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Hybridization and rapid differentiation after secondary contact between the native green anole (Anolis carolinensis) and the introduced green anole (Anolis porcatus)

Abstract In allopatric species, reproductive isolation evolves through the accumulation of genetic incompatibilities. The degree of divergence required for complete reproductive isolation is highly variable across taxa, which makes the outcome of secondary contact between allopatric species unpredictable. Since before the Pliocene, two species of Anolis lizards, Anolis carolinensis and Anolis porcatus, have been allopatric, yet this period of independent evolution has not led to substantial species‐specific morphological differentiation, and therefore, they might not be reproductively isolated. In this study, we determined the genetic consequences of localized, secondary contact between the native green anole, A. carolinensis, and the introduced Cuban green anole, A. porcatus, in South Miami. Using 18 microsatellite markers, we found that the South Miami population formed a genetic cluster distinct from both parental species. Mitochondrial DNA revealed maternal A. porcatus ancestry for 35% of the individuals sampled from this population, indicating a high degree of cytonuclear discordance. Thus, hybridization with A. porcatus, not just population structure within A. carolinensis, may be responsible for the genetic distinctiveness of this population. Using tree‐based maximum‐likelihood analysis, we found support for a more recent, secondary introduction of A. porcatus to Florida. Evidence that ~33% of the nuclear DNA resulted from a secondary introduction supports the hybrid origin of the green anole population in South Miami. We used multiple lines of evidence and multiple genetic markers to reconstruct otherwise cryptic patterns of species introduction and hybridization. Genetic evidence for a lack of reproductive isolation, as well as morphological similarities between the two species, supports revising the taxonomy of A. carolinensis to include A. porcatus from western Cuba. Future studies should target the current geographic extent of introgression originating from the past injection of genetic material from Cuban green anoles and determine the consequences for the evolutionary trajectory of green anole populations in southern Florida.

