Journal of Herpetology, Vol. 49, No. 2, 230–236, 2015
Copyright 2015 Society for the Study of Amphibians and Reptiles
Successful Establishment of a Non-Native Species after an Apparent Single Introduction
Event: Investigating ND4 Variability in Introduced Black Spiny-Tailed Iguanas
(Ctenosaura similis) in Southwestern Florida
ANDREA M. NACCARATO,1,2 JAN B. DEJARNETTE,1
AND
PHIL ALLMAN1
1
Department of Biological Sciences, Florida Gulf Coast University, Fort Myers, Florida 33965 USA
ABSTRACT.—Recent studies on invasive species have led to the development of an apparent paradox when trying to explain how populations
succeed after experiencing genetic bottlenecks in their new environments. Many introduced populations retain genetic diversity from multiple
introduction events, but others that resulted from a single introduction event are expected to have low genetic diversity and low evolutionary
potential. Introduced Black Spiny-Tailed Iguanas (Ctenosaura similis) on a barrier island (Keewaydin Island [KI]) in subtropical Florida are
thought to be the result of a single introduction of a small founder group, although this population has expanded significantly since its
founding in 1995. We investigated the presence of this genetic paradox by determining the genetic variation of this introduced population. We
extracted DNA from muscle tissue samples (N = 21) and sequenced a region of the ND4 gene to allow for comparison with previously described
native Ctenosaura populations. We documented a single haplotype from KI, which means this iguana population likely descended from a single
introduction event and one geographic source population (Honduras). If this single haplotype represents an overall reduction in genetic
diversity, then this population demonstrates that genetic variability is not always necessary for a species to become established in a new
ecological range. This interpretation may have strong implications for invasive species management.
The anthropogenic spread of animal and plant species
continues to disturb native ecosystems and impact global
biodiversity. Although such translocations are increasingly
common (Ricciardi et al., 2000; Floerl et al., 2009), the likelihood
of establishment after a dispersal event depends primarily on
the biology of the invader species and its new environment
(East et al., 1999; Koop, 2004; Lloret et al., 2005). Previous
studies have shown species with high reproductive output and
tolerance of environmental extremes can establish new populations quickly (Reed, 2005; Guzman et al., 2012). The successful
establishment of the Cuban Treefrog (Osteopilus septentrionalis)
in Florida is due to high fecundity, broad habitat requirements,
and a broad diet (Schwartz and Henderson, 1991; Meshaka,
1996, 2001). Factors associated with abiotic and biotic interactions within the new environment may also influence the
success of the introduced species (Williams et al., 1990; Mack et
al., 2000; Theoharides and Dukes, 2007; Sih et al., 2010).
Some minimal level of genetic diversity is also typically
required for an introduced species to sustain a population (Le
Roux and Wieczorek, 2009). Since small founding populations
are expected to have reduced genetic diversity, the repeated
success of species introductions has led to the recognition of a
genetic paradox (Kolbe et al., 2004; Frankham, 2005; Roman and
Darling, 2007). Populations with limited founders are expected
to suffer founder effects and therefore have a high risk for
inbreeding and extirpation (Frankham, 2005). But recent studies
indicate that introduced populations of certain species can
maintain genetic diversity that is equal to, or higher than, the
source populations (Cabe, 1998; Lockwood et al., 2005; Kelly et
al., 2006). Roman and Darling (2007) found that only 16 (37%) of
43 aquatic species reviewed showed significant reduction of
genetic diversity in instances of introductions with few
founders. Furthermore, 10 (63%) of the 16 were species capable
of reproducing without sexual recombination. These findings
indicate that successful introduced species appear to overcome
founder effects associated with reduced genetic diversity.
The size and number of introductions into an area seem to
mediate genetic diversity of the founding population. Intro2
Corresponding Author. E-mail: [email protected]
DOI: 10.1670/13-060
duced Brown Anole (Anolis sagrei) populations in Florida have
higher genetic diversity than do populations in Cuba as a result
of at least eight introduction events (Kolbe et al., 2004). Similar
evidence has been provided for many plant and animal species,
indicating that multiple introduction events elevate genetic
diversity and enhance ability to succeed and evolve (Sakai et al.,
2001; Henshaw et al., 2005; Dlugosch and Parker, 2008; Pairon et
al., 2010; Bouchard et al., 2011).
