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2009 SOUTHEASTERN NATURALIST 8(4):709–722
Effects of Corridors on Genetics of a Butterfl y in a
Carrie N. Wells1,2, Ray S. Williams1,*, Gary L. Walker1,
and Nick M. Haddad3
Abstract - To investigate the possible role of landscape connectivity on the genetic
structure of isolated populations, we examined the effects of habitat corridors on
the population genetics of a vagile butterfl y species, Junonia coenia, within a largescale,
experimental system. Using allozyme electrophoresis, a total of nine loci were
identified and scored, six of which exhibited polymorphism. Our data demonstrated
consistently higher levels of expected (He) and observed (Ho) heterozygosity in butterfl
ies sampled from patches connected by corridors compared to unconnected patches.
A t-test comparing He and Ho in connected versus unconnected patches found a marginally
significant difference in one locus, the glycolytic enzyme phosphoglucose
isomerase (PGI). Connected patches exhibited overall lower FST values compared to
unconnected patches, indicating potentially increased levels of gene fl ow due to corridors.
Our results support previous investigations on dispersal and population size for J.
coenia, and show that higher dispersal through corridors promotes genetic variability
at a locus (PGI) implicated in dispersal and fitness in butterfl ies.
The fragmentation and outright loss of natural habitats are currently
thought to be the most serious threats to global biodiversity (Fischer and
Lindenmayer 2007, Saunders et al. 1991, Solé et al. 2004, Wilcove et al.
1998). Fragmentation of large contiguous areas into smaller habitat islands
often results in the geographic isolation of populations, in turn limiting
movement of individuals between populations (Bierregaard et al. 1992,
Harris 1984). A popular management strategy to counter this type of fragmentation
often involves attempts to connect habitat remnants with a corridor
of similar habitat (Mann and Plummer 1995, Merriam and Saunders
1993, Noss 1992). Habitat corridors have been shown to facilitate dispersal
of diverse taxa (Berggren et al. 2002, Haas 1995, Haddad 1999a, Haddad
et al. 2003, Machtans et al. 1996, Sutcliffe and Thomas 1996, Tewksbury et
al. 2002, Zhang and Usher 1991), reduce local extinction rates (Fahrig and
Merriam 1985, Noss 1991), and increase levels of gene flow (Aars and Ims
1999, Hale et al. 2001, Mech and Hallett 2001). While the use of habitat
corridors presents a potential benefit for species in isolated habitats, many
uncertainties about their importance for use as a conservation strategy
1Department of Biology, PO Box 32027, 572 Rivers Street, Appalachian State University,
Boone, NC 28608. 2Current address - Department of Biological Sciences,
132 Long Hall, Clemson University, Clemson, SC 29634-0315. 3 Department of
Zoology, 2104 North Gardner, Box 7617, North Carolina State University, Raleigh,
NC 27695. *Corresponding author - email@example.com.
710 Southeastern Naturalist Vol. 8, No. 4
remain. Of particular concern is, even if corridors increase dispersal rates,
does more movement have impacts on populations, including population
Population genetics provides a powerful approach to investigate population
dynamics in a landscape context (Manel et al. 2003). Smaller, isolated
habitats could be expected to contain fewer individuals than larger, more
contiguous areas (see Vandewoestijne and Baguette 2004), affecting genetic
diversity within species (Schmitt and Seitz 2002). Therefore, studies that
examine the effects of connectivity on maintaining genetic variation are
timely. Since new alleles appear in a gene pool through both the process of
mutation and immigration of individuals from separated populations (Mettler
et al. 1988, Wallace 1981), both mechanisms contributing to genetic
variation within populations need to be considered. In the short-term, gene
fl ow via immigration tends to increase genetic diversity within a given local
population by offsetting the loss of alleles due to inbreeding and drift
(Hoole et al. 1999, Peterson and Denno 1998, Slatkin 1985). Habitat degradation
limits gene fl ow by reducing the movement of individuals and their
alleles between fragmented, isolated populations (Hänfl ing et al. 2004; Nei
1973, 1987; Slatkin 1985). In addition to fewer alleles being introduced, the
isolation of unconnected populations would likely reduce both heterozygosity
and the total number of alleles in the population through the process of
random genetic drift and inbreeding (Slatkin 1985, Van Rossum et al. 2004,
Wright 1978). Recent work suggest distinct correlations between fitness
and genetic diversity (Reed and Frankham 2003), with particular attention
paid to molecular polymorphism in the glygolytic enzyme phosphoglucose
isomerase (PGI), as this locus has been shown to affect fitness in butterfl ies.
Polymorphism at the PGI locus has specifically been shown to enhance the
fl ight performance and fitness of Colias butterfl ies (Watt 2003), as well as
to act to increase metabolic rate, fecundity, and population growth in the
Melitaea cinxia L. (Glanville Fritillary) (Haag et al. 2005, Hanski and Saccheri
2006). With the potential for habitat isolation to alter genetic structure
at some loci and perhaps not others, a consideration of the role habitat connectivity
plays in affecting the population genetics of previously isolated
populations is important.
