Dimorphism and Divergence in Island and Mainland Anoles

Biological Journal of the Linnean Society, 2016, 118, 852–872. With 8 ﬁgures.

Dimorphism and divergence in island and mainland Anoles

HUGO H. SILICEO-CANTERO1, ANDRES GARCIA2, R. GRAHAM REYNOLDS3, GUALBERTO PACHECO4 and BRADFORD C. LISTER5*

1Posgrado en Ciencias Biologicas, Instituto de Biologıa, Universidad Nacional Autonoma de Mexico, Mexico City, Mexico 2Estacion de Biologıa Chamela, Instituto de Biologıa, Universidad Nacional Autonoma de Mexico, Mexico City, Mexico 3Department of Organismic and Evolutionary Biology & Museum of Comparative Zoology, Harvard University, Cambridge, MA, 02138, USA 4Posgrado en Ciencias Biologicas, Instituto de Ecologıa, Universidad Nacional Autonoma de Mexico, Mexico City, Mexico 5Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA

Received 21 August 2015; revised 23 December 2015; accepted for publication 23 December 2015

Relative to the West Indies, the ecology and evolution of anoles inhabiting islands off Central and South America have received little attention. The paucity of studies on continental islands has limited our ability to generalize and extend results based on the West Indian paradigm, as well as our understanding of the profound differences between the adaptive radiations of continental vs. Greater Antillean anoles. Here we compare the morphological, ecological, behavioural and genetic divergence between Anolis nebulosus populations inhabiting a small island in the Bay of Chamela, Mexico, and a nearby mainland forest. Notably, the two populations exhibit intra-sexual dimorphism with respect to head and limb sizes, the ﬁrst such polymorphism documented for an Anolis species. We also compare the shape of island and mainland A. nebulosus with each other, the six West Indian ecomorphs and a hypothetical generalist species. Finally, we address the generalist convergence hypothesis for anoles on single species islands. We conclude that convergence on a generalist morphology is widespread among solitary anoles in the West Indies. We present data on a limited sample of solitary anoles with mainland ancestors that suggest a parallel convergence on a similar generalist morphology, probably due to similar adaptive landscapes shaped by selective forces common to small island environments. © 2016 The Linnean Society of London, Biological Journal of the Linnean Society, 2016, 118, 852–872.

KEYWORDS: convergence – ecomorph – generalist morphology – niche expansion – polymorphism.

INTRODUCTION The diversity of the genus (nearly 400 species in the West Indies, Central and South America), combined Since Darwin landed in the Galapagos and Wallace with the sheer number of islands harbouring at least explored the Malay Archipelago, islands have played one resident anole, provides a rich array of natural a major role in elucidating the mechanisms of evolu- experiments that biologists have mined to address a tionary change. In keeping with this tradition, Anolis broad range of questions in ecology and evolution lizards inhabiting islands in the West Indies have (Losos, 2009). emerged as model organisms for studies of evolution Relative to the West Indies, anoles inhabiting at both the micro- and the macro-evolutionary scale. islands off the coasts of Central and South America have received little attention. Besides the research reported here, and related work by Senczuk et al. *Corresponding author. E-mail: [email protected]

(2014), Siliceo-Cantero & Garcia (2014) and Hernan- expansion on single species islands did not entail dez-Salinas, Ramirez-Bautista & Pavon (2014), only diversifying selection for specialized phenotypes two other studies have compared the adaptive diver- and a concomitant increases in adaptive varia- gence of an island Anolis population with an ances- tion, as predicted by the niche variation hypoth- tral mainland population (Klutsch et al., 2007; esis (Van Valen, 1965; Bolnick et al., 2007). Logan et al., 2012). Both of these latter studies Rather, solitary species of West Indian anoles focused on Anolis lemurinus in the Bay Islands of appeared to converge on a generalist phenotype Honduras, and morphological comparisons were lim- capable of exploiting a variety of perch sites, ited to dewlap and body size. The paucity of studies thermally diverse habitats and a broad range of on continental islands has limited our ability to gen- prey sizes. If widely replicable, this pattern eralize and extend results based on West Indian would represent a reversal of evolutionary direc- anoles, as well as our understanding of the pro- tion and an example of deterministic, microevo- found differences between the adaptive radiations of lutionary convergence at the species level continental vs. West Indian species (Irschick et al., (Losos, Irschick & Schoener, 1994; Wollenberg 1997). Here we compare the morphological, ecologi- et al., 2013). cal, behavioural and genetic differentiation between an Anolis nebulosus population inhabiting the small, land bridge island of San Agustın in the Bay of METHODS Chamela, Mexico, and a nearby mainland population, with the aim of addressing the following ques- STUDY AREAS AND FOCAL SPECIES tions. Our mainland site was located within the Chamela- Cuixmala Biosphere Reserve (Ch-CBR) in the state 1. To what extent has the island population of of Jalisco on the west coast of Mexico (19°22003″– A. nebulosus diverged morphologically and genet- 19°35011″N and 104°56013″–105°03025″W). The Ch- ically from the ancestral mainland population, CBR encompasses 13 142 ha of tropical dry forest. and is the adaptive significance of any changes The study area was approximately 1 km from the interpretable in the light of ecological and beha- National Autonomous University of Mexico’s Esta- vioural differences between the populations?As cion de Biologia Chamela. many mainland predators and competitors are Our second study site was the small island of San absent from San Agustın, we would expect shifts Agustın located 0.47 km from the coast and 5.5 km in resource exploitation, behaviour and morphol- from the mainland study area. The island has a ogy similar to those found amongst anoles inhab- length of 280 m, a width of 230 m and an area of iting small islands in the West Indies (Schoener, 3.3 ha. All the islands in the Bay of Chamela are 1969; Lister, 1976a, b; Andrews, 1979; Losos & de land bridge islands and were separated from the Queiroz, 1997). On the other hand, the West mainland during a rapid rise in sea level that took Indian paradigm might not hold for island popu- place between 7000 and 8000 years BP (Ramı́rez- lations with different phylogenies that may exhi- Herrera, Kostoglodovb & Urrutia-Fucugauchi, 2004). bit clade-specific responses to insularity, and Compared with nearby islands, San Agustın’s vegeta- evolutionary constraints that limit the degree of tion is more open, less scrubby and consists of tropi- divergence from ancestral populations (Meiri, cal dry forest which is much more similar in 2008; Schaad & Poe, 2010). structure to the mainland forest rather than to that 2. Does the morphology of the island or mainland of other islands in the bay (see Fig. 6). The focal spe- population of A. nebulosus correspond to any of cies of this research, Anolis nebulosus, is a small the six West Indian ecomorphs? The morphology tree-dwelling anole, endemic to western Mexico and of A. nebulosus has not been compared with widely distributed from eastern Sonora to southern either West Indian or mainland species, and Guerrero. Mainland males average approximately understanding its relative position with respect 42 mm in snout–vent length (SVL), with a maximum to the two major adaptive radiations of anoles SVL of 49 mm and a mean body weight of 1.6 g could shed light on the evolutionary trajectories (3.0 g maximum). Females are smaller, with a mean of both island and mainland populations. SVL of 36 mm (maximum 45 mm), and mean body 3. In the absence of most mainland competitors, has weight of 1.0 g (maximum 1.6 g; Supporting Infor- the island population of A. nebulosus undergone mation, Table S1). niche expansion and evolved towards a more gen- All permissions for fieldwork and specimen collec- eralist phenotype? In a study of niche expansion tions associated with this study were carried out and morphological variation in island Anolis pop- under a licence to A. Garcia from the National ulations, Lister (1976b) concluded that niche Autonomous University of Mexico.