Although the presence of multiple introductions may solve
the genetic diversity paradox for many species, successful
introductions are known to result from a single or small
introduction event. Bai et al. (2012) reported that American
Bullfrogs, Lithobates (= Rana) catesbeianaus, established several
introduced populations in China from small founder populations. Successful populations that exhibit low genetic diversity
(Wang et al., 2005; Geng et al., 2007) are afforded an opportunity
to purge deleterious alleles that would otherwise restrict
evolutionary fitness and population growth.
In 1995, an island resident introduced a small number (5–30)
of Black Spiny-Tailed Iguanas (Ctenosaura similis; Gray, 1831) to
Keewaydin Island (KI) in southwestern Florida (Krysko et al.,
2003). The population’s apparent establishment and rapid
growth might indicate the high reproductive fitness typically
associated with high genetic diversity. In order to confirm that
this population of C. similis is the result of a single introduction,
we measured its genetic diversity using the nicotinamide
adenine dehydrogenase subunit 4 (ND4) mitochondrial gene,
compared the diversity to populations throughout the species
native range, and used haplotype matching to identify the
source population(s). Others have demonstrated that the ND4
gene displays an appropriate level of sensitivity (Sunnucks,
2000) for studying genetic structure within and among
Ctenosaura populations (Hasbún et al., 2005; Zarza et al., 2008;
Pasachnik et al., 2009).
MATERIALS
AND
METHODS
Specimen Collection.—We surveyed five sites on KI for C. similis.
Keewaydin Island is a mostly undeveloped barrier island (Fig. 1)
managed by Rookery Bay National Estuarine Research Reserve
GENETIC STRUCTURE OF CTENOSAURA SIMILIS IN FLORIDA
(RBNERR) in Collier County, Florida. The island is approximately 6.4 km long by 0.32 km wide (Florida Department of
Environmental Protection, 2010) and has primarily beach dune,
coastal strand, and mangrove habitats. Habitat disturbance on KI
occurs from residential construction activities, tropical storms, or
invasive species (e.g., Wild Boar [Sus scrofa]).
We collected 21 specimens from three sites in November 2009
by noose pole capture, opportunistic hand capture, or as
donations from invasive species eradication efforts by RBNERR
or the Florida Fish and Wildlife Conservation Commission
(FWC). We stored specimens in a subzero freezer at Florida Gulf
Coast University (FGCU) until tissue extraction. Samples
included individuals of both sexes (10 females, 11 males) and
all age classes (10 juveniles, 2 subadults, 9 adults).
DNA Sequencing.—We removed 21 muscle tissue samples from
a rear leg or the base of the tail and preserved these samples in
95% ethanol at 48C. Proteinase K digested samples for 3 h at 558C
and the ZR Genomic DNA-Tissue MiniPrep Kite (Zymo
Research Corp.) extracted DNA. We assessed DNA quantity
and quality by 1% agarose gel electrophoresis and UV-imaging
on an Alpha Imager HP (AlphaInnotech).
We amplified a 609-base pair (bp) fragment of the ND4 region
using primers ND4 and ND4Rev (Arévalo et al., 1994) obtained
from Eurofins MWG Operon. The PCR was composed of Taq 2·
MasterMix (New England BioLabs), 0.2 M trehalose, 1.5 lM
MgCl2, 0.4 lM ND4 forward primers, 0.4 lM ND4Rev reverse
primers, and 1 lg of DNA. Thermal cycling began with an
initial denaturation at 948C for 5 min followed by 40 cycles of
denaturation at 948C for 30 sec, annealing at 528C for 40 sec, and
extension at 728C for 1 min, with a final extension step at 728C
for 5 min (after Zarza et al., 2008). We assessed amplified PCR
products with 2% agarose gel electrophoresis, isolated these
products with GeneCatche (Epoch BioLabs, Inc), and then sent
the DNA samples to Eurofins MWG Operon (Huntsville, AL)
for sequencing (GenBank accession number KJ629171).