Allozyme electrophoresis has been a useful tool for investigating the
genetic structure of numerous insect populations, including nymphalid butterfl
ies (Britten and Brussard 1992, Britten et al. 1994, Brittnacher et al. 1978,
Porter and Mattoon 1989). Allozymes are considered to be a valuable but
conservative measure of genetic variation and are interpretable in terms of
Mendelian inheritance for specific loci (Avise 1975, 1994). Numerous investigators
have successfully used both DNA and allozyme analyses to examine
the effects of habitat fragmentation on the genetic structure of various insects,
including beetles (Britten and Rust 1996, Knutsen et al. 2000, Six et al. 1999),
crickets (Berggren et al. 2002), and butterfl ies (Baguette et al. 2003, Johannesen
et al. 1997, Keyghobadi et al. 1999, Kronforst and Fleming 2001, Meglécz
et al. 2004, Vandewoestijne et al. 1999, Williams et al. 2003).
2009 C.N. Wells, R.S. Williams, G.L. Walker, and N.M. Haddad 711
The main objective of this study was to examine if habitat corridors
facilitated gene fl ow in an open-habitat specialist butterfl y, Junonia coenia
Hübner (Common Buckeye), within a large-scale experimental system at
Savannah River Site (SRS), SC. This system was established within native
Pinus taeda L. (Loblolly Pine) forest and contained replicate connected and
unconnected habitat patches. Several previous studies with J. coenia at SRS
(Haddad 1999a, 1999b, 2000; Haddad and Tewskbury 2005) showed that
the establishment of corridors increased movement of butterfl ies, resulting
in higher population densities in habitat patches that were experimentally
connected by corridors (Haddad and Baum 1999, Tewksbury et al. 2002).
Because the facilitation of movement between patches via corridors was
previously established, we asked if the genetic structure of butterfl ies within
isolated and connected patches would be differentially affected via connectivity.
We expected that increased levels of gene fl ow in habitat patches
connected to each other by a corridor would cause J. coenia populations
in connected habitat patches to have higher genetic diversity than those in
unconnected patches. We also collected butterfl ies from outside the experiment
to establish a background or “source” level of variation. These
butterfl ies also provided insight into the development of genetic structure
within the experiment since the original colonization by the butterfl y after
experimental creation. The main questions addressed were: (1) do Common
Buckeye butterfl ies sampled from patches connected by a corridor have a
different genetic structure than butterfl ies sampled from isolated unconnected
patches and, (2) does the model-corridor system at SRS refl ect the
natural genetic structure of J. coenia?
Materials and Methods
We examined the genetic structure of J. coenia in an experimental-model
system established in the winter of 1999–2000 within the 1240-km2 Savannah
River Site. Eight 50-ha experimental blocks were created to examine the
effects of corridors on plant and animal dispersal. Each block contained five
open habitat patches within a dense matrix of pine forest. Patches are being
restored to Pinus palustris P. Mill. (Longleaf Pine) savannah through restoration
of Longleaf Pine and other herbaceous species, and through regular
controlled fire. In each block, a central 1-ha patch was surrounded by four
other patches, each 150 m away (Fig. 1). This central patch was connected
to only one of the peripheral patches (also 1 ha) by a 25- X 150-m corridor.
The three remaining patches were unconnected and equal to the size of the
connected patch plus the corridor (i.e., 1.375 ha; Fig. 1). For a complete description
of the site and establishment of plots, see Tewksbury et al. (2002).
This model system was ideal for our questions because a previous study
found movement of butterfl ies from the central patch to peripheral patches
was greater with a corridor present (Tewksbury et al. 2002).
712 Southeastern Naturalist Vol. 8, No. 4
Butterfl y Collection
The Common Buckeye is a multi-voltine, open-habitat butterfl y that occurs
throughout the southeastern United States (Opler 1998). Because the
Common Buckeye in the southern US produces more than one generation per
year, we estimate that at the time of our collection (summer 2002), from 6–7
generations could have developed within experimental patches since plot
colonization in 2000. A total of 111 butterfl ies were collected from experimental
patches during May and June of 2002. We choose this period to collect
because populations likely peak in May and June in the deep south (N.M.
Haddad, pers. observ.; Pyle 1981). In total, 53 butterfl ies were collected from
patches connected by a corridor, while 58 butterfl ies were collected from unconnected
peripheral patches (for the number of individuals collected in each
patch refer to Table 1). No butterfl ies were sampled within the boundaries of
an actual corridor. After collection, we determined that two blocks yielded too
few butterfl ies (n = 7) and thus were excluded from analysis. Therefore, our
Figure 1. Patch arrangement in a single experimental block at the Savannah River Site.
2009 C.N. Wells, R.S. Williams, G.L. Walker, and N.M. Haddad 713
genetic analysis was based on comparisons between and among six blocks,
using a total of 104 butterfl ies. While sample size was a potentially limiting
factor in our experiment, based on the numbers of alleles identified and the
amount of genetic variation found across sites (see below), we feel there was
a sufficient number of specimens collected to adequately address our primary
questions. In order to assess genetic variation in long established populations
of J. coenia at SRS, we sampled two populations (n = 18, n = 20) from outside
of the experiment. These large, panmictic butterfl y populations were sampled
from wide, open power-line right-of-ways. Since butterfl ies from surrounding
habitats founded patches within the experimental blocks, the genetic structure
of “source” butterfl ies provides insight into mechanisms responsible for observed
genetic variation within the experiment. In all cases, butterfl ies were
captured with a handheld net and stored on ice in the field for no longer than
2 h. Individuals were then frozen and transported to Appalachian State University
(ASU), Boone, NC for analysis.
