46 14 78 0.42

6 CHELONIAN CONSERVATION AND BIOLOGY, Volume 7, Number 1 – 2008

CP2. This differentiation is not surprising given the much

greater geographic separation between these 2 localities

(Fig. 1).

We suggest 3 factors that may explain the lack of

subdivision within the Hawkeye populations (Greencastle

and Mallard Pools) despite mark–recapture data to the

contrary as well as the genetic differences between the

HWA and Muscatine populations: 1) recent colonization,

2) recent population subdivision, and 3) gene flow within

HWA that has been undetected by field studies.

Recent Colonization. — What could explain why FST

and RST analyses indicated that Muscatine and HWA were

not genetically distinct populations? One potential expla-

nation is the relatively recent colonization of ornate box

turtles in Iowa following the Wisconsinan glaciation,

which likely would result in low levels of genetic

variability (Hewitt 1996).

Fossil evidence places Terrapene south and west of

Iowa prior to the Pleistocene (Milstead 1967; Moodie and

Van Devender 1978; Dodd 2001); during the Wisconsinan

glaciation, only a small portion of central Iowa was

glaciated (Prior 1991). As the ice retreated north (approx.

14,000 years ago), wetlands, sandy plains, and prairie

vegetation replaced tundra and forests. These new niches

provided opportunities for fauna (Pielou 1991), and this

was probably the time that ornate box turtles migrated into

Iowa, initially into southern Iowa.

By the early 1800s, surveys reported that nearly 70%–

80% of Iowa was prairie, suitable habitat for T. ornata

ornata (Dinsmore 1994). Brumfiel (1919) found numerous

turtles in Johnson County which he described as the ‘‘dry

land tortoise,’’ and soil characteristics suggest that ornate

box turtles potentially could have been widely distributed

throughout the county, especially the northern third where

HWA is located. In addition, small, fragmented, remnant,

extant populations in Iowa indicate a probable wider past

distribution, especially across the southern half of the state.

However, the relatively recent time frame that box turtles

have inhabited Iowa, Illinois, and Wisconsin may not have

allowed for allopatric differentiation of the turtle popula-

Table 3. Hardy-Weinberg probability test using 3 loci indicating no significant deviation from equilibrium within the 3 studypopulations of Terrapene ornata ornata.

p value 3 loci SE 3 loci p value 2 loci SE 2 lociFixation

index

Muscatine, Mallard Pools, GreencastleCP3

Muscatine 0.9403 0.0022 0.9419 0.0024 �0.117Mallard Pools 0.3410 0.0060 0.3545 0.0060 �0.027Greencastle 0.2985 0.0050 0.3001 0.0052 0.130

CP2Muscatine 0.1114 0.0019 0.1105 0.0019 0.307Mallard Pools 0.2738 0.0022 0.2756 0.0022 0.063Greencastle 0.1628 0.0017 0.1639 0.0016 0.026

CP10Muscatine 0.0682 0.0029 0.071Mallard Pools 0.0001 0.0000 0.554Greencastle 0.0001 0.0000 0.544

Muscatine, HawkeyeCP3

Muscatine 0.9447 0.0021 0.9439 0.0023 �0.117Hawkeye 0.3541 0.0070 0.3560 0.0069 0.041

CP2Muscatine 0.1089 0.0019 0.1127 0.0020 0.307Hawkeye 0.4132 0.0026 0.4127 0.0027 0.040

CP10Muscatine 0.0657 0.0027 0.071Hawkeye 0.0001 0.0000 0.548

Mallard Pools, GreencastleCP3

Mallard Pools 0.3578 0.0058 0.3414 0.0059 �0.027Greencastle 0.3024 0.0051 0.2991 0.0049 0.130

CP2Mallard Pools 0.2727 0.0021 0.2737 0.0023 0.063Greencastle 0.1593 0.0017 0.1658 0.0018 0.026

CP10Mallard Pools 0.0001 0.0000 0.554Greencastle 0.0001 0.0000 0.544

RICHTSMEIER ET AL. — Migration, Gene Flow, and Genetic Diversity in Terrapene ornata 7

tions. Detection of allopatric differentiation may be

hindered by the relative longevity, high nest predation,

and juvenile mortality of box turtles (Dodd 2001; Bowen

et al. 2004).