Data Analysis.—We used MEGA software (version 5) to
analyze ND4 sequences for each specimen (Kumar et al., 2008;
Tamura et al., 2011). ClustalW function in MEGA aligned
sequences with default parameters, and then we visually
inspected the sequences. We defined unique ND4 haplotypes
by at least one nucleotide alteration as compared with other
sequences. GenBank was our source for additional Ctenosaura
ND4 sequences (Benson et al., 2011; Table 1). MEGA determined
nucleotide composition, number of variable sites, parsimonyinformative sites, 4-fold degenerate sites, and nondegenerate sites
for all C. similis sequences. Pairwise genetic distances determined
nucleotide diversity between haplotypes. We constructed a
cladogram using MEGA to determine relationships between
haplotypes and species.
We determined nucleotide diversity within C. similis by
creating pairwise genetic distances (p-distances) matrices,
where each p-distance value is the proportion of nucleotide
positions that differ when compared with a given paired
sequence. One p-distances matrix used a pairwise deletion
mechanism to provide a more-specific gauge of dissimilarity
between sequence pairs. A second p-distances matrix used a
complete deletion mechanism, in which only overlapping
nucleotide positions common to all sequences were analyzed,
to be consistent with the cladogram analysis.
Model test function in MEGA determined that the general
time reversible + gamma distribution was the most suitable
nucleotide substitution model for the Ctenosaura sequences. The
model test suggests the best substitution model based on
231
FIG. 1. Map displaying the general location of Keewaydin Island in
southwestern Florida and the specific outline of the island in Collier
County, Florida.
Bayesian score and Akaike information criteria. MEGA constructed a consensus cladogram using the maximum likelihood
(ML) method with complete deletion mechanism and statistical
bootstrapping (1,000 pseudoreplicates; Felsenstein, 1985) to
obtain a measure of branch support. Additionally, a haplotype
network was created using Maximum Spanning Tree function in
SplitsTree Software (Huson and Bryant, 2006) using NeighborNet (Bryant and Moulton, 2004) and ML algorithms. The Green
Iguana (Iguana iguana) was used as an outgroup because
Ctenosaura share many iguanid traits and represent a sister
clade to Iguana (Wiens and Hollingsworth, 2000).
RESULTS
ND4 sequences from 21 iguanas captured on KI were
analyzed to determine the level of genetic diversity in this
introduced population. All specimens possessed identical
nucleotide sequences (609 bp) at the ND4 locus, suggesting
the presence of a single haplotype. Because all sequences were
identical, all p-distances equaled zero (data not shown). For this
ND4 haplotype, the proportions of each of the four nucleotides
are as follows: A = 0.338; C = 0.330; G = 0.103; T = 0.228.
Considering all C. similis sequences (i.e., KI and those
downloaded from GenBank), 147/895 sites were variable and
37 sites were parsimony-informative. Of the 895 sites, 105 were
4-fold degenerate and 595 were nondegenerate. The transition :
transversion ratio (R) equaled 1.131 (based on a maximum
composite likelihood estimate). Based on p-distance analysis
232
A. M. NACCARATO ET AL.
TABLE 1. List of Ctenosaura species (and outgroup) used for Ctenosaura data analyses and cladogram construction along with the number of
sequences (sample size) acquired for each species, their GenBank accession numbers, and authors who submitted these sequences.
Iguana species
Sample Size
C. acanthura
C. bakeri
C. flavidorsalis
2
4
11
C. hemilopha
C. melanosterna
C. oaxacana
5
3
4
C.
C.
C.
C.
oedirhina
palearis
pectinata
quinquecarinata
C. similis
Iguana iguana
GenBank accession numbers
EU246718, EU246733
EU271876–77, EU271879; GU331998
AF417076, AF417083, AF417086; AY730645–49,
AY730651–52
EU246694–97, EU246705
GU332008, GU332011–12
AF417091, AF417096, AF417098; AY730655
5
6
78
4
GU331999–2001, GU906221–22
GU332002–04, GU906219–20
EU246698–755, EU24757–780
AF417103, AF417105, AF417106; AY730660
21
EU246704, EU271880, EU407508–11; EU407513–
25; GU331994; U66228
1
U66230
with pairwise deletion mechanism (895 bps; Table 2), the ND4
haplotype discovered on KI matched the C. similis haplotype
from the island of Utila, Honduras (Pasachnik et al., 2009) as
well as additional sequences from San Lorenzo, San Patricio,
and the island of Utila, Honduras (Gutsche and Köhler, 2008).