Individuals were dissected to determine gender. The thorax was then
partitioned into two equal-sized parts, with one half stored at -80 °C, and
the remaining half ground in 2.5 ml of simple grinding buffer solution described
by Werth (1985). Homogenized tissue was partitioned into two equal
aliquots and stored at -80 °C until used in electrophoresis.
Nine allozyme loci were resolved on 13% starch gels (Sigma Chemical
Company) using three gel/electrode buffer systems following Werth (1985)
and Selander et al. (1971). Four loci, Aspartate aminotransferase (AAT) (EC
184.108.40.206), Malate dehydrogenase (MDH) (EC 220.127.116.11), Malic enzyme (ME)
(EC 18.104.22.168), and Aldolase (ALD) (EC 22.214.171.124), were found to be consistently
scorable on a Tris-borate-EDTA pH 9.1 gel/electrode buffer system
with gel run for 6 hours at 50 mA. Phosphoglucomutase (PGM) (EC 126.96.36.199),
Phosphoglucoisomerase (PGI) (EC 188.8.131.52), and Isocitrate dehydrogenase
(IDH) (EC 184.108.40.206) were consistently scorable on a discontinuous Triscitrate
pH 6.3/6.7 gel/electrode buffer system with gel run for 5 hours at
100 mA. Triosephosphate isomerase (TPI) (EC 220.127.116.11) and Glycerol-3-
Phosphate dehydrogenase (G3PDH or α-GPDH) (EC 18.104.22.168.) were scorable
on a discontinuous Tris-citrate pH 8.2/8.7 gel/electrode buffer system with
gel run for 5 hours at 100 mA. Stains were prepared according to Cardy et al.
(1983), Soltis et al. (1983), and Werth (1985). The fastest-tracking allele was
always scored as number one, with all subsequent alleles ordered sequentially.
Zymograms were drawn immediately following staining to record all
observed banding patterns. Stained gel slices were digitally photographed
using a Nikon Coolpix 3500 digital camera.
Standard measures of genetic diversity, including observed heterozygosities
(Ho; Levene 1949), expected heterozygosities (He; Nei 1978) and Wright’s
F-statistics (Wright 1951) were calculated using Popgene-32 (Yeh et al. 1997)
and F-stat (Goudet 2001). Observed heterozygosity and He across all loci were
calculated using a correction for small sample size (Nei 1978). Differences
714 Southeastern Naturalist Vol. 8, No. 4
between Ho and He in connected versus unconnected patches were tested for
significance at each locus using a paired t-test (Proc GLM, SAS Institute,
2001). Butterfl ies were pooled in either connected or unconnected patches
in an experimental block and the data analyzed using the block as a replicate
(n = 6). All values were arcsine transformed to normalize the data. Expected
and observed heterozygosities were tested for deviation from Hardy-Weinberg
expectations using a chi-square goodness-of-fit analysis. Wright’s F-statistics,
specifically FST, was used to examine relative amounts of reductions in
heterozygosity at a given level of the population structure relative to another
more inclusive level of population structure (see Wright 1965, 1978). Results
where 0.10 > P > 0.05 are reported as marginally significant.
Of the 111 butterfl ies sampled within the model system, eighteen females
(16.2%) and 93 males (83.8%) were collected (male:female ratio of 5.2:1).
The source populations contained a total of 38 butterfl ies, with 8 females
(21.1%) and 30 males (78.9%) (male:female ratio of 3.8:1). We believe that
the skewed sex ratio is caused by lower detection probabilities of females
(males are pseudo-territorial and highly visible; N.M. Haddad, pers. observ.).
Allele frequencies and numbers of butterfl ies collected in each experimental
unit for the polymorphic loci are presented in Table 1. Of the nine
enzyme systems used for analysis, six displayed polymorphism at a 95%
confidence level. The loci ALD, α-GPDH, and ME were all fixed for a single
allele and were therefore considered monomorphic. While only two blocks
(in connected patches) had butterfl ies polymorphic for MDH, this locus was
included in the analysis (Table 1). There was considerable variation in the
numbers of J. coenia collected in the different blocks.
We found that four loci from connected patches and two loci from the
unconnected patches were not in Hardy-Weinberg equilibrium (Table 2).
When comparing observed and expected heterozygosity across treatments
using a student t-test, only one locus, PGI, differed between connected and
unconnected patches (marginally significant; Table 2). Without considering
other factors such as wing wear and changes due to fl ight season, this result
possibly demonstrates an effect on some loci but not others and suggests,
based on other studies with PGI, that a locus implicated in dispersal ability
of butterfl ies is affected by connectivity between habitats.
There was considerable variation in Ho and He within blocks in both
connected and unconnected patches (Table 3). Overall, Ho, He, and percent
polymorphic loci were higher in connected than unconnected patches for five
of the six blocks sampled. One word of caution regarding sample size: in
some plots, the disproportionate number of butterfl ies collected in connected
versus unconnected patches could have biased our results with respect to
calculated Ho and He (see Tables 1 and 3). However, the consistent observation
of higher heterozygosites in connected versus unconnected patches
2009 C.N. Wells, R.S. Williams, G.L. Walker, and N.M. Haddad 715
Table 1. Allele frequencies for connected (C) and unconnected (U) patches in six polymorphic loci in sequentially numbered experimental blocks. n = number of
Common Buckeye butterfl ies collected in patches. See Materials and Methods for a description of the loci.
