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1 Journal of Herpetology, Vol. 49, No. 2, , 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 ALLMAN 1 1 Department of Biological Sciences, Florida Gulf Coast University, Fort Myers, Florida 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 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. 2 Corresponding Author. DOI: / 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. Introduced 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

2 GENETIC STRUCTURE OF CTENOSAURA SIMILIS IN FLORIDA 231 (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 MgCl 2, 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 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 Neighbor- Net (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 = 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 (based on a maximum composite likelihood estimate). Based on p-distance analysis

3 232 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 GenBank accession numbers Authors C. acanthura 2 EU246718, EU Zarza et al., 2008 C. bakeri 4 EU , EU271879; GU Pasachnik et al., 2009; Pasachnik et al., 2010 C. flavidorsalis 11 AF417076, AF417083, AF417086; AY , AY Hasbun et al., unpubl. data; Hasbun et al., 2005 C. hemilopha 5 EU , EU Zarza et al., 2008 C. melanosterna 3 GU332008, GU Pasachnik et al., 2010 C. oaxacana 4 AF417091, AF417096, AF417098; AY Hasbun et al., unpubl. data; Hasbun et al., 2005 C. oedirhina 5 GU , GU Pasachnik et al., 2010 C. palearis 6 GU , GU Pasachnik et al., 2010 C. pectinata 78 EU , EU Zarza et al., 2008 C. quinquecarinata 4 AF417103, AF417105, AF417106; AY Hasbun et al., unpubl. data; Hasbun et al., 2005 C. similis 21 EU246704, EU271880, EU ; EU ; GU331994; U66228 Gutsche and Köhler 2008; Pasachnik et al., 2010; Pasachnik et al., 2009; Sites et al., 1996 Iguana iguana 1 U66230 Sites et al., 1996 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) 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.

4 GENETIC STRUCTURE OF CTENOSAURA SIMILIS IN FLORIDA 233 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. Species, location (ID) C. similis, KI, Florida, USA C. similis, Mexico (H11) C. similis, Honduras (H1) C. similis, Honduras (H7) C. similis, Honduras (213339) C. similis, Honduras (213332) C. similis, Honduras (213127) C. similis, Honduras (213126) C. similis, Honduras (213098) C. similis, Honduras (BYU39457) C. similis, Honduras (213333) C. similis, Honduras (213329) C. similis, Honduras (213328) C. similis, Honduras (213327) C. similis, Honduras (213123) C. similis, Honduras (213121) C. similis, Honduras (213120) C. similis, Honduras (213118) C. similis, Honduras (AG 30) C. similis, Honduras (AG 27) C. similis, Honduras (AG 26) C. similis, Honduras (AG 25) I. iguana

5 234 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.

6 GENETIC STRUCTURE OF CTENOSAURA SIMILIS IN FLORIDA 235 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 ). LITERATURE CITED ARÉVALO, E., S. K. DAVIS, AND J. W. 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