MORPHOLOGY Testors acrylic model airplane paint. The SVL of cap- From February to October 2011, and in January tured lizards was also measured to the nearest 2014, 183 A. nebulosus individuals were captured, 78 0.5 mm. Quadrats were censused twice a day until in the mainland study area and 105 on San Agustın. six consecutive censuses yielded no unmarked The sex of each individual was identified based on lizards. At this point, we considered the anoles dewlap size. Body weight was measured using a within the quadrat to be close to completely marked Pesola 5-g scale, and several other body measure- out. On the mainland, we censused A. nebulosus ments were taken using Vernier calipers. We also along existing trails through the forest. We marked recorded the perch height and perch diameter used the locations of every lizard encountered on a map, by all individuals. To avoid resampling the same indi- and then estimated the area searched on either side viduals, captured lizards were marked by toe clipping of the trails to determine the approximate number of 2 and released at the point of capture. While no studies individuals per 225 m . to date have demonstrated reduced survival due to To estimate relative rates of predation, we teth- this technique (Losos, 2009), we minimized any ered at a height of 2.0 m ten adult A. nebulosus to potential effects on clinging ability by clipping just trees in the mainland and island forests over 12 days the tip of the toe, an area free of lamellae. during the summer wet season and 10 days during We measured ten morphological variables for 46 the winter dry season. The tethers were 0.5-m insular females, 66 insular males, 35 mainland lengths of black 6-pound test fishing line tied around females and 43 mainland males: SVL from the tip of the waist of the lizards directly above the rear legs the snout to the anterior end of the cloaca; tail and secured to the tree trunks with a small nail. length (TaL) from the anterior of the cloaca to the Lizards were checked each day during the morning tip of the tail; snout length (SL) from the tip of the and afternoon, and any missing individuals were snout to the skull base; mandibular width (MW), the recorded. Every 3 days, remaining lizards were widest part of the jaw; upper arm length (UAL) from replaced with newly caught individuals. The data the point at which the forelimb enters the body to were analysed using the survival analysis routines the apex of the elbow; forearm length (FAL) from the in IBM SPSS 21. apex of the elbow to the centre of the wrist; hand length (HL) from the centre of the wrist to the tip of VEGETATION STRUCTURE forehand digit IV; femur length (FeL) from the insertion in the body wall to the apex of the knee; tibia Variation in local vegetation structure can confound length (TiL) from the apex of the knee to the centre comparisons of perch site utilization and microhabi- of the ankle; and paw length (PL) from the centre of tat niche breadth between different habitats (John- the ankle to the tip of toe IV. Upper limb length son et al., 2006). The greater the similarity in (ULL) was calculated as UAL + FaL, and lower limb habitat structure, the greater the probability that length (LLL) as FeL + TiL. ULL in anoles is often differences in microhabitat use between populations measured from the distal end of the longest toe to are due to differences in selectivity and behaviour. the point of insertion of the limb in the body wall. To compare the vegetation of the island and main- Here the correlation between our measure of ULL land forests, we measured the heights and diameters and ULL + HL averaged 0.98 for male morphs and of all woody plants within 1 m to the left and right 0.97 for female morphs. Our results are the same of two 50-m transects through both habitats. regardless of which measure is employed to represent upper limb length. GENETIC DATA AND ANALYSES We obtained 23 tail tips from individuals on San BEHAVIOURAL OBSERVATIONS, INSECT SAMPLES, Agustın and 27 from the mainland study area, and POPULATION DENSITIES AND PREDATION preserved the samples in 95% EtOH. We extracted EXPERIMENTS whole genomic DNA using the Promega Wizard SV Methods employed for behavioural observations and DNA purification system according to the manufac- insect sampling were the same as those described by turer’s protocol, and stored the extracts at 20 °C. Lister & Garcia (1992). Density estimates and beha- We used PCRs to amplify a portion of the mitochon- vioural observations were conducted every 6 months drial locus ND2 (NADH subunit II, conditions as from July 1990 to July 1991. On the island, popula- specified by Revell et al., 2007), and purified and tion densities of anoles were estimated in 15-m by sequenced products in both directions on an auto- 15-m quadrats. Captured lizards were weighed to the mated sequencer (ABI 3730XL) at Massachusetts nearest 0.1 mg with a Pesola spring balance, and General Hospital DNA Core Facility, Cambridge, marked on the dorsal surface with fast drying MA. We assembled contigs and manually verified

© 2016 The Linnean Society of London, Biological Journal of the Linnean Society, 2016, 118, 852–872 DIMORPHISM/DIVERGENCE IN ISLAND AND MAINLAND ANOLES 855 ambiguous base calls using GENEIOUS 7.1.2 (Biomat- reads. All sequencing reads are then mapped to the ters). We then aligned sequences using the CLUSTALW reference with an alignment identity threshold of 2.1 (Larkin et al., 2007) algorithm implemented in 93% (BBMap, http://sourceforge.net/projects/bbmap/). GENEIOUS with reference sequences and default Genotypes were called using Samtools and bcftools parameters. We deposited representative sequences using the following flags: (samtools mpileup –gu –Q from all loci in GenBank (accession numbers 10 –t DP,DPR –f ref.fasta –b anolis.align_samples | KU757434-KU757453). We estimated genetic varia- bcftools call –cv – > anolis.vcf). We converted the tion within populations as nucleotide (p) and haplo- resulting variant-call format (.vcf) genotype file to type (h) diversity, as well as ΦST between subsequent input files using PDGSpider (Lischer & populations. We conducted the above analyses, as Excoffier, 2012) after excluding loci with only miss- well as inferred haplotypes and connection distances ing data and non-polymorphic loci, as well as SNPs between haplotypes, in ARLEQUIN 3.5.1.3 (Excoffier & with Phred scores less than 10. This resulted in Lischer, 2010). We determined the significance of 34 543 polymorphic SNPs among 18 018 loci. We fur-