Locus Allele 1-C 1-U 2-C 2-U 3-C 3-U 4-C 4-U 5-C 5-U 6-C 6-U
n 10 3 6 3 5 6 21 13 4 21 5 7
AAT 1 0.083 0.047 0.154 0.024 0.100
2 1.000 1.000 0.917 1.000 1.000 1.000 0.929 0.808 1.000 0.976 0.900 1.000
3 0.024 0.039
TPI 1 0.024 0.192 0.125 0.024 0.143
2 1.000 1.000 1.000 1.000 1.000 0.976 0.769 0.875 0.976 0.900 0.857
3 0.039 0.100
PGI 1 0.100 0.083 0.120 0.077
2 0.450 0.500 0.417 0.500 0.500 0.250 0.452 0.462 0.625 0.452 0.100 0.357
3 0.500 0.500 0.500 1.000 0.300 0.583 0.405 0.423 0.375 0.548 0.900 0.929
4 0.050 0.083 0.100 0.083 0.024 0.039 0.071
PGM 1 0.050 0.167 0.024
2 0.250 0.167 0.250 0.024 0.115 0.125 0.143 0.100
3 0.700 0.677 0.583 0.833 0.800 0.667 0.050 0.577 0.625 0.738 0.900 0.928
4 0.250 0.167 0.083 0.800 0.667 0.214 0.269 0.250 0.119 0.071
5 0.083 0.039
IDH 1 0.050 0.083 0.083 0.039 0.024
2 0.850 0.833 0.833 0.500 0.800 0.917 0.833 0.962 0.875 0.905 0.900 0.857
3 0.100 0.167 0.083 0.500 0.200 0.167 0.125 0.048 0.100 0.143
MDH 1 0.200 0.125
2 1.000 1.000 1.000 1.000 0.800 1.000 1.000 1.000 0.875 1.000 1.000 1.000
716 Southeastern Naturalist Vol. 8, No. 4
with near equal numbers of butterfl ies collected supports our stated result.
Averaging blocks, observed heterozygosity was 20% higher in the connected
(Ho = 0.1928) than unconnected (Ho = 0.1547) patches. Similarly, expected
heterozygosity increased by 23% in connected patches, while Wrights FST
was 37% lower in connected than unconnected patches when blocks were
averaged (Table 3). Finally, source-population butterfl ies were substantially
and consistently higher in Ho, He, and percent polymorphic loci compared to
butterfl ies collected within the experiment. Average values of Wrights FST
for the source populations were more comparable to butterfl ies collected in
connected than unconnected patches (Table 3).
Table 3. Mean ± standard deviation (SE) of observed (Ho) and expected (He) heterozygosity
within each block for connected (C) and unconnected (U) patches and source (S) populations.
Also presented are mean ± SE for all blocks (1–6) and source populations (S1–S2) combined
for Ho, He, percent polymorphic loic (% Ploci) and Wrights F statistic (FST). See Materials and
Methods for a description of the analyses.
Block Ho (mean ± SE) He (mean ± SE) % Ploci FST
1 C 0.2393 ± 0.2827 0.2163 ± 0.2660 55.6 0.0299
U 0.1746 ± 0.3228 0.1914 ± 0.2555 55.6 0.0913
2 C 0.1333 ± 0.1414 0.1330 ± 0.1518 55.6 0.0816
U 0.1270 ± 0.1667 0.1202 ± 0.1652 44.4 0.0973
3 C 0.2500 ± 0.3062 0.1840 ± 0.2064 55.6 0.0462
U 0.1270 ± 0.1813 0.1319 ± 0.1942 55.6 0.0943
4 C 0.1778 ± 0.2906 0.1778 ± 0.2325 44.4 0.1354
U 0.1481 ± 0.2561 0.1358 ± 0.2329 33.3 0.1681
5 C 0.1778 ± 0.3073 0.1394 ± 0.2209 33.3 0.0908
U 0.2222 ± 0.3727 0.1420 ± 0.2224 33.3 0.1001
6 C 0.2037 ± 0.3203 0.1775 ± 0.2474 44.4 0.0510
U 0.1481 ± 0.3379 0.0864 ± 0.1803 22.2 0.0783
1–6 C 0.1928 ± 0.0177 0.1690 ± 0.0126 47.4 ± 3.72 0.0643 ± 0.0156
U 0.1547 ± 0.0147 0.1310 ± 0.0139 38.7 ± 5.51 0.1017 ± 0.0130
S1 0.3210 ± 0.3411 0.2454 ± 0.2544 55.6 0.0185
S2 0.3278 ± 0.3392 0.2517 ± 0.2452 55.6 0.0185
S1–S2 0.3244 ± 0.0034 0.2485 ± 0.0032 55.6 ± 0.0 0.0185 ± 0.0
Table 2. Statistical results from Hardy-Weinberg Equilibrium analysis (χ2 and P value) for connected
and unconnected patches and Ho and He analysis using a t-test (t and P value) for each
locus. See Materials and Methods for a description of the analyses and loci. * = P < 0.05, **
= P < 0.1.