Empirical studies that document genetically cryptic hybridization patterns are rare (James & Abbott, 2005;Keller, Fields, Berardi, & Taylor, 2014;Kronforst, Young, Blume, Gilbert, & McMillan, 2006;Lavretsky, Engilis, Eadie, & Peters, 2015;Mims, Darrin Hulsey, Fitzpatrick, & Todd Streelman, 2010), and strong inferences often require sampling of reference populations of parental species as well as cytoplasmic and nuclear markers. Reference populations are necessary because repeated recombination and backcrossing can homogenize nuclear genotypes, which makes it challenging to distinguish hybrid populations from subpopulations of parental species (Della Croce, Poole, & Luikart, 2016). Non-recombining cytoplasmic markers retain parental genotypes and thus can help to distinguish hybrid populations from population structure (Della Croce et al., 2016). A mismatch between nuclear genotypes and cytoplasmic haplotypes is often the first step to identifying hybrid populations (Toews & Brelsford, 2012). Subsequent population genetic analyses are then needed to distinguish between cytoplasmic introgression, past (including ancient) hybridization, and incomplete lineage sorting (Della Croce et al., 2016). Identifying past hybridization events between introduced and native species is particularly challenging since the geographic location of the source population is often unknown or includes multiple source locations, and pure native populations might be genetically swamped by introduced genotypes (Caracristi & Schlötterer, 2003;Della Croce et al., 2016;Kolbe et al., 2004Kolbe et al., , 2007Kronforst et al., 2006). In this study, we aimed to reconstruct the invasion history of the Cuban green anole, Anolis porcatus, and determine the genetic consequences of localized, secondary contact with the native green anole, Anolis carolinensis, in southern Florida (USA). We use multilocus nuclear genotypes and mitochondrial haplotypes to distinguish between contemporary and past gene flow, allowing us to test whether secondary contact has eroded putative species boundaries or whether the two sister species are reproductively isolated and coexist as genetically distinct taxonomic units.
Anolis porcatus and A. carolinensis are allopatric species and have been geographically separated for an estimated 6-12 million years, since before the Pliocene (Campbell-Staton et al., 2012;Manthey, Tollis, Lemmon, Moriarty Lemmon, & Boissinot, 2016;Tollis & Boissinot, 2014). Anolis carolinensis is nested within a clade of A. porcatus from western Cuba, making the latter species paraphyletic Glor, Losos, & Larson, 2005). After the initial colonization of the Florida Peninsula, A. carolinensis has undergone substantial range expansion and differentiation resulting in five major mitochondrial clades. The current distribution ranges from southern Florida to North Carolina and west to Texas (Campbell-Staton et al., 2012;Manthey et al., 2016;Tollis & Boissinot, 2014).
The introduction of A. porcatus in Florida was first suggested in the 1990s based on morphological characters (Meshaka, Clouse, Butterfield, & Hauge, 1997) and later confirmed genetically (Kolbe et al., 2007). Two individuals collected in Miami were genetically similar to A. porcatus in western Cuba, indicating the putative source population of the introduction (Kolbe et al., 2007). Since the 1940s, seven other non-native anole species from various locations in Cuba and in the Caribbean have established in Miami, leading to admixture among genetically distinct source populations in several cases (Kolbe et al., 2007). Despite widespread intraspecific admixture, hybridization between recognized species is considered rare among anoles (Losos, 2009). A few cases are documented between closely related species, including A. porcatus × Anolis allisoni in central Cuba  and Anolis pulchellus × Anolis krugi in Puerto Rico (Jezkova, Leal, & Rodríguez-Robles, 2013).
Hybridization between A. carolinensis and A. porcatus has been suggested repeatedly, mainly because the two species have no species-specific morphological characters despite considerable divergence time (Camposano, 2011;Kolbe et al., 2007;Tollis, 2013).
Sufficient evidence for reproductive isolation or lack thereof has not been shown.
In this study, we examine whether A. porcatus and A. carolinensis are reproductively isolated species, and characterize the genetic consequences of secondary contact in South Miami. We used one mtDNA marker and 18 nuclear microsatellite loci to test whether K E Y W O R D S approximate Bayesian computation, cytonuclear discordance, reproductive isolation hybridization has occurred between the two species. We distinguished between contemporary and historic gene flow and estimated the timing of the admixture event. Discordance between nuclear and cytoplasmic markers is characteristic of hybridization and commonly used to identify hybrid individuals (Toews & Brelsford, 2012). If the two species interbreed in South Miami, we expect a high frequency of individuals with mismatches between nuclear genotypes and mtDNA haplotypes. To distinguish between contemporary, ongoing gene flow (such as expected in an active hybrid zone) versus a limited, historic gene flow event (such as common in human-mediated introductions), we used a genetic cluster analysis and a tree-based analysis allowing for migration between previously separated lineages. In the case of contemporary gene flow, we expect genetic clusters that reflect the two parental lineages and an admixed cluster in which individuals carry nuclear DNA from both parental lineages. In the case of an historic gene flow event, genetic admixture is likely erased, leading to an independent genetic cluster for the hybrid population in which the genetic variation is the result of historic, but not ongoing gene flow.

| Sample collection
We sampled 32 A. carolinensis individuals from the J.W. Corbett Wildlife Management in southern Florida, which is ~135 km north of Miami, 92 green anole individuals from the putative hybrid population in South Miami, and 54 A. porcatus individuals from western Cuba (Supporting Information Table S1). Genomic DNA was extracted from tail tips and liver tissue using a modified ethanol precipitation protocol.

| Molecular methods
We amplified a region of 343-571 bp of the mtDNA NADH dehydrogenase subunit two using primers from Campbell-Staton et al. of 25 mM MgCl, 1.0 µl of 10 µM primer, 0.1 µl of 5 units/µl Taq polymerase (New England Biolabs® Inc.), and 4 µl of 50 ng/µl genomic DNA. Cycles started with initial denaturation at 94°C for 2 min, followed by 35 cycles of 94°C for 45 s, T m for 45 s, 72°C for 1 min, and a final elongation step at 72°C for 10 min. PCR products were purified and sequenced at the Rhode Island Genomics and Sequencing Center.
We amplified 18 microsatellite markers using PCR with fluorescently labeled primers. We used seven newly designed primers (Supporting Information Table S1)