ΦST values via the maximum number of permuta- ther trimmed this dataset to include only the first tions in ARLEQUIN, and visualized haplotype connec- SNP from each locus in order to retain only presum- tions using HapStar (Teacher & Griffiths, 2010). ably independent loci for clustering analyses (Pritch- We additionally selected a subset of ten individuals ard, Stephen & Donnelly, 2000). (five mainland, five island) for RAD (restriction site We assessed potential genetic structure between associated DNA) sequencing followed by single populations with the Bayesian clustering algorithm nucleotide polymorphism (SNP) genotyping. We used STRUCTURE (Pritchard et al., 2000), which uses a the nextRAD (Nextera – tagmented reductively Markov chain Monte Carlo approach to cluster K amplified DNA) genotyping-by-sequencing (GBS) groups based on individual genotype profiles. We approach as implemented by SNPsaurus (Institute of used the admixture model and five replications of Molecular Biology, Eugene, OR, USA). Briefly, nex- K = 1–4, with each replication having a run length of tRAD uses selective primers to amplify fragments 100 000 generations and a burn-in of 20%. We across the genome, as opposed to using restriction selected values of K using the DK method of Evanno, enzymes followed by size selection as in similar Regnaut & Goudet (2005) in STRUCTURE HARVESTER RADseq approaches (Etter & Johnson, 2012). These (Earl & von Holdt, 2012). We also used a discrimi- fragments are initially generated using Illumina nant analysis of principal components (DAPC; Jom- Nextera tagmentation, followed by selective PCR bart, Devillard & Balloux, 2010) implemented in the amplification and ligation of sequencing adapters R 3.1.2 (R Institute for Statistical Computing) ade- and barcoded indices, as in Russello et al. (2015). We genet package (Jombart, 2008) to identify genotypic quantitated each of our genomic DNA (gDNA) sam- clusters. This method attempts to maximize genetic ples using a Qubit 2.0 system prior to library prepa- differentiation between groups, and minimize varia- ration. Genomic DNA was fragmented using the tion within groups, by clustering individual geno- Nextera reagent (Illumina), which also ligates short types with a principal components transformation of adapter sequences to the ends of each fragment. This the genetic data prior to discriminant analysis. We reaction calls for 15 ng of gDNA per sample, used a Bayesian information criterion approach to although 22.5 ng of gDNA was included to compen- obtain the predicted number of clusters between sate for degraded DNA in our samples. These frag- K = 1 and 20 after retaining all PCs. To perform the ments were then amplified using a selective primer DAPC, we selected the optimal number of PCs using complementary to the adapter sequences, which also optim.a.score() in adegenet with 1000 replications, includes a 9-bp selective sequence (GTGTAGAGC). resulting in retention of the first two PCs and the Amplification was conducted using PCR at 73 °C for first eigenvalue in the analysis. We calculated sum- 26 cycles, during which only fragments of gDNA that mary statistics using ARLEQUIN for each of the group- were able to hybridize with the selective sequence ings delimited in the above analyses, and we were amplified. estimated FST between groups in ARLEQUIN via the Sequencing was performed on an Illumina Next- maximum number of permutations. Seq 500 at the Institute of Molecular Biology, Eugene, OR, using a high-output run to generate ~400 million single-end sequencing reads 75 bp in STATISTICAL ANALYSES length. Reads were quality-filtered resulting in a Prior to statistical analysis, measurements of all mean of 3656 509 reads per individual and SNPs morphological variables were transformed by taking were called using the SNPsaurus nextRAD pipeline. natural logarithms. To comply with the linearity This pipeline consists of custom scripts (SNPsaurus, assumptions of the statistical procedures employed, LLC) that create a de novo reference from abundant we used the cube root of individual body weight

(CMASS) rather than raw body weight measure- for snout length, mandibular width, upper arm ments (Butler & Losos, 2002). In the initial multi- length and forearm length. Each sex in each location variate morphological space comparisons, we were has a large-headed, short-limbed morph, and a interested in the actual size differences within and small-headed, long-limbed morph (Supporting Infor- between the island and mainland populations, and mation, Table S1; Fig. 1). On the island, the SVLs of used the log-transformed morphological data without the large-headed, small-limbed male and female size adjustment. For comparisons of shape diver- morphs were significantly higher than the SVLs of gence using multivariate analysis of variance (MAN- the corresponding small headed, long-limbed morphs OVA), principal components analysis (PCA) and (Mann–Whitney U test, P < 0.01). The scatterplots in discriminant analysis (DA), we employed Mosimann’s Figure 1 illustrate the dimorphism by plotting snout (1970) geometric mean method to adjust the log- length against forearm length for island and main- transformed data for size differences (Butler & land males. The bimodal histograms for snout Losos, 2002). Unless otherwise noted, statistical lengths, mandibular widths and forearm lengths are analyses were carried out in IBM SPSS 21. given in Supporting Information (Fig. S1). As Sup- porting Information (Table S1) shows, the large- headed, short-limbed males and females were the most common morphs, comprising 75 and 77% of the RESULTS island and mainland male samples, and 87 and 91% To assess morphological divergence between the of the island and mainland female samples, respec- island and mainland populations, we first analysed tively. differences in intra- and intersexual dimorphism, Numerous studies have shown that Anolis limb overall size and the multivariate morphological space dimensions are correlated with the structural occupied by the two populations. We then analysed features of the microhabitat that a given species divergence in shape and compared the morphology of characteristically occupies (Losos et al., 1994; Losos, male A. nebulosus with the six canonical West Warheit & Schoener, 1997). In particular, relative Indian ecomorphs and a hypothetical generalist phe- hind limb length increases as perch diameter notype. Finally, we documented ecological, beha- increases, both within and between species (Losos, vioural and genetic differences between island and 2009). In mainland species, forelimb length is nega- mainland A. nebulosus. tively correlated with perch height, while no such relationship is found in Greater Antillean anoles (Irschick et al., 1997). Given these patterns, we INTRASEXUAL SIZE DIMORPHISM analysed the relationship between the means of the Our exploratory analysis of the morphological data size-adjusted lower and upper limb lengths and the for male and female A. nebulosus included his- average logarithms of the perch heights and perch tograms of all the measured variables, as well as diameters utilized by the island and mainland bivariate plots of all variables against SVL. These morphs. There was no significant correlation graphs revealed that both males and females from between height and limb length. However, as Fig- the mainland and island populations were dimorphic ure 2 illustrates, there was a positive association

16.0 A Island B Mainland

(mm) 12.0

8.0

Snout length 4.0 4.0 6.0 8.0 10.0 12.0 14.0 4.0 6.0 8.0 10.0 12.0 14.0 Forearm length (mm)

Figure 1. Snout length vs. forearm length for adult male A. nebulosus from the island (A) and mainland (B) populations. Circles: large-headed, short-limbed morph; triangles: small-headed, long-limbed morph.