Connected Unconnected Ho He
Locus χ2 P χ2 P t P t P
AAT 9.310 0.030* 10.31 0.020* 0.58 0.595 0.22 0.595
TPI 0.030 0.990 1.800 0.610 0.83 0.443 0.94 0.390
PGI 13.33 0.040* 21.42 0.002* 2.11 0.059** 1.15 0.275
PGM 7.050 0.720 8.080 0.620 0.51 0.621 0.37 0.718
IDH 14.27 0.003* 0.850 0.990 0.95 0.364 0.56 0.583
MDH 34.33 0.001*
2009 C.N. Wells, R.S. Williams, G.L. Walker, and N.M. Haddad 717
We found that J. coenia had higher levels of genetic variation in connected
patches as measured by percent polymorphic loci, observed and
expected heterozygosity, and lower FST values, strongly suggesting that
increased dispersal between populations infl uences the genetic structure of
populations. Furthermore, differences in genetic variation at the PGI locus
occur due to connectivity, demonstrating that variation at loci implicated for
importance in fitness and dispersal in butterfl ies is likely due to increased
connectivity provided by corridors. Our study supports a previous multi-year
study conducted at the SRS that found a greater number of marked butterfl ies
released in a central patch are recaptured in patches connected by a corridor
(Tewksbury et al. 2002). In combination with those results, our evidence
suggests that movement between isolated populations is important for maintaining
genetic diversity of J. coenia.
By a number of metrics, our data suggest that habitat connectivity promotes
greater genetic variability, as J. coenia had overall higher level of
heterozygosity and polymorphic loci when corridors were present. The fixation
index (i.e., FST) indicates moderate levels of genetic differentiation (see
Wright 1978 for description) in populations collected within habitat patches,
regardless of connectivity. Butterfl ies collected in connected patches did,
however, have an overall lower FST than those in unconnected patches
(Table 3), indicating less genetic divergence, likely because of increased
gene fl ow. We base this conclusion in part on the fact that our results correspond
with data on J. coenia movement within the same experimental
corridor system that has shown increased movement of this butterfl y in
patches connected by corridors (Tewksbury et al. 2002). Although other investigators
have examined the effects of habitat fragmentation on the genetic
structure of insect populations (Baguette et al. 2003, Johannesen et al. 1997,
Kronforst and Fleming 2001, Meglécz et al. 2004, Williams et al. 2003), few
have attempted to link a genetic analysis to movement results, providing a
mechanistic explanation as to why genetic changes occur (Castellón and
Sieving 2006, Keyghobadi et al. 1999).
Our results also support the notion that habitat connectivity promotes
polymorphism at a particular locus. Populations of J. coenia exhibited higher
overall genetic variation at the PGI locus in connected habitats, refl ected in the
marginally significantly higher Ho in connected versus unconnected patches
(Table 2). This result is extremely interesting in light of recent studies linking
the PGI locus to dispersal capability in butterfl ies (Hanski and Saccheri 2006).
Relevant to our investigation, patch area and spatial connectivity of habitat
have been demonstrated as being important in maintaining polymorphism
of PGI (Hanski et al. 2004). Our results support the conclusion that spatial
configuration of habitat can contribute to the maintenance of molecular polymorphisms,
even in vagile taxa like butterfl ies. This is especially relevant in the
experimental system used in this study, which accounted for the size of the corridor,
and from previous work that determined the shape of the habitat patches
718 Southeastern Naturalist Vol. 8, No. 4
had no effect on the ability to capture butterfl ies (Tewksbury et al. 2002). Thus,
in this system, there is no evidence the corridors acted as drift fences with respect
to butterfl y movement. Although somewhat speculative, we conclude that
variation at the PGI locus provides evidence that gene fl ow is the primary mechanism
shaping the genetic structure of J. coenia in connected habitat patches.
To better understand possible mechanisms contributing to the genetic
differentiation observed in our experiment, we asked how well the model
system refl ected source-population genetic differentiation. This is relevant to
consider since the population genetic structure within experimental patches
was ultimately dependent on butterfl ies colonizing from nearby source populations.
We found that source populations had much higher genetic variation
than butterfl ies in the experiment (Table 3). The substantially lower FST in these
populations indicate higher levels of gene fl ow, resulting in genetic panmixia.
Lower values of heterozygosity and FST values observed in our experiment are
likely the result of a combination of founder effects and reduced gene fl ow (see
Baughman et al. 1990, Nice and Shapiro 2001) compared to butterfl ies in the
open environment outside the experimental units, which have likely passed
through many generations. With no prior knowledge of genetic structure in
founding butterfl ies, it is somewhat speculative to conclude that the genetic
variation we found was due mostly to gene fl ow and not genetic drift (possibly
due to a small population size in patches compared to the surrounding area).
Nevertheless, provided that colonizing butterfl ies in connected and unconnected
patches had similar levels of genetic variation after patches were created, the
lower FST and higher heterozygosity in the connected patches two years later
provide evidence that since the time of establishment, populations of J. coenia
have differentiated more when a corridor connected patches.