| Phylogenetic analysis and haplotype divergence
To determine the species identity of mtDNA haplotypes for indi-  Table S3). We combined previously published sequences with newly sampled individuals (see Supporting   Information Table S3) resulting in 111 individuals of A. porcatus from eastern and western Cuba spanning the entire native range, 83 individuals of A. carolinensis sampled throughout Florida and 86 individuals from South Miami ( Figure 1a). Anolis loysiana was used as outgroup taxon. Sequences were aligned and visually inspected for accuracy using the MUSCLE plugin in Geneious v7.1.9 (Kearse et al., 2012). We collapsed individual sequences into distinct haplotypes using DNAcollapser implemented in FaBox v1.41 (Villesen, 2007).
To retain individuals with short mtDNA sequences, we generated two separate alignments. One alignment consisted of 571 bp for 200 individuals, resulting in 156 haplotypes. The second alignment was 343 bp long and included all 280 samples resulting in 182 haplotypes. We used RAxML v8.0 (Stamatakis, 2006) implemented in the CIPRES Science Gateway v3.3 (Miller, Pfeiffer, & Schwartz, 2011) to generate maximum-likelihood phylogenies. Bootstrap values were obtained from 1,000 iterations using rapid bootstrapping.
We used pairwise sequence divergence to determine the degree of nucleotide divergence between native Cuban and introduced Florida A. porcatus haplotypes. We identified the genetically most similar individuals based on the fewest number of pairwise nucleotide differences. Pairwise sequence divergence was calculated as the number of nucleotide differences divided by the sequence length.

| Population structure and differentiation
First, we performed a Bayesian cluster analysis with STRUCTURE v2.3.4 (Rosenberg, 2004), using the admixture model and correlated allele frequencies. We allowed for gene flow among populations and modeled six different clustering scenarios, sequentially increasing the number of clusters from K = 1-6. We conducted 10 independent runs for each scenario with a burn-in of 500,000 and 1,000,000 MCMC iterations. We used delta K to determine the most likely number of clusters following the Evanno method (Evanno, Regnaut, & Goudet, 2005) implemented in STRUCTURE HARVESTER v0.6.94 (Earl & Vonholdt, 2012). We combined the genotype proportions of each cluster (q-matrix) from 10 independent runs with CLUMPP (Jakobsson & Rosenberg, 2007) and visualized the results with the R package ggplot2 v2.1.0 (Wickham, 2011). We repeated the Bayesian cluster analysis with population pairs (SFL-MIA and WCU-MIA) as well as WCU separately to identify potential population substructure. Model parameters were used as described above. Second, we used discriminant analysis of principal components (DAPC) to determine the degree of differentiation between clusters using the R package adegenet (Jombart, 2008). To characterize and find genetic clusters, DAPC uses a multivariate approach and PCA transformed allele frequencies. In contrast to the Bayesian clustering approach, DAPC does not rely on specific population model assumptions, such as HWE. The number of clusters (K) was sequentially increased starting with one cluster. The model fit for K clusters was determined with the Bayesian information criterion (BIC).

| Maximum likelihood and ABC modeling of historic admixture
To detect historic gene flow, we used a tree-based maximum-likelihood approach with the program TreeMix (Pickrell & Pritchard, 2012). This approach uses allele frequencies to model relatedness among populations as a non-bifurcating tree. Migration edges are added as additional branches to a bifurcating tree allowing for population ancestry from more than one parental population. Migration edges are added stepwise to the tree model until the covariance of the model best matches the covariance of the data. Residual matrices were used to determine the model fit. Positive residuals indicate greater genetic variation in the population than explained by the simple tree model suggesting admixture (Pickrell & Pritchard, 2012).
The model assumes migration within a single generation. The fraction of alleles derived from migration is represented as weight of the migration edge.
To infer the timing of the admixture event, we used approximate Bayesian computation to model the demographic history of the three populations using DIY-ABC v2.0 (Cornuet et al., 2014). A set of summary statistics was used to assess the fit between simulated datasets and empirical data. We used mean number of alleles, mean genetic diversity, pairwise F ST values and the maximum-likelihood coefficient of admixture λ (Choisy, Franck, & Cornuet, 2004). The demographic scenario simulates divergence between SFL and WCU and a more recent admixture event that gave rise to the MIA population. The prior for the divergence between SFL and WCU was set between (6,000,000-13,000,000) generations, based on previous divergence time estimates (Campbell-Staton et al., 2012;Tollis & Boissinot, 2014). We set the prior for the effective population size as (100-10,000) using a uniform prior distribution. To estimate timing of the admixture event, we used a prior of (1-4,000) generations assuming one generation per year. We simulated 1,000,000 datasets and used the 1,000 datasets with the smallest Euclidean distance to the empirical data for parameter estimation.