However, the statistical results of the more sensitive

genotypic distribution analysis between Muscatine and

HWA indicated that the Muscatine population was

significantly different in genotypic distribution and was

isolated from the HWA. This alternative could have

resulted from a recent bottleneck of either the Muscatine or

HWA populations or, alternatively, from either a greater or

an earlier isolation of the 2 populations than outlined

above.

Population Differentiation. — In contrast to recent

colonization, the populations at HWA and Muscatine were

just beginning to differentiate, possibly resulting from

fragmentation of panmictic populations in which gene

flow was recently hindered by rivers, roads, and/or farms

(e.g., Curtin 1997).

Hunting, trapping, and the destruction of habitat led to

declines, extirpation, and extinction of many species by

the end of the 19th century (Dinsmore 1994), and habitat

destruction continues to threaten ornate box turtles today

(Iowa Department of Natural Resources 2002).

Approximately 0.12% of original Iowa prairie remains

in isolated fragments (Dinsmore 1994). Although HWA

has both natural and reconstructed prairies, the area around

HWA has been farmed for most of the last 100 years, and

turtles regularly utilize nearby agricultural fields in daily

movements and during reproduction (Bernstein and Black,

unpubl. data, 2000). However, we also noted turtle

mortality from farm machinery, and long-term farming

in an area can most likely extirpate a population.

In addition to farming, mortality on roads can create

barriers to gene flow by fragmenting populations (e.g.,

Doroff and Keith 1990). Muscatine and HWA have

increasingly been separated by roads and major highways

(Iowa Department of Transportation 1999). According to

Keller and Largiader (2003), fragmentation caused by

roads reduced gene flow and genetic variability in ground

beetles (Carabus violaceus), and Gibbs (1998) observed

restricted amphibian movements because of roads in

southern Connecticut. Anderson (1956) reported a 10-

year decline in ornate box turtles in Missouri as traffic

increased on a highway, and similar to observations on

desert tortoises (Gopherus agassizii) in California (Boar-

man et al. 1997), we have observed ornate box turtles at

HWA killed by cars.

However, given the long life span of ornate box turtles

and the short period of time that farming and roads have

been in Iowa, it seems unlikely that these modern activities

alone would be the current cause of Muscatine’s isolation.

Rivers can also be boundaries or barriers to dispersal and

gene flow, and although ornate box turtles are capable of

swimming, they are poor swimmers and less likely to cross

the several large rivers between HWA and Muscatine (Fig.

1). In addition, distance may also be a limiting factor: 3.3

km is the maximum homing distance for T. ornata noted

by Metcalf and Metcalf (1970). Although Schwartz and

Schwartz (1982) noted one Terrapene carolina triunguis(transient) that traveled 10.0 km in a straight line, crossing

highways and, after much hesitation, the Moreau River in

Missouri, Bernstein et al. (2007) documented much

smaller home ranges and movements at HWA. Therefore,

rivers and distance, in conjunction with habitat fragmen-

tation and roads, are the likely causes of any genetic

isolation between Muscatine and HWA. However, neither

of the first 2 hypotheses explains gene flow within HWA.

Gene Flow Within HWA. — If HWA is a large central

population, then no genetic differences would be found

between individuals in different locations. Radiotracking

Table 4. Genotypic linkage disequilibrium for 3 loci in the 3study populations of Terrapene ornata ornata with the onlysignificant difference between CP2 and CP10 at Mallard Poolsand Greencastle.

Locus 1 Locus 2 p value SE

Muscatine, Mallard Pools, GreencastleMuscatine CP3 CP2 0.0655 0.00130Muscatine CP3 CP10 0.1953 0.00392Muscatine CP2 CP10 0.5843 0.00293Mallard Pools CP3 CP2 0.1795 0.00409Mallard Pools CP3 CP10 0.9788 0.00242Mallard Pools CP2 CP10 0.0195 0.00172Greencastle CP3 CP2 0.2464 0.00502Greencastle CP3 CP10 0.5589 0.00938Greencastle CP2 CP10 0.0199 0.00182