© 2016 The Linnean Society of London, Biological Journal of the Linnean Society, 2016, 118, 852–872 DIMORPHISM/DIVERGENCE IN ISLAND AND MAINLAND ANOLES 857 between size-adjusted forelimb and hind limb length significantly larger than their mainland counterparts and perch diameter. The Spearman correlation (ANOVA, P < 0.000001, Tamahane post-hoc test, between the size-adjusted upper limb lengths and P < 0.00001). SVLs of the small-headed, long-limbed mean perch diameters was significant (q = 0.833, island males were also significantly larger than those P = 0.010), while the correlation between the size- of the corresponding mainland morph (ANOVA, adjusted lower limb lengths and mean perch diame- P < 0.001; Tamahane post-hoc test, P = 0.014). Sam- ters was marginally insignificant (q = 0.667, ple sizes were insufficient to test for differences in P = 0.071). We also conducted t-tests for the differ- body size between the island and mainland small- ences between the logarithms of the perch diameters headed, long-limbed female morphs. occupied by the male and female morphs. In all four The average sexual size dimorphisms (SSDs) of comparisons the small-headed, long-limbed morph male morphs to female morphs were similar on island utilized perches with larger diameters. However, and mainland for both the ratio of mean SVLs (1.16 none of the t-tests was significant (P > 0.35). vs. 1.10) and the ratio of maximum SVLs (1.12 vs. 1.17; Supporting Information, Table S2). To add per- spective to these differences, the histograms on the SIZE AND SEXUAL SIZE DIMORPHISM left of Figure 3 show the distribution of SSDs (mean On the island, body sizes (LnSVL) of both male male SVL/mean female SVL) for 102 Greater Antil- morphs were significantly larger than the corre- lean species compared with 28 solitary species inhab- sponding female morphs (Supporting Information, iting smaller islands in the West Indies. The Table S1; ANOVA, P < 0.0001, followed by Tama- histograms on the right show the SSDs for 60 main- hane post-hoc tests, P < 0.01). On the mainland, the land anoles vs. four solitary species from islands off body size of the large-headed, short-limbed male Central and South America that are derived from morph was also significantly larger than the large- mainland ancestors, including A. nebulosus on San headed, short-limbed female morph (ANOVA, Agustın. The median SSDs for the Greater Antillean P < 0.00001, Tamahane post-hoc test, P < 0.00001). species (1.16) and the West Indian solitary anoles Sample size of the mainland small-headed, long- (1.32) were significantly different (Mann–Whitney U limbed female morph was insufficient to test for dif- test, P < 0.000001). The median SSD for the mainland ferences in body size. species was 1.03 compared with 1.29 for the solitary Overall, island morphs were larger than the corre- island species with mainland ancestors, and these sponding mainland morphs (Supporting Information, medians were also significantly different (Mann– Table S1). Taking ratios of mean SVLs, island male Whitney U test, P = 0.00013). morphs were 1.26 and 1.17 times larger than the corresponding mainland morphs, and the large-headed, short-limbed female morph was 1.29 times larger MORPHOLOGICAL SPACE COMPARISONS than the corresponding mainland female morph. For Intraspecific divergence in size and shape between both island males and females, body sizes (LnSVL) of sexes is common in anoles, and can significantly the large-headed, short-limbed morphs were increase both morphological and ecological diversity

.92 .90 MORPH 1 2 .90 .80 3 4 5 .88 .70 6 7 .86 .60 8

.84 .50 Mean SA ULL Mean SA LLL .82 .40

.80 .30

1.0 1.5 2.0 2.5 3.0 1.0 1.5 2.0 2.5 3.0 Mean Lnperchdiam Mean Lnperchdiam

Figure 2. Plots of mean size-adjusted lower limb length (SA LLL), and mean size-adjusted upper limb lengths (SA ULL), against the average natural logarithm of perch diameter for each of the mainland and island morphs. The least squares regression lines are shown (see text). Numbers adjacent to the plotting symbols correspond to the eight island and mainland morphs: 1–2, island male morphs; 3–4, island female morphs; 5–6, mainland male morphs; 7–8, mainland female morphs.

A C

B D Percent 0 10 20 30 40 50 0 10 20 30 40 50 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 Sexual size dimorphism

Figure 3. A, distribution of sexual size dimorphism (SSD) indices for 102 Anolis species from the four large islands of the Greater Antilles. The dark grey bars delineate species classified as trunk-ground and trunk-crown ecomorphs. The light grey bars represent anoles from the other four ecomorphs. B, distribution of SSD indices for 28 solitary Anolis populations inhabiting smaller islands in the West Indies (data from Schoener, 1977). C, distribution of SSD indices for 60 mainland Anolis species. D, histogram of the SSD indices of four solitary species with mainland ancestors from islands off Central and South America. The SSD of A. nebulosus on San Agustın is represented by the leftmost histobar in D. Histobars on the right represent SSDs for three other solitary anoles with continental ancestors: A. pinchoti (1.19), A. concolor (1.33) and A. agassizi (1.33). within Anolis communities (Schoener, 1967; Butler, area is defined as that part of a given morph’s PC space Sawyer & Losos, 2007). Here we analyse the that does not overlap with another morph. On island joint impact of dimorphism within and between and mainland, males and females each contributed an sexes on the area of multivariate morphological average of 50% to the total morphological space occu- space occupied by the island and mainland pied by a given morph. Hence, within morphs, sexual populations. dimorphism approximately doubles the total morpho- Figure 4 illustrates the results of a PCA based logical space. On the island, the combined area of the on the natural logarithms of seven of the measured male and female large-headed, short-armed morphs morphological characters. The first two PCs was 2.09 vs. 1.93 PC units2 for the small-headed, accounted for 86 and 78% of the variation in the long-armed male and female morphs, respectively, giv- data for the island and mainland populations, ing a total morphospace area of 4.02 PC units2.Onthe respectively. The characters were not size adjusted, mainland, the combined area occupied by the male and and hence the multivariate space represents differ- female large-headed, short-armed morphs was 1.90 vs. ences in absolute size rather than shape. 1.89 PC units2 for the small-headed, long-armed male We defined the area of the PCA morphospace occu- and female morphs, respectively, giving a total mor- pied by each of the island and mainland morphs by con- phospace area of 3.79 units2. Hence, in each environ- necting the points in Figure 4 that delimited the outer ment, the existence of a second morph essentially boundaries of the PC scores. We then calculated the doubles the total morphological breadth of the respec- areas of the resulting polygons. The total and unique tive A. nebulosus population. areas for each morph, as well as the total morphospace In continuously growing species such as anoles, occupied by all four morphs in each environment, are variance and coefficients of variation of SVLs are given in Supporting Information (Table S3). Unique highly correlated with average SVL (Lister &