In conclusion, our investigation demonstrates the value of addressing genetic
questions when the role of habitat connectivity on species in fragmented
landscapes is being examined. We demonstrated greater levels of genetic differentiation
in butterfl y populations connected by corridors and, importantly,
provided evidence that variation in a key locus implicated in the fitness and
dispersal of butterfl ies is affected by connectivity. Data from source populations
suggest effects of both gene fl ow and possibly genetic drift acting in this
system, pointing to a need to examine both population and genetic data in conservation
studies. Though we studied a common species, our study suggests a
need for similar experimental approaches in cases where species become rare
or imperiled due to habitat fragmentation.
We sincerely thank Ellen Damschen for her help at SRS. We would like to thank
all field assistants, especially Jeff White, for help with the butterfl y collections and
Aaron Kennedy and Matt Estep (Appalachian State University), who provided valuable
assistance in the allozyme analysis. We thank the anonymous reviewers for their
helpful comments that improved this manuscript. The Cratis D. Williams Graduate
School, the Biology Graduate Student Association, and the Graduate Student
Association Senate supported this research at Appalachian State University. The
2009 C.N. Wells, R.S. Williams, G.L. Walker, and N.M. Haddad 719
creation of the experimental sites at SRS was funded by a collaborative grant from
the National Science Foundation and by an interagency agreement with the Department
of Energy Operations Office through the US Forest Service/Savannah River
Aars, J., and R.A. Ims. 1999. The effect of habitat corridors on rates of transfer and
interbreeding between vole demes. Ecology 80:1648–1655.
Avise, J.C. 1975. Systematic value of electrophoretic data. Systematic Zoology
Avise, J.C. 1994. Molecular Markers, Natural History, and Evolution. Chapman and
Hall, New York, NY.
Baguette, M., G. Mennechez, S. Petit, and N. Schtickzelle. 2003. Effect of habitat
fragmentation on dispersal in the butterfl y Proclossiana eunomia. Comptes Rendus
Baughman, J.F., P.F. Brussard, P.R. Ehrlich, and D.D. Murphy. 1990. History, selection,
drift, and gene fl ow: Complex differentiation in checkerspot butterfl ies.
Canadian Journal of Zoology 68:1967–1975.
Berggren, A., B. Birath, and O. Kindvall. 2002. Effect of corridor and habitat edges
on dispersal behavior, movement rates, and movement angles in Roesel’s Bush
Cricket (Metrioptera roeseli). Conservation Biology 16:1562–1569.
Bierregaard, R.O., T.E. Lovejoy, V. Kapos, A. Augusto dos Santos, and R.W. Hutchings.
1992. The biological dynamics of tropical rainforest fragments: A prospective
comparison of fragments and continuous forest. Bioscience 42:859–866.
Britten H.B., and P.F. Brussard. 1992. Genetic divergence and the Pleistocene history
of alpine butterfl ies (Nymphalidae) and the endangered Nymphalidae in western
North America. Zoology 70:539–548.
Britten, H.B., and R.W. Rust. 1996. Populaiton structure of a sand dune-obligate
beetle, Eusattus muricatus, and its implications for dune management. Conservation
Britten H.B., P.F. Brussard, D.D. Murphy, and G.T. Austin. 1994. Colony isolation
and isozyme variability of the Western Seep Fritillary, Speyeria nokomis
apacheana (Nymphalidae), in the Western Great Basin. Great Basin Naturalist
Brittnacher, J.G., S.R. Sims, and F.J. Ayala. 1978. Genetic differentiation between species
of the genus Speyeria (Lepidoptera: Nymphalidae). Evolution 32:199–210.
Cardy, B.J., C.W. Stuber, J.F. Wendel, and M.M. Goodman. 1983. Techniques for
starch gel electrophoresis of enzymes from maize (Zea mays L.). Institute of
Statistics Monograph Series No.1317R, North Carolina State University, Raleigh,
Castellón, T.D., and K.E. Sieving. 2006. An experimental test of matrix permeability
and corridor use by an endemic understory bird. Conservation Biology
Fahrig, L., and G. Merriam. 1985. Habitat patch connectivity and population survival.
Fischer, J., and D.B. Lindenmayer. 2007. Landscape modification and habitat fragmentation:
A synthesis. Global Ecology and Biogeography 16:265–280.
Goudet, J. 2001. F-STAT user’s Manual: A Program to Estimate and Test Gene Diversities
and Fixation Indices (version 2.9.3). Available online at http://www.unil.
ch/izea/softwares/fstat.html. Accessed September 2003.
720 Southeastern Naturalist Vol. 8, No. 4
Haag C.R., M. Saastamoinen, J.H. Marden, and I. Hanski. 2005. A candidate locus for
variation in dispersal rate in a butterfl y metapopulation. Proceedings of the Royal
Society B 272:2449–2456.
Haas, C.A. 1995. Dispersal and use of corridors by birds in wooded patches on an agricultural
landscape. Conservation Biology 9:845–854.
Haddad, N.M. 1999a. Corridor and distance effects on interpatch movements: A landscape
experiment with butterfl ies. Ecological Applications 9:612–622.
Haddad, N.M. 1999b. Corridor use as predicted from behaviors at habitat boundaries.
American Naturalist 153:215–227.
Haddad, N.M. 2000. Corridor length and patch colonization by a butterfl y, Junonia
coenia. Conservation Biology 14:738–745.
Haddad, N.M., and K.A. Baum. 1999. An experimental test of corridor effects on butterfl
y densities. Ecological Applications 9:623–633.