There is a distinctly higher level of dissimilarity between
haplotype 1 (H1) and all other C. similis sequences.
According to the ML consensus cladogram (Fig. 2) and
haplotype network (Fig. 3), the KI haplotype clusters with other
C. similis sequences from Honduras. The C. similis haplotype
from Mexico branches separately from all Honduran sequences
(with 99% bootstrap support). One C. similis haplotype (H1)
from Honduras branches even more distinctly from the rest of
the C. similis sequences (Fig. 2). Ctenosaura similis H1 clusters
with a different species, namely the endangered Yellowback
Spiny-Tailed Iguana (Ctenosaura flavidorsalis). Such separation in
the consensus cladogram is consistent with high dissimilarity
values in the p-distances matrix. The overall branching in the
haplotype network (Fig. 3) is in agreement with the species
relationships shown in Fig. 2, including the placement of H1
with C. flavidorsalis.
DISCUSSION
The goal of this study was to explore the genetic diversity of a
Black Spiny-Tailed Iguana (Ctenosaura similis) population
introduced to Keewaydin Island, Florida. We measured the
genetic diversity of KI’s population, compared its haplotype
diversity to Ctenosaura populations throughout their native
range, and used haplotype matching to identify the source
population. Although the entire native range of C. similis has yet
to be characterized at the ND4 locus, at least seven haplotypes
have been recognized (Table 2). The observation of one ND4
haplotype on KI, which may represent one maternal lineage,
suggests that a single introduction event occurred from one area
of the native range, specifically the Caribbean coast of
Honduras or a nearby island (i.e., Utila). This interpretation
supports the prediction that this introduced population conforms to the paradox of established, nonnative populations that
descended from single introduction events.
Other researchers have matched haplotypes or alleles
between populations to suggest the original source of exotic
or invasive species. For example, Eales and Thorpe (2010)
Authors
Zarza et al., 2008
Pasachnik et al., 2009; Pasachnik et al., 2010
Hasbun et al., unpubl. data; Hasbun et al.,
2005
Zarza et al., 2008
Pasachnik et al., 2010
Hasbun et al., unpubl. data; Hasbun et al.,
2005
Pasachnik et al., 2010
Pasachnik et al., 2010
Zarza et al., 2008
Hasbun et al., unpubl. data; Hasbun et al.,
2005
Gutsche and Köhler 2008; Pasachnik et al.,
2010; Pasachnik et al., 2009; Sites et al.,
1996
Sites et al., 1996
discovered a population of introduced Brown Anoles (Anolis
sagrei) on St. Vincent Island, Lesser Antilles that possessed a
single haplotype that was characteristic of another introduced
population in Tampa, Florida. The ability to determine the
source population from which an invasive species originates is
important when designing appropriate actions to hinder
additional translocations.
Despite evidence for lower genetic diversity, the C. similis
population on KI noticeably expanded from the point of
introduction in <10 years (Krysko et al., 2003). Typically, it is
surmised that founding populations with lower genetic diversity will suffer inbreeding depression, and eventual extirpation,
similar to the inbreeding vortex experienced by endangered
species (Beebee and Rowe, 2008). Therefore, successful establishment and expansion after a genetic bottleneck is interpreted
as paradoxical (Frankham, 2005; Pérez et al., 2006), especially if
the possibility of multiple introductions has been rejected
(Frankham, 2005).