A B PC 2

Island Mainland –3 –2 –1 0 1 –3 –2 –1 0 1 2 3 –3 –2 –1 0 1 2 –2 –1 0 1 2 3 PC 1 PC 1

Figure 4. Morphological space deﬁned by principal components 1 and 2 for (A) the island male and female morphs, and (B) the mainland male and female morphs. The principal components analysis was conducted utilizing the natural logarithms of tail length, snout length, mandibular width, upper arm length, forearm length, lower limb length and SVL without size adjustment. Blue circles in both graphs represent the large-headed, short-armed male morphs. Red circles are the small-headed, long-armed male morphs. Green triangles in both graphs are for the large-headed, short-armed female morphs. Black triangles are the small-headed, long-armed female morphs.

McMurtrie, 1975). Given the larger SVLs of island Table S5). All tests of between-morph effects for the A. nebulosus, we would expect the CV of their SVLs size-adjusted variables were also significant to be greater than their mainland counterparts. The (P < 0.0001). means and standard deviations of SVLs for all The first two discriminant axes accounted for morphs and sexes combined were 42.89 (6.84) and 98.6% of the variation in the discriminant scores, 34.1 (3.98) mm for the island and mainland, respec- and both functions were significant (Wilks’ k = 0.011 tively. The CV of the SVLs for all the island morphs and 0.374, P < 0.00001). The loadings of the size- and sexes combined was 0.159 vs. 0.116 for all the adjusted variables on each discriminant axis are mainland morphs and sexes. The difference was sig- given in Supporting Information (Table S4). The dis- nificant (P = 0.0037) via a likelihood ratio test criminant analysis classified 63% of all individuals (Miller, 1991). We also calculated Shannon diversity correctly with respect to morph group. The discrimi- indices (H) for the total SVL distributions of the nant scores for all morphs are plotted in two-dimen- island and mainland populations. The H values for sional discriminant space in Supporting Information the mainland and island were 2.57 and 2.12, respec- (Fig. S3). tively. Hutcheson’s t-test for Shannon diversity indices (Hutcheson, 1970) rejected the null hypothe- – sis (P < 0.002), and we conclude that the size diver- ISLAND MAINLAND DIVERGENCE IN SHAPE sity of the island population was significantly greater To further analyse island and mainland populations than that of the mainland population. with respect to shape, we conducted a PCA utilizing the same eight size-adjusted variables employed in the discriminant analyses. Figure 5 shows the rela- DISCRIMINATION AND CLASSIFICATION OF THE tive positions of the male and female morphs in the MORPHOLOGICAL GROUPS morphological space defined by the first two PC axes. To determine if differences in shape as well as size Supporting Information (Fig. S4) illustrates the load- could be used to define and classify the various ings of the size-adjusted values on these axes. The morphs, we conducted a discriminant function analy- size-adjusted values for size (CMASS), snout length, sis (DFA) using eight size-adjusted variables (Sup- mandibular width and upper limb length were all porting Information, Table S4). In the preliminary highly correlated with the first PC axis, while tail one-way MANOVA, with morpho-group as the main length, hand length and rear foot length were most factor and the size-adjusted morphological measure- highly correlated with the second axis. ments as the dependent variables, all tests for over- Following a significant one-way MANOVA (Sup- all differences in the size-adjusted means were porting Information, Table S5), a Tukey’s post-hoc significant (P < 0.00001; Supporting Information, test was conducted to assess the significance of the

ABMale morphs Isl-ML Female morphs Isl-ML PC 2 –3 –2 –1 0 1 2 –2.5 –1.5 –.5 0 1.5 2.5 –2 –1 0 1 2 3 –2 –1 0 1 2 3 4 PC 1 PC 1

Figure 5. A, individual scores on the first two PC axes for the island and the mainland male morphs. Blue circles and green diamonds – island large-headed, short-armed and small-headed, long-armed male morphs, respectively. Red circles and orange diamonds – mainland large-headed, short-armed and small-headed, long-armed male morphs, respectively. B, individual scores on the first two PC axes for the island and mainland female morphs. Blue circles and green diamonds – island large-headed, short-armed and small-headed, long-armed female morphs, respectively. Red circles and orange diamonds – mainland large-headed, short-armed and small-headed, long-armed female morphs, respectively. The filled circles are the centroids of the PC scores for each morph that define the relative morphological differences between matching island and mainland phenotypes. Euclidean distances between the centroids of the island and mainland morphs are given in Supporting Information (Table S6). differences between equivalent mainland and island ecomorphs with anoles from one- and two-species morphs with respect to the eight size-adjusted vari- islands in the Lesser Antilles and Caribbean. Utiliz- ables. Both mainland male morphs had proportion- ing the same data set, we conducted a PCA that ally longer snouts and wider mandibles than their included corresponding morphological data for island counterparts. The mainland female morphs A. nebulosus large-headed, short-limbed males from also had consistently longer size-adjusted snout the island and mainland habitats. Our aim was to lengths and wider mandibles than the corresponding determine if males from the island and mainland island morphs. However, only the difference in rela- populations could be classified as one of the West tive mandibular width between the island and main- Indian ecomorphs, and whether island males were land small headed, long-armed female morphs was more similar to the generalist phenotype than main- significant (Tukey’s post-hoc test, P = 0.0004; Sup- land males. porting Information, Table S6). Differences in size- Figure 6 compares the positions, in the same PC adjusted upper limb lengths between the island and space utilized by Losos & de Queiroz (1997), of island mainland morphs were marginally insignificant and mainland males with each other and with the (P = 0.09). As a measure of the morphological diver- six canonical Anolis ecomorphs. Morphologically, gence between the island and mainland populations, both A. nebulosus populations are clearly separated the Euclidean distances between the centroids shown from the West Indian ecomorphs, as well as from the in Figure 5 were calculated (Supporting Information, putative generalist morphology, and are more similar Table S7), and ranged from 0.75 to 1.16. Compara- to each other than to any of the West Indian species. tive interpretation of these distances is hampered by The Euclidean distances from the centroids of the the use of different morphological variables in differ- island and mainland males to the ecomorph cen- ent studies. troids (Supplementay Information, Table S8) indicate that the centroid for the trunk ground ecomorph was closest to the centroids for both mainland (4.12 PC COMPARISON WITH WEST INDIAN ECOMORPHS AND units) and island (3.98 PC units) males. In compar- THE HYPOTHETICAL GENERALIST ANOLE ison, the average distance to the nearest ecomorph In an investigation of the generalist convergence centroid for the 33 West Indian species in the Losos hypothesis for solitary anoles, Losos & de Queiroz & de Queiroz (1997) study was 1.36 PC units. The (1997) compared the morphology of male anoles distance to the postulated centroid of the generalist belonging to the six canonical Greater Antillean anole was also much lower for the West Indian spe-