| Phylogenetic analysis and mtDNA haplotype divergence
The 571-bp alignment resulted in a total of 155 haplotypes, 41 from southern Florida, 43 from central and northern Florida, 52 from western Cuba, and 19 from eastern Cuba. Anolis porcatus from western Cuba was sister to the monophyletic A. carolinensis (Supporting Information Figure S1). Since the 343-bp alignment resulted in an overall similar tree topology (Supporting Information Figure S2 (Figure 1a). Branches within the clade were not well supported (bootstrap <95), which limits our ability to identify a more specific source location(s) for the introduction. Average sequence divergence between introduced A. porcatus haplotypes and the genetically most similar ones sampled from Cuba ranged from 0.0% to 1.75% divergence (mean = 1.14% ± 0.68%; Table 1).
One individual from South Miami (MIA640) shared the same haplotype with one individual from Havana (JJK2796).

| Genetic diversity and differentiation using nuclear microsatellite loci
Genetic structure and diversity were assessed for populations from WCU, SFL, and MIA using nuclear microsatellite markers. Three microsatellite loci (Ac2, F06, g01) deviated significantly (p < 0.05) from HWE. Excluding those loci from the analysis did not affect the results and we thus included them in subsequent analyses. Allelic richness was similar across populations (mean A R = 10.62 ± 0.55; Table 2).

| Population structure and differentiation
The Bayesian cluster analysis using STRUCTURE recovered three distinct genetic clusters (Figure 2b  When the number of clusters was set to K = 2 in the STRUCTURE analysis, the MIA population remained a distinct cluster while SFL and WCU formed a single genetic cluster (Figure 2b). Analysis of separate population pairs recovered all three populations and did not suggest population substructure (Supporting Information Figure S6).
Similarly, genotypes from five sampling locations in WCU showed no evidence for population structure when analyzed separately (Supporting Information Figure S7).

| Maximum likelihood and ABC modeling of historic admixture
Tree-based maximum-likelihood analysis of microsatellite markers supported one migration event between WCU and MIA (Figure 2a).
The weighted migration edge suggested that ~33% of the nuclear genetic ancestry in the MIA population is derived from WCU. Including the migration edge significantly improved the fit of the model as compared to a strictly bifurcating tree model (p < 0.001, Supporting Information Table S4). The migration model explained 99% of the total variance in the data, whereas the strictly bifurcating tree model accounted for only 80% (Supporting Information Figure S8).  generations (median T A = 887, mode T A = 554, 95% CI 245-2,670;  Figure 3). The median rate of admixture was R A = 0.24 (95% CI 0.14-0.35), which is similar to the maximum-likelihood coefficient of admixture λ = 0.31 obtained from the summary statistics (Supporting Information Table S5). Estimates for the remaining parameters used in the model are shown in Supporting Information Table S5. Summary statistics generated from the posterior probability distribution show similar values compared to the observed data and were largely nonsignificant, suggesting that modeled parameters provide a good fit for the data (Supporting Information Table   S5). Performance measures for parameter estimates were consistently low, indicating accurate estimates (Supporting Information   Table S6).