Several hypotheses attempt to explain this paradox. Upon
introduction, the initial bottleneck may reduce genetic diversity,
which may remove deleterious alleles and allow the population
to expand (Bossdorf et al., 2005; Frankham, 2005; Roman and
Darling, 2007). A founding population may possess some genes
fortuitously advantageous or neutral in the new environment
(Suarez and Tsutsui, 2008) or even a single ‘‘general-purpose
genotype’’ that imparts fitness to the population without genetic
diversity (Bossdorf et al., 2005; Roman and Darling, 2007).
Certain life history traits may allow introduced species to
exploit new environments. Such traits include tolerance of
disturbed land, an opportunistic diet, high reproductive output,
and polygamous reproductive strategies. For example, exotic
Puerto Rican Crested Anole (Anolis cristatellus) individuals on
the island of Dominica were able to expand rapidly from a small
founding population because females likely had stored sperm
from multiple males (Eales et al., 2008). Future study may reveal
if C. similis is capable of such polygamous reproductive
strategies that provide additional genetic diversity; however,
these iguanas possess the other three life history traits that help
small founder groups become established (Henderson, 1973;
Fitch and Henderson, 1978).
Ctenosaura similis is a species that evolved in an environment
prone to tropical disturbances. If the species is represented by
alleles that result in ecological generalism or plasticity, then C.
1 C. similis, KI, Florida,
USA
2 C. similis, Mexico (H11)
3 C. similis, Honduras
(H1)
4 C. similis, Honduras
(H7)
5 C. similis, Honduras
(213339)
6 C. similis, Honduras
(213332)
7 C. similis, Honduras
(213127)
8 C. similis, Honduras
(213126)
9 C. similis, Honduras
(213098)
10 C. similis, Honduras
(BYU39457)
11 C. similis, Honduras
(213333)
12 C. similis, Honduras
(213329)
13 C. similis, Honduras
(213328)
14 C. similis, Honduras
(213327)
15 C. similis, Honduras
(213123)
16 C. similis, Honduras
(213121)
17 C. similis, Honduras
(213120)
18 C. similis, Honduras
(213118)
19 C. similis, Honduras
(AG 30)
20 C. similis, Honduras
(AG 27)
21 C. similis, Honduras
(AG 26)
22 C. similis, Honduras
(AG 25)
23 I. iguana
Species, location (ID)
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
0.000 0.013 0.117 0.000 0.006 0.007 0.003 0.004 0.000 0.007 0.006 0.006 0.006 0.006 0.004 0.004 0.004 0.003 0.000 0.000 0.000
0.013
0.128 0.128 0.147 0.144 0.129 0.131 0.131 0.129 0.131 0.125 0.129 0.129 0.129 0.129 0.129 0.129 0.129 0.131 0.131 0.131 0.131 0.131
0.000 0.013
0.000 0.000 0.013
0.000 0.000 0.000 0.013
0.000 0.013 0.117 0.000 0.006 0.007 0.003 0.004 0.000 0.007 0.006 0.006 0.006 0.006 0.004 0.004 0.004 0.003 0.000 0.000
0.000 0.013 0.117 0.000 0.006 0.007 0.003 0.004 0.000 0.007 0.006 0.006 0.006 0.006 0.004 0.004 0.004 0.003 0.000
0.000 0.013 0.117 0.000 0.006 0.007 0.003 0.004 0.000 0.007 0.006 0.006 0.006 0.006 0.004 0.004 0.004 0.003
0.002 0.002 0.002 0.002 0.013
0.001 0.002 0.002 0.002 0.002 0.013
0.000 0.001 0.002 0.002 0.002 0.002 0.013
0.004 0.015 0.117 0.004 0.006 0.007 0.000 0.001 0.003 0.007 0.006 0.006 0.006 0.006 0.001 0.001 0.001
0.007 0.018 0.116 0.006 0.007 0.008 0.001 0.000 0.004 0.008 0.007 0.007 0.007 0.007 0.000 0.000
0.007 0.018 0.116 0.006 0.007 0.008 0.001 0.000 0.004 0.008 0.007 0.007 0.007 0.007 0.000