2.0 A lizard-eating mammals including the white nosed TC coati (Nasua narica), the Tayra (Eira barbara), the TR 1.0 pygmy spotted skunk (Spilogale pygmaea) and the raccoon (Procyon lotor). Over the past 25 years we 0.0 TW X TG have not encountered any snakes on San Agustın, or any mammals. Given the paucity of predators, the PC 4 PC –1.0 survivorship of resident A. nebulsous on San Agustın GB was expected to be signiﬁcantly greater than on the NM –2.0 mainland. Both observational and experimental data NI support this hypothesis. –3.0 On the mainland, the average survival time of adult males and females was 2.4 ( 0.7) months, and 3.0 B only 16% of the lizards alive at the beginning of the year were still alive after 6 months (A. Ramirez-Bau- 2.0 CG tista, pers. comm.). Our census data indicate that on TC 1.0 San Agustın, average survival time of adult males and females was 7.1 ( 1.1) months, and 60% of the 0.0 X TG individuals alive in January were still alive after

PC 2 TW –1.0 GB 6 months. Overall, survivorship was significantly TR lower on the mainland (Mantel-Cox log rank test, –2.0 P = 0.00001). As our survivorship data utilized NI cohorts of marked adult lizards, life expectancy at –3.0 NM birth could not be calculated. In general, the life –4.0 –3.0 –2.0 –1.0 0.0 1.0 2.0 span of mainland anoles rarely exceeds a year PC 1 (Andrews & Nichols, 1990), while island anoles consistently have longer life expectancies (Lister, 1981; Figure 6. Position of the centroids for the island (grey Schoener & Schoener, 1982). Our tethering experi- diamond) and mainland (black diamond) male A. nebulo- ments also suggest that predation rates are consider- sus compared with the six canonical West Indian eco- ably greater on the mainland. During both wet and morphs within the morphological space defined by PC dry seasons, survivorship of the tethered lizards was axes 1 and 4 (A), and 1 and 2 (B). The X indicates the significantly higher on San Agustın (Supporting position of the hypothetical generalist anole, intermediate Information, Fig. S2; Wilcoxon test, P = 0.0001 dry in morphology with respect to the six canonical eco- season; P = 0.003 wet season). morphs. The polygons outline the centroids of species belonging to the same ecomorph. TG, trunk-ground; TC, To gain insight into relative levels of intraspecific trunk-crown; TR, trunk; CG, crown-giant; GB, grass- competition, we also estimated the densities of the bush; TW, twig; NI, A. nebulosus island males; NM, island and mainland A. nebulosus populations, and A. nebulosus mainland males. Data for West Indian measured resource levels in the wet and dry seasons anoles provided by Jonathan Losos. using sticky traps (Fig. 7). Combined densities of males and females in the 15-m by 15-m census quadrat on San Agustın totalled 128 individuals. This is cies (0.866) than for island (3.53) and mainland over 60 times higher than the mainland density of (3.57) males. about two individuals per 225 m2, which in turn is somewhat lower than the density of ten individuals per equivalent area found in a mainland forest in ECOLOGICAL AND BEHAVIOURAL DIVERGENCE Nayarit, Mexico, 360 km north of the Ch-CBR forest Predation and competition (Jenssen, 1970). The density of A. nebulosus on San Predators are known to have a major impact on the Agustın is equivalent to Anolis densities on islands survival of island and mainland anoles (Schoener, in the West Indies. For example, estimated Anolis Losos & Spiller, 2005). Like other tropical mainland density per 225 m2 is 140 individuals in the Luquillo habitats (Losos, 2009), the Ch-CBR forest abounds rainforest (Lister, 1981; B. C. Lister & A. Garcia, with predators. Of the 108 resident bird species, at unpubl. data), 150 on Bimini, 119 on St. Croix and least 15 are documented lizard predators, 16 of the 80 on Dominica (Stamps, Losos & Andrews, 1997). 24 most common snake species are known to include The biomass of arthropods caught in sticky traps lizards in their diet, and six of the 15 resident was consistently higher on the mainland, ranging lizard species prey upon other lizards, including from 4.3- to 8.8-fold greater depending on the season A. nebulosus. The mainland also harbours several (Fig. 7). The mainland results are similar to those