Muscatine, HawkeyeMuscatine CP3 CP2 0.0655 0.00130Muscatine CP3 CP10 0.1916 0.00411Muscatine CP2 CP10 0.5883 0.00297Hawkeye CP3 CP2 0.2172 0.00555Hawkeye CP3 CP10 0.9686 0.00351Hawkeye CP2 CP10 0.0739 0.00429

Mallard Pools, GreencastleMallard Pools CP3 CP2 0.1856 0.00434Mallard Pools CP3 CP10 0.9749 0.00287Mallard Pools CP2 CP10 0.0203 0.00178Greencastle CP3 CP2 0.2502 0.00516Greencastle CP3 CP10 0.5669 0.00946Greencastle CP2 CP10 0.0205 0.00199

Table 5. Genotypic differentiation for 2 loci for the 3 studypopulations of Terrapene ornata ornata indicating no significantdifferences.

Locus 1 Locus 2 p value SE

Muscatine, Mallard Pools, GreencastleMuscatine CP3 CP2 0.0645 0.00131Mallard Pools CP3 CP2 0.1936 0.00432Greencastle CP3 CP2 0.2565 0.00507

Muscatine, HawkeyeMuscatine CP3 CP2 0.0662 0.00130Hawkeye CP3 CP2 0.2087 0.00533

Mallard Pools, GreencastleMallard Pools CP3 CP2 0.1841 0.00421Greencastle CP3 CP2 0.2461 0.00500

8 CHELONIAN CONSERVATION AND BIOLOGY, Volume 7, Number 1 – 2008

and mark–recapture studies may not detect migrants and

transients between the subpopulations, and gene flow

could be maintained by matings between individuals

within the subpopulations and transients (Kiester et al.

1982) and/or consecutive matings across overlapping

home ranges between Mallard Pools and Greencastle.

The latter conclusion is supported by the knowledge of

unmarked turtles found between the 2 subpopulations

(Bernstein and Black, unpubl. data, 2000) along with

evidence of home ranges that both overlap between

individuals and span multiple habitats (Bernstein et al.

2007). These data suggest a larger, panmictic population

of ornate box turtles outside of our study areas at HWA, a

conclusion supported by observations and reports of ornate

box turtles outside of Mallard Pools and Greencastle.

Also, similarities in allelic distribution may be due to

the age of the turtles as blood was collected from 15–30-

year-old adults. Furthermore, because each area has

abundant resources as well as nesting and overwintering

sites, long-range movements between sites may not occur.

However, the distinct alleles found in the 3 turtles at

the Mallard Pools (1 turtle with allele 145 at CP3 and 2

turtles with allele 225 at CP10), may indicate only slight

contact with the Greencastle site. Newman and Squire

(2001) suggested that subtle differentiation between wood

frogs (Rana sylvatica), separated by small distances of a

few kilometers, indicated that the populations acted as a

metapopulation with only slight interactions. Thus, Mallard

Pools and Greencastle could drift to fixation for 1 of the 2

new alleles if there is only slight contact between the sites.

Extreme weather events can also affect ornate box

turtle home ranges and may bring ornate box turtles

together at HWA. In 1993, much of the Greencastle

subpopulation was isolated onto high points as floodwaters

inundated the area, a situation partially repeated in spring

2005. In contrast, we also noted extensive movements

between wetlands during a drought period, and these

atypical dispersions are similar to the hypothesis of Rowe

et al. (2000) that Natterjack toads (Bufo calamita) may

Table 6. Results of genotypic distribution analysis for the 3 studypopulations of Terrapene ornata ornata. Significant p valuesindicate population differentiation.

Population pair

2 loci

p value SE

Muscatine, Mallard Pools, GreencastleMuscatine, Mallard Pools

CP3 0.2016 0.0026CP2 0.0437 0.0001

Muscatine, GreencastleCP3 0.0474 0.0012CP2 0.0348 0.0009

Mallard Pools, GreencastleCP3 0.6340 0.0035CP2 0.9239 0.0008

Muscatine, HawkeyeCP3 0.0914 0.0021CP2 0.0244 0.0009

Mallard Pools, GreencastleCP3 0.6350 0.0035CP2 0.9249 0.0009

Table 7. FST and RST statistics for the 3 study populations of Terrapene ornata ornata indicating no significant differences. FST wasgenerated by Genepop, RST by FSTAT, and the last 2 columns of RST by SPAGeDI.