0.000 0.000 0.001 0.002 0.002 0.002 0.002 0.013
0.003 0.003 0.003 0.003 0.003 0.003 0.003 0.003 0.013
0.000 0.003 0.003 0.003 0.003 0.003 0.003 0.003 0.003 0.013
0.007 0.018 0.116 0.006 0.007 0.008 0.001 0.000 0.004 0.008 0.007 0.007 0.007 0.007
0.009 0.013 0.116 0.008 0.000 0.001 0.006 0.007 0.006 0.001 0.000 0.000 0.000
0.009 0.013 0.116 0.008 0.000 0.001 0.006 0.007 0.006 0.001 0.000 0.000
0.000 0.000 0.003 0.003 0.003 0.003 0.003 0.003 0.003 0.003 0.013
0.000 0.000 0.000 0.003 0.003 0.003 0.003 0.003 0.003 0.003 0.003 0.013
0.001 0.001 0.001 0.001 0.003 0.003 0.003 0.003 0.003 0.003 0.003 0.003 0.011
0.009 0.013 0.116 0.008 0.000 0.001 0.006 0.007 0.006 0.001 0.000
0.009 0.013 0.116 0.008 0.000 0.001 0.006 0.007 0.006 0.001
0.011 0.011 0.105 0.010 0.001 0.003 0.007 0.008 0.007
0.003 0.003 0.003 0.003 0.003 0.002 0.002 0.002 0.002 0.000 0.000 0.000 0.000 0.013
0.002 0.003 0.003 0.003 0.003 0.003 0.000 0.000 0.000 0.001 0.002 0.002 0.002 0.002 0.013
0.001 0.002 0.003 0.003 0.003 0.003 0.003 0.001 0.001 0.001 0.000 0.002 0.002 0.002 0.002 0.013
0.000 0.013 0.117 0.000 0.006 0.007 0.003 0.004
0.007 0.018 0.116 0.006 0.007 0.008 0.001
0.004 0.015 0.117 0.004 0.006 0.007
0.003 0.003 0.003 0.002 0.001 0.001 0.001 0.001 0.003 0.003 0.003 0.003 0.003 0.003 0.003 0.003 0.013
0.001 0.003 0.003 0.003 0.001 0.000 0.000 0.000 0.000 0.003 0.003 0.003 0.003 0.003 0.003 0.003 0.003 0.013
0.004 0.004 0.003 0.003 0.000 0.004 0.004 0.004 0.004 0.004 0.003 0.003 0.003 0.003 0.000 0.000 0.000 0.000 0.014
0.011 0.015 0.114 0.009 0.001
0.009 0.013 0.116 0.008
0.000 0.013 0.108
0.012 0.014 0.014 0.014 0.014 0.014 0.012 0.014 0.014 0.014 0.014 0.014 0.014 0.014 0.014 0.014 0.014 0.014 0.014 0.014
0.005 0.012 0.000 0.004 0.005 0.003 0.004 0.000 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.003 0.000 0.000 0.000 0.000 0.014
0.012 0.005 0.006 0.006 0.006 0.007 0.006 0.004 0.006 0.006 0.006 0.006 0.007 0.007 0.007 0.006 0.006 0.006 0.006 0.006 0.014
2
0.099 0.090
0.012
1
TABLE 2. Pairwise genetic distances (pairwise deletion) for Ctenosaura similis and outgroup (Iguana iguana). Numbers below the diagonal represent proportion of dissimilarity and those above the
diagonal represent standard error.
GENETIC STRUCTURE OF CTENOSAURA SIMILIS IN FLORIDA
233
234
A. M. NACCARATO ET AL.
FIG. 2. Ctenosaura maximum likelihood consensus cladogram
(topology only) for ND4 gene with Iguana iguana as an outgroup. All
C. similis and C. flavidorsalis sequences are shown. Numbers shown
above nodes represent bootstrap values (1,000 pseudoreplicates).
similis would be a candidate invasive species even under
conditions of very low genetic diversity. Ctenosaura similis can
thrive during dynamic conditions because it can exploit
disturbed or edge habitats, take refuge in burrows or crevices,
eat a varied diet, grow rapidly, and reproduce copiously
(Henderson, 1973; Fitch and Henderson, 1978; Van Devender,
1982; McKercher, 2001; Gier, 2003; Pianka and Vitt, 2003; Krysko
et al., 2009; Funck and Allman, 2012). Keewaydin Island is
subject to disturbance from hurricanes, invasive plants and
animals, and human activity. The island is depleted of large
predators that would limit population growth of C. similis and
has an abundance of habitat (e.g., tortoise burrows) and food
resources that may promote rapid expansion on the island.