variables, and location as the independent variable. 595 The MANOVA was significant (Wilk’s k = 0.958, 413 P = 0.001). There were also significant univariate main effects for perch diameter differences between the island and mainland vegetation, (P = 0.007), but not for perch height (P = 0.247). Supporting Information (Fig. S5B) shows the distribution of perch heights and perch diameters occu- Biomass (mg) pied by the island and mainland populations (males 128 43 47 plus females). A MANOVA indicated significant dif- 10 ferences in the mean logarithms of perch sites 0 200 400 600 800 1000 between the island and mainland populations (Wilks July March June k = 0.892, P = 0.000039). There were significant uni- Time period variate main effects for perch height (P = 0.000007), but not for perch diameter (P = 0.93). The mean Figure 7. Biomass of arthropods captured per day in perch height of island males was 111 cm vs. 78 cm sticky traps placed on the ground in the mainland (dia- for mainland males, and the mean perch heights of monds) and island (circles) study areas. The average biomass caught per trap per day is entered above each time island and mainland females were 98 and 71 cm, period. respectively. An ANOVA with a post-hoc Tamahane test was employed to test the null hypothesis of no difference amongst the island and mainland male obtained by Lister & Garcia (1992) in the same Ch- and female morphs with respect to perch height. The CBR study area, and, as these authors point out, ANOVA was significant (P = 0.00008), and the represent one of the highest seasonal fluctuations in Tamahane post-hoc test indicated that island males arthropod resources reported to date within a tropi- (both morphs combined) perched significantly higher cal ecosystem. Such large reductions in food than the combined mainland male morphs resources suggest that interspecific competition on (P = 0.005), and the combined mainland female the mainland and intraspecific competition on the morphs (P = 0.003), but not significantly higher than island may be particularly pronounced during pro- island females. There was also no significant differ- longed, El Nino-driven dry seasons. ence in perch height between mainland males and Siliceo-Cantero & Garcia (2014) recently published females (P > 0.90). The similarity of the vegetation a comparison of the San Agustın and mainland popu- on island and mainland suggests that the observed lations of A. nebulosus with respect to growth rate, shifts are not artefacts of differences in available body condition, habitat use and food availability. perch sites. While habitat use matched the data presented here, results for arthropod abundance are not comparable Niche expansion due to differences in methodology. In 2011 and 2015, As a measure of niche width, the Simpson diversity two major hurricanes, Jova and Patricia, made land- index was calculated for the utilization of height, fall near the Ch-CBR forest and the Bay of Chamela perch diameter, and perch height and diameter com- and impacted habitat structure as well as the popu- bined by the male and female island and mainland lation density and survivorship of anoles on San populations. There were no notable changes in the Agustın (H. H. Siliceo-Cantero and A. Garcia, diversity of perch diameters utilized by island com- unpubl. results). These data are being prepared for pared with mainland A. nebulosus. However, perch publication. height diversity of island males was almost twice that of the mainland males (9.8 vs. 5.2, Supplemen- Niche shifts tary Information, Table S9). The Simpson niche Analysis of the vegetation sampling data revealed width of 9.8 for heights utilized by island males considerable overlap in the structure of the Ch-CBR is greater than the Simpson indices for height and San Agustın forests (Supporting Information, utilization found amongst male anoles on several Fig. S5A). The average height of woody plants within single-anole islands in the West Indies. For example, the island transects was 351 cm vs. 394 cm for the the perch height diversities for A. sagrei on Swan mainland transects. Woody plant diameter averaged Island, Cayman Brac and Duck Key are 7.2, 7.8 and 3.9 and 2.9 cm, respectively, on the island and main- 5.9, and 7.8 for A. monensis on Mona Island (Lister, land. To test for differences in vegetation height and 1976a). diameter, a MANOVA was conducted with the logs The diversity of the combined utilization of perch of vegetation height and diameter as the dependent heights and diameters by all morphs was also higher

© 2016 The Linnean Society of London, Biological Journal of the Linnean Society, 2016, 118, 852–872 DIMORPHISM/DIVERGENCE IN ISLAND AND MAINLAND ANOLES 863 on the island (3.46 vs. 2.78). Excluding the small GENETIC DIVERGENCE male and female morphs, the diversity of the com- We obtained a maximum of 758 bp of mitochondrial bined perch heights and diameters utilized by the ND2 sequence data [partial coding sequence (cds) large male and female morphs was 2.84. Hence, the plus some tRNAs] from 23 island and 27 mainland small male and female morphs increased microhabi- individuals of A. nebulosus, resulting in a total of 20 tat diversity by a substantial 18%. On the mainland haplotypes. Genetic diversity was low on San Agustın the small male and female morphs contribute 10% to (h = 0.50; p = 0.0007) relative to the mainland – the overall perch height perch diameter diversity of (h = 0.95; p = 0.006), and the populations were fairly 2.78. Employing the methods of Roughgarden (1974), divergent (FST = 0.34; Fig. 8A). We selected five perch site niche width was partitioned into within- mainland and five island individuals for RADseq, morph (WMC) and between-morph (BMC) compo- including island individuals sharing mtDNA haplo- nents. BMC measures the proportion of total niche types with the mainland population. Our reduced width that is due to niche differences between the dataset for clustering analyses consisted of 16 382 morphs, while WMC gives the proportion of total SNPs, resulting from the first SNP from each locus. niche width due to differences in resource utilization Our mainland dataset consisted of an average of 92% among individuals in the population. WMC on San genotypic coverage per locus (SD = 12%) and 8.3% Agustın was 75.6% and BMC was 24.4%. WMC for missing data (missing alleles across loci). For San the mainland was 82.8% and BMC was 17.2%. The Agustın, we obtained 91% coverage per locus higher BMC on the island suggests that overlap in microhabitat is less amongst the island morphs, which would be expected if intraspecific competition A is higher on the island.

Behaviour Between 1989 and 1993 we observed adult males of A. nebulosus on San Agustın for a total of 84 h and adult females for 78 h. We also accumulated 73 h of observation on adult males within the Ch-CBR forest over the same period, which, when combined with the observations of Lister & Garcia (1992), yielded a total observation time of 231 h for mainland males and 89 h for mainland females. From these detailed observations, we calculated the per cent of time spent in social behaviour and in resting/hiding by B insular and mainland males. During the wet (breed- Island ing) season, island males spent much more time in social behaviour, mainly courting females, than mainland males (95 vs. 49%), and far less time rest- Mainland ing and hiding (1 vs. 44%). There was no difference in the number of moves per hour between island and mainland males during the dry season (2.5 vs. 2.6 h1). Over two wet seasons, the average number of movements per hour for island males (37.2) was 3.8-fold higher than for mainland males over five wet seasons (9.7), and feeding rates were 5–10 times 0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.1 0.2 higher than those of mainland males (Supporting –30 –20 –10020 10 Information, Figs S7, S8). The maximum dewlap rate Discriminant function 1 during the breeding season was also close to five 1 times higher for island males (72 h ) than for main- Figure 8. A, mitochondrial haplotype network showing 1 land males (15 h ). Only A. carolinensis males at haplotypes private to the island and mainland popula- 1 75–78 displays h (Johnson & Wade, 2010) match tions, as well as shared haplotypes in grey. Note that the the display rate of male A. nebulosus on San mainland population has significantly more genetic diver- Agustın. Like mainland females, island females are sity than the island population. B, results from the dis- considerably more sedentary than island males, criminant analysis of principal components analysis for especially during the wet season (4.9 vs. 37.2 moves the 16 382 SNP loci. The mainland and island popula- h 1). tions are readily recovered.