Haddad, N.M., and J.J. Tewksbury. 2005. Low-quality habitat corridors as movement
conduits for two butterfl y species. Ecological Applications 15:250–257.
Haddad, N.M., D.R. Bowne, A. Cunningham, B. Danielson, D. Levey, S. Sargent, and
T. Spira. 2003. Corridor use by diverse taxa. Ecology 84:609–615.
Hale, M.L., P.W. Lurz, M.D. Shirley, S. Rushton, R.M. Fuller, and K. Wolff. 2001. Impact
of landscape management on the genetic structure of red squirrel populations.
Hänfl ing, B., W. Durka, and R. Brandl. 2004. Impact of habitat fragmentation on genetic
population structure of roach, Rutilus rutilus, in a riparian ecosystem. Conservation
Hanski, I., and I. Saccheri. 2006. Molecular-level variation affects population growth
in a butterfl y metapopulation. PLoS Biology 4: e129.
Hanski, I., C. Erälahti, M. Kankare, O. Ovaskainen, and H. Sirén. 2004. Variation in
migration rate among individuals maintained by landscape structure. Ecology Letters
Harris, L.D. 1984. The Fragmented Forest: Island Biogeographic Theory and Preservation
of Biotic Diversity. University of Chicago Press, Chicago, IL.
Hoole, J.C., D.A. Joyce, and A.S. Pullin. 1999. Estimates of gene fl ow between populations
of the Swallowtail Butterfl y, Papilio machaon, in Broadland, UK and implications
for conservation. Biological Conservation 89:293–299.
Johannesen, J., U. Schwing, W. Seufert, A. Seitz, and M. Veith. 1997. Analysis of gene
fl ow and habitat-patch network for Chazara briseis (Lepidoptera: Satyridae) in an
agricultural landscape. Biochemical Systematics and Ecology 25:419–427.
Keyghobadi, N., J. Roland, and C. Strobeck. 1999. Infl uence of landscape on the population
genetic structure of the alpine butterfl y Parnassius smintheus (Papilionidae).
Molecular Ecology 8:1481–1495.
Knutsen, H., B.A. Rukke, P.E. Jorde, and A.I. Rolf. 2000. Genetic differentiation
among populations of the beetle Bolitophagus retculatus (Coleoptera: Tenebrionidae)
in a fragmented and continuous landscape. Heredity 84:667–676.
Kronfrost, M.R., and T.H. Fleming. 2001. Lack of genetic differentiation among
widely spaced subpopulations of a butterfl y with home-range behavior. Heredity
Levene, H. 1949. On a matching problem arising in genetics. Annals of Mathematics
and Statistics 20:91–94.
Machtans, C.S., M. Villard, and S.J. Hannon. 1996. Use of riparian buffer strips as
movement corridors by forest birds. Conservation Biology 10:1366–1379.
Manel, S., M.K. Schwartz, G. Luikart, and P. Taberlet. 2003. Landscape genetics:
Combining landscape ecology and population genetics. Trends in Ecology and
2009 C.N. Wells, R.S. Williams, G.L. Walker, and N.M. Haddad 721
Mann, C.C., and M.L. Plummer. 1995. Are wildlife corridors the right path? Science
Mech, S.G., and J.G. Hallett. 2001. Evaluating the effectiveness of corridors: A genetic
approach. Conservation Biology 15:467–474.
Meglécz, E., G. Nève, K. Pecsenye, and Z. Varga. 2004. Genetic variations in space
and time in Parnassius mnemosyne (L.) (Lepidoptera) populations in northeast
Hungary: Implications for conservation. Biological Conservation 89:251–259.
Merriam, G., and D.A. Saunders. 1993. Corridors in restoration of fragmented landscapes.
Pp. 71–87, In D.A. Saunders, R.J. Hobbs, and P.R. Ehrlich (Eds.). Nature
Conservation III: Reconstruction of Fragmented Ecosystems. Surrey Beatty and
Sons, Chipping Norton, New South Wales, Australia.
Mettler, L.E., T.G. Gregg, and H.E. Schaffer. 1988. Population Genetics and Evolution,
2nd Edition. Patience Hall, Englewood Cliffs, NJ.
Nei, M. 1973. Analysis of gene diversity in subdivided populations. Proceedings of
the National Academy of Science 70:3321–3323.
Nei, M. 1978. Estimation of average heterozygosity and genetic distance from a
small number of individuals. Genetics 89:583–890.
Nei, M. 1987. Molecular Evolutionary Genetics. Columbia University Press, New
Nice, C.C., and A.M. Shapiro. 2001. Population genetic evidence of restricted gene
fl ow between host races in the butterfl y genus Mitoura (Lepidoptera: Lycaenidae).
Annals of the Entomological Society of America 94:257–267.
Noss, R.F. 1991. Landscape connectivity: Different functions at different scales.
Pp. 27–39, In W.E. Hudson (Ed.). Landscape Linkages and Biodiversity. Island
Press, Washington, DC.
Noss, R.F. 1992. Wildlife corridors. Pp. 76–113, In D. Smith and P. Hellmund (Eds.).
Ecology of Greenways. University of Minnesota Press, Minneapolis, MN.
Opler, P.A. 1998. A Field Guide to Eastern Butterflies. Houghton Mifflin, New
Peterson, M.A., and R.F. Denno. 1998. Life-history strategies and the genetic structure
of phytophagous insect populations. Pp. 263–322, In M. Mopper and S.Y.