Epigenetics may also play a role in allowing introduced
species with low genetic diversity to become invasive in new
environments. Epigenetic processes modify gene expression
without changing the DNA sequence (e.g., gene silencing due to
methylation; Pérez et al., 2006; Jirtle and Skinner, 2007).
Epigenetic responses to the new environment may lead to
short-term phenotypic plasticity that could increase the likelihood of a successful invasion (Pérez et al., 2006). For example,
Gao et al. (2010) used alligator weed (Alternanthera philoxeroides), an invasive plant in China, to elucidate epigenetic
regulation in invasive species. These authors demonstrated that
1) wild plants in different introduced habitats (aquatic or
terrestrial) had signature methylation patterns, and 2) methylation patterns changed in individual plants when environmental conditions changed. Therefore, methylation-induced
modification to gene expression may possess the rapid
flexibility required for introduced species to succeed under
novel conditions.
The importance of conducting genetic analyses of Ctenosaura
species is illustrated by the difficulty in distinguishing species
by morphological descriptions alone. For example, C. similis
shares significant resemblance with Ctenosaura pectinata and has
been physically misidentified in Florida (Krysko et al., 2003;
McKercher, 2001). Our analyses revealed that C. similis may
have been misidentified by Pasachnik et al. (2010) because their
C. similis sequences (H1) clustered with C. flavidorsalis with
strong branch support and displayed the highest dissimilarity
values between other C. similis sequences. Correct differentiation between these two species is important, as C. similis is a
species of least concern and C. flavidorsalis is an endangered
species according to the International Union for Conservation of
Nature Red List (IUCN, 2012). This situation may serve as
another instance in which advances in genetic techniques can
clarify relationships when morphological identification is
difficult.
In the future, the C. similis population on KI should be
monitored for demographic and morphological changes, ana-
FIG. 3. Haplotype network of ND4 gene sequence from Ctenosaura with Iguana iguana as an outgroup.
GENETIC STRUCTURE OF CTENOSAURA SIMILIS IN FLORIDA
lyzed for genetic variation at additional neutral and proteincoding genes, and screened for epigenetic activity over time and
under various environmental conditions. Although mitochondrial DNA likely accumulates a greater number of synonymous
mutations than does nuclear DNA (Lee and Wei, 2007),
additional genes should be explored to uncover potential
genetic diversity not found in this study. Long-term monitoring
is important because a population crash following impressive
expansion may occur, as evidenced by the attempts at recovery
of Gray Wolves (Canis lupus) in Finland (Jansson et al., 2012) or a
failed introduction of Topmouth Gudgeon (Pseudorasbora parva)
in England (Copp et al., 2007). Currently, it seems C. similis
belongs to an infamous group of organisms who overcame the
obstacle of low genetic diversity upon colonization and have
exploited new lands outside their historical ecological ranges.
Acknowledgments.—We acknowledge E. Quintero from Ave
Maria University for generous assistance with DNA amplification procedures and funding for sequencing, as well as R.
Bullens of FGCU for providing laboratory space and materials
for dissections. We acknowledge E. Everham and J. Jackson of
FGCU for editing earlier versions of this manuscript and
serving on the project committee. We thank W. Gurley of the
U.S. Fish and Wildlife Service for assistance with GIS mapping.
We are grateful to the students of the FGCU Herpetology
Research Lab for their assistance in the field and lab: D. De
Witt, J. Donini, S. Funck, L. Hamilton, B. Jackson, J. Knott, and
J. Ross. Finally, this project would not have been possible
without the good-natured cooperation from staff at the
Conservancy of Southwest Florida, FWC, and RBNERR. The
Institutional Animal Care and Use Committee at Florida Gulf
Coast University approved the methods used in this study
(IACUC protocol no. 0910-04).
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Accepted: 15 June 2014.
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