(SD = 12%) and 10.5% missing allelic data. Island other Anolis species inhabits the Ch-CBR forest, or and mainland individuals grouped cleanly (K = 2, most other parts of A. nebulosus’ range in western from our DK analysis) in both STRUCTURE and Mexico (Garcia & Ceballos, 1994), A. nebulosus is an DAPC analyses (Fig. 8B), with individuals showing unusual example of a solitary mainland anole. This 100% posterior support in STRUCTURE for member- absence of congeners may have resulted in vacant ship in their respective populations (Island, Main- niches and the evolution of different morphs to fill land). We further found evidence for divergence them. between these two populations (FST = 0.18), as well Several studies suggest that the densities and as lower genetic diversity on the mainland (h = 0.38) behaviour of mainland anoles are primarily shaped relative to San Agustın (h = 0.32; Supplementary by predation (Andrews, 1979; Lister & Garcia, 1992). Information, Table S10). In theory, such top-down control should alleviate intense inter- and intraspecific competition, and con- sequently reduce the effects of density-dependent, disruptive selection. Under these conditions, competi- DISCUSSION tion for enemy-free space rather than food could be Given the above results, we return to our initial the driving force behind the evolution of mainland questions. polymorphism (Jeffries & Lawton, 1984). Like resource competition, competition for enemy-free space could promote shifts in behaviour and diver- 1. TO WHAT EXTENT HAS THE ISLAND POPULATION gence in microhabitat use. While not as common as OF A. NEBULOSUS DIVERGED MORPHOLOGICALLY those arising from competitive interactions, polymor- AND GENETICALLY FROM THE ANCESTRAL phisms resulting from predator-driven disruptive MAINLAND POPULATION? selection are well known and include the green and brown colour morphs of the Pacific tree frog (Morey, Retention of intrasexual dimorphism 1990), the dorsal pattern polymorphism of female The island population has retained the same dimor- A. sagrei (Calsbeek & Cox, 2011) and the behaviour– phisms in relative head and limb size that evolved in coloration polymorphism in garter snakes (Brodie, the mainland population. Such intrasexual polymor- 1992). phisms involving morphology, coloration, behaviour While specification of the selective forces underly- and life history have been reported for a variety of ing intrasexual dimorphism in A. nebulosus awaits vertebrate taxa including birds, reptiles, fish and further research, previous studies suggest that mul- amphibians (Knudsen et al., 2006; Huber et al., tiple selection pressures may shape the direction and 2007; Martin & Pfennig, 2009; Higham et al., 2015). degree of such polymorphisms. Intersexual differ- In anoles, research to date has focused on polymor- ences in microhabitat use are common in anoles and phisms in female dorsal pattern (Paemelaere, Guyer often associated with shape dimorphisms similar to & Dobson, 2011). To our knowledge, the only docu- those found amongst the morphs of A. nebulosus. mented example of non-sexual, morphological dimor- Many male anoles, for example, perch higher than phism in an anole other than A. nebulosus is for females, and often have relatively longer legs and Anolis agassizi on Malpelo Island, an oceanic island larger heads (Butler et al., 2007). Manipulative field off the west coast of Colombia (Lopez-Victoria, experiments have also demonstrated that intraspeci- Herron & Botello, 2011). Here male A. agassizi have fic differences in limb length in A. sagrei result from two morphs which differ in maximum SVL (84 vs. density-dependent, disruptive selection for separate 110 mm) and coloration. performance optima on narrow and broad surfaces The evolution of intrasexual dimorphism in a (Calsbeek & Smith, 2008). mainland anole is somewhat surprising, given that Our current understanding of adaptive radiation increases in ecomorphological diversity often occur in anoles is based on a paradigm of ecological and on islands and other areas where reduced competi- allopatric speciation due to evolutionary change in tion can lead to niche expansion and character continuously varying traits driven by interspecific release (Lister, 1976a, b; Butler et al., 2007). For competition and niche divergence. The existence of example, the degree of sexual size dimorphism in intraspecific dimorphisms in A. nebulosus and Caribbean anoles is inversely related to the number A. agassizi raises the possibility that sympatric spe- of co-occurring congeners (Schoener, 1977). In the- ciation, driven by intraspecific competition, predation ory, as the number of sympatric species increases, or sexual selection, may have played a role in the size differences between males and females contract origin of new Anolis species, as is apparently the to lessen the impact of interspecific competition for case in a variety of other taxa (Knudsen et al., 2006; food and other resources. However, given that no Huber et al., 2007; Takahashi, Takahashi & Parris,

2010; Winkelmann et al., 2014). Butler et al. (2007) Smith, 2003). While we do not know when San conclude that intersexual size and shape dimorphism Agustın was colonized, evolution of shape in response have played an important role in the adaptive radia- to local conditions can occur rapidly in insular anoles tion of anoles. We would expect the same conclusion (Stuart et al., 2014), and limited divergence in shape to hold for intrasexual dimorphism, assuming that due to limited adaptation time is unlikely. Transloca- its existence in A. nebulosus and A. agassizi is not tion experiments within and between islands have unique in the evolutionary history of anoles. also shown that the magnitude of divergence of experimental populations from the source population Island–mainland divergence in shape is a function of the degree of difference in vegetation The characters that separate phenotypes on island (Malhotra & Thorpe, 1991; Losos et al., 1997). As our and mainland are known to be ecologically relevant Supporting Information (Fig. S5) indicates, the struc- and adaptively important in anoles. Species with tures of the island and mainland habitats are quite longer limbs, such as trunk-ground anoles, run faster similar, and this may have contributed to the mini- on broad surfaces than short-limbed species, while mal shape divergence between the two populations. species with shorter legs are more agile on thinner perches (Losos et al., 1998). These differences in per- Divergence in size formance are functionally significant in escaping from The major morphological difference between island predators, foraging and reproducing (Calsbeek & and mainland A. nebulosus is the larger size of the Irschick, 2007). The positive relationship between island morphs. Anoles and other island-dwelling size-adjusted limb length and perch diameter docu- lizards often show a size-diversity effect in which mented here among A. nebulosus morphs is consistent size increases as the number of other lizard species with a general pattern that holds across the Greater inhabiting the same island decreases (Schoener, Antillean ecomorphs, as well as within species such as 1970; Meiri, 2008). The traditional explanation of A. sagrei and A. carolinensis (Losos, 2009). this trend is that interspecific competition declines in The ecology and evolution of head size have been direct proportion to the number of co-occurring spe- extensively studied in anoles and other lizards. Head cies, which in turn leads to ecological release and size and prey size covary in anoles, and even differ- niche expansion. Phenotypes, including larger bodies, ences as small a 1 mm in head size are associated jaws and beaks, that facilitate utilization of a with significant differences in prey size distributions broader set of resources are then selected for (Owens (Schoener & Gorman, 1968). Within and between et al., 2006). Available evidence suggests strongly species, larger head size is also highly correlated that on San Agustın competition with other saurian with greater bite force, and individuals with greater and avian insectivores should be considerably bite force consume larger, harder prey items (Herrel reduced compared with the adjacent mainland. In et al., 2006; Runemark, Sagonas & Svensson, 2015; the mainland forest, A. nebulosus coexists with 14 Van Kleeck, Chiaverano