Strauss (Eds.). Genetic Structure and Local Adaptation in Natural Insect Populations.
Chapman and Hall, New York, NY.
Porter, A.H., and S.O. Mattoon. 1989. A new subspecies of Coenonympha tullia
(Müller) (Nymphalidae: Satyrinae) confined to the coastal dunes of northern
California. Journal of the Lepidopterists’ Society 43:229–238.
Pyle, R.M. 1981. National Audubon Society Field Guide to North American Butterfl
ies. Alfred A. Knopf, New York, NY.
Reed, D.H., and R. Frankham. 2003. Correlation between fitness and genetic diversity.
Conservation Biology 17:230–237.
Saunders, D.A., J.A. Hobbs, and C.R. Margules. 1991. Biological consequences of
ecosystem fragmentation: A review. Conservation Biology 5:18–32.
SAS Institute. 2001. PROC User’s Manual, version 8.12., Cary, NC.
Schmitt, T., and A. Seitz. 2002. Infl uence of habitat fragmentation on the genetic
structure of Polyommatus coridon (Lepidoptera: Lycaenidae): implications for
conservation. Biological Conservation 107:291–297.
Selander, R.K., M.H. Smith, S.Y. Yang, W.E. Johnson, and J.B Gentry. 1971. Biochemical
polymorphism and systematics in the genus Peromyscus: Variation in
the Oldfield Mouse (Peromyscus polionotus). University of Texas Publication
722 Southeastern Naturalist Vol. 8, No. 4
Six, D.L., T.D. Paine, and J.D. Hare. 1999. Allozyme diversity and gene fl ow in the
bark beetle, Dendroctonous jeffreyi (Coleoptera: Scolytidae). Canadian Journal
of Forest Restoration 29:315–323.
Slatkin, M. 1985. Gene fl ow in natural populations. Annual Review of Ecology and
Solé, R.V., D. Alonso, and J. Saldana. 2004. Habitat fragmentation and biodiversity
collapse in neutral communities. Ecological Complexity 1:65–75.
Soltis, D.E., C.H. Haufl er, D.C. Darrow, and G.J. Gastony. 1983. Starch gel electrophoresis
of ferns: A compilation of grinding buffers, gel and electrode buffers,
and staining schedules. American Fern Journal 73:9–27.
Sutcliffe, O.L., and C.D. Thomas. 1996. Open corridors appear to facilitate dispersal
of Ringlet Butterfl ies (Aphantopus hyperantus) between woodland clearings.
Conservation Biology 10:1359–1365.
Tewksbury, J.J., D.J. Levey, N.M. Haddad, S. Sargent, J.L. Orrock, A. Weldon, B.J.
Danielson, J. Brinkerhoff, E.I. Damschen, and P. Townsend. 2002. Corridors affect
plants, animals, and their interactions in fragmented landscapes. Proceedings
of National Academy of Science 99:12923–12926.
Vandewoestijne, S., and M. Baguette. 2004. Genetic population structure of the vulnerable
Bog Fritillary butterfl y Hereditas 141:199–206.
Vandewoestijne, S., G. Nève, and M. Baguette. 1999. Spatial and temporal population
genetic strucuture of the butterfl y Aglais urticae L. (Lepidoptera: Nymphalidae).
Molecular Ecology 8:1539–1543.
Van Rossum, F., S.C. De Sousa, and L. Triest. 2004. Genetic consequences of
habitat fragmentation in an agricultural landscape on the common Primula
veris, and comparison with its rare congener, P. vulgaris. Conservation Genetics
Wallace, B. 1981. Basic Population Genetics. Columbia University Press, New
Watt, W.B. 2003. Mechanistic studies of butterfl y adaptation. Pp. 319–352, In C.L.
Boggs, W.B. Watt, and P.R. Ehrlich, (Eds.). Butterfl y Ecology and Evolution Take
Flight. University of Chicago Press, Chicago, IL.
Werth, C.R. 1985. Implementing an isozyme laboratory at a field station. Virginia
Journal of Science 36:53–76.
Wilcove, D.S., D. Rothstein, J. Dunbow, A. Phillips, and E. Losos. 1998. Quantifying
threats to imperiled species in the United States. Bioscience 48:607–615.
Williams, B.L., J.D. Brawn, and K.N. Page. 2003. Landscape-scale genetic effects
of habitat fragmentation on a high gene-fl ow species: Speyeria idalia (Nymphalidae).
Molecular Ecology 12:11–20.
Wright, S. 1951. The genetical structure of populations. Annals of Eugenics
Wright, S. 1965. The interpretation of population genetic structure by F-statistics
with special regard to systems of mating. Evolution 19:395–420.
Wright, S. 1978. Variability Within and Among Natural Populations. Volume 4. The
University of Chicago Press, Chicago, IL.
Yeh, F.C., R.C. Yang, T.B. Boyle, Z.H.Ye, and J.X. Mao. 1997. Popgene-32: The
user-friendly shareware for population genetic analysis. Molecular Biology and
Biotechnology Centre, University of Alberta, Edmonton, Alberta, Canada.
Zhang, Z., and M.B. Usher. 1991. Dispersal of Wood Mice and Bank Voles in an
agricultural landscape. Acta Theriological 36:239–245.