| D ISCUSS I ON
In an effort to characterize the genetic consequences of secondary contact between the native A. carolinensis and the closely related introduced A. porcatus, our data show evidence for past hybridization followed by differentiation of the hybrid population. We found discordance between nuclear microsatellite markers and mtDNA haplotypes in the South Miami population, which is indicative of hybridization (Hailer et al., 2012;Miller et al., 2012;Roy et al., 2015).
Thirty-five per cent of mitochondrial haplotypes in the South Miami population are derived from A. porcatus from western Cuba and 65% from the native A. carolinensis in southern Florida, verifying the introduction of A. porcatus from Cuba and producing secondary contact with native A. carolinensis in southern Florida. Genetic cluster analyses of nuclear markers show that the South Miami population is homogeneous and genetically distinct from populations of both parental species, which is characteristic of hybrid ancestry rather than ongoing hybridization (James & Abbott, 2005;Keller et al., 2014;Kronforst et al., 2006;Lavretsky et al., 2015;Mims et al., 2010). Tree-based maximum-likelihood analysis confirms that ~33% of the nuclear genetic ancestry is derived from western Cuba via secondary gene flow. This proportion of nuclear ancestry (~33%) is strikingly similar to the proportion of A. porcatus mtDNA haplotypes in South Miami (35%). Thus, reproductive barriers between A. porcatus and A. carolinensis appear weak or absent despite divergence in allopatry since before the Pliocene. Thus, secondary contact after species introduction has led to hybridization and formed a genetically distinct green anole population of hybrid origin.
Time estimates from ABC analyses suggest that hybridization occurred between 245 and 2,670 generations ago with a skewed distribution toward the present (Figure 3), suggesting relatively recent introduction and rapid differentiation of the hybrid population.
Surprisingly, the differentiation of the hybrid population in South Miami from both parental species is similar in magnitude to the differentiation between the parental species, A. porcatus and A. carolinensis. Potential factors facilitating differentiation include reduced gene flow with populations of the parental species (Hasselman et al., 2014;James & Abbott, 2005;Roy et al., 2015;Schumer, Cui, Powell, Rosenthal, & Andolfatto, 2016), assortative mating of hybrid individuals (Mavárez et al., 2006), increased hybrid fitness (e.g., heterosis; Schwarz, Matta, Shakir-Botteri, & McPheron, 2005), and genome incompatibility (Schumer, Cui, Rosenthal, & Andolfatto, 2015 (Gray, 1840;Powell, 1992;Voigt, 1831 until described as a distinct taxonomic unit (Powell, 1992). However, a thorough evaluation of morphological differences between the species concluded that morphological characters are inadequate for species delimitation (Camposano, 2011). Genetic evaluation of the A. carolinensis species complex revealed paraphyly of A. porcatus, dividing this species into eastern and western clades in Cuba, with the western clade being sister to A. carolinensis (Glor et al., , 2005. Our study provides a genetic perspective on species boundaries between A. carolinensis and A. porcatus. According to the biological species concept, populations of distinct species are incapable of effectively interbreeding with one another (Mayr, 1982), which is inconsistent with the findings of our study. Thus, genetic evidence for successful hybridization, as well as morphological similarities between the two species (Camposano, 2011), supports revising the taxonomy of the clade of A. porcatus from western Cuba, which should be subsumed into the earlier named A. carolinensis.
Several Anolis species have been introduced to Florida and some from multiple native-range source populations in Cuba (Kolbe et al., 2007). In agreement with previously collected A. porcatus haplotypes from Miami (Kolbe et al., 2007), our phylogenetic analysis identified sampling sites located near Havana in western Cuba as potential source of the introduction. Haplotypes from the western Cuba locations sampled in our study did not show evidence of geographic structure (Figure 1a), which is consistent with a single western Cuban population based on microsatellite data (Supporting Information Figure S6). Thus, the source locations likely resemble a single panmictic population. However, a more comprehensive sampling approach of western Cuban populations is needed to clarify whether population structure exists and whether the introduction of A. porcatus involves a single or multiple independent introductions.  (Gray, 1840;Powell, 1992;Voigt, 1831). A major finding was that a temporally restricted hy-

CO N FLI C T O F I NTE R E S T
None declared. interpreting data analyses and results, and by playing a critical role throughout the manuscript revision process.

DATA ACCE SS I B I LIT Y
The authors confirm that data supporting the findings of this study will be made available on GenBank and on GitHub (https://github.