FST, RST statistics for each microsatellite locus

FST RST

3 Loci 2 Loci 3 Loci 2 Loci 3 Loci 2 Loci

Genepop Genepop FSTAT FSTAT SPAGeDI SPAGeDI

Muscatine, Mallard Pools, GreencastleCP3 0.0162 0.0162 �0.012 �0.005CP2 0.0205 0.0205 �0.004 �0.004CP10 0.0233 0.012All 2 loci 0.0178 �0.0003All 3 loci 0.0199 �0.0120

Muscatine, HawkeyeCP3 0.0469 0.0469 �0.014 �0.014CP2 0.0686 0.0686 0.005 0.006CP10 0.0618 0.055All 2 loci 0.0553 0.0036All 3 loci 0.0578 0.0167

Mallard Pools, GreencastleCP3 �0.0044 �0.0044 �0.01 �0.01 �0.0124 �0.0124CP2 �0.0108 �0.0108 �0.01 �0.01 �0.0081 �0.0081CP10 �0.0035 �0.016 �0.0157All 2 loci �0.0069 �0.0098 �0.0121All 3 loci �0.0057 �0.0127 �0.0140

RICHTSMEIER ET AL. — Migration, Gene Flow, and Genetic Diversity in Terrapene ornata 9

cross areas at low tide and maintain gene flow. However, it

is unclear if any of these weather-induced dispersal events

affected mating patterns of ornate box turtles at HWA.

Therefore, these data can possibly be explained by

using all 3 hypotheses. The recent glaciations may not

have allowed HWA and Muscatine populations to

significantly differentiate; although, the process of sepa-

ration may have begun due to natural barriers and

fragmentation of landscapes by human activity. Finally,

the high degree of gene flow within HWA was indicative

of one central panmictic population.

Future Research and Management Implications. —Analysis of more microsatellite loci might reveal a clearer

picture of gene flow because the null allele plus the nearly

monomorphic CP2 allele could have affected results, and

CP3 showed polymorphism. In addition, specific primers

for ornate box turtles might eliminate the problems of the

null allele. An alternative for future study would be to

examine mitochondrial DNA (mtDNA) markers to deter-

mine mating patterns within the HWA (Balloux et al.

2000; Johnson et al. 2003; Bouzat and Johnson 2004).

Of course, more study using microsatellites or mtDNA

may only confirm that turtles from the 2 sites at HWA are

currently part of a single panmictic population with gene

flow within a large, dispersed population. The population

at HWA appears to extend beyond the boundaries of the

studied sites, and gene flow within this large population

may promote genetic diversity and long-term stability

under current conditions (Kuo and Janzen 2004, 2007).

However, as Dodd et al. (1994) and Bowen et al. (2004)

recommended for box turtles in other locations, we

recommend that all efforts be made to provide continuous

habitat throughout the range of the ornate box turtles at

HWA to prevent edge effects and the mortality that results.

Additionally, the turtles should remain protected from

collection and other unnatural causes of mortality.

ACKNOWLEDGMENTS

D. Richtsmeier, S.L. O’Kane, Jr., T.A. Spradling, and

J.W. Tamplin provided advice, technical assistance, and

encouragement throughout the project, and an anonymous

reviewer provided helpful comments. The Monsanto

Company, MidAmerican Energy, and the Iowa Depart-

ment of Natural Resources (IDNR) allowed access to

turtles for blood collection, and the project was funded by

a GRASP award through the University of Northern Iowa.

Funding was also received by the Mount Mercy College

Department of Biology and Faculty Development Pro-

gram, the Cornell College Department of Biology, the

Associated Colleges of the Midwest, the Iowa College

Foundation of the R. J. McElroy Trust, the Iowa Wildlife

Diversity program of the IDNR, R. Rhodes II, and the

Iowa Natural History Association. We thank them all. This

research was carried out under a State of Iowa Scientific

Collector Permit, SC95, to N.P. Bernstein.

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Received: 11 February 2006

Revised and Accepted: 21 April 2007

RICHTSMEIER ET AL. — Migration, Gene Flow, and Genetic Diversity in Terrapene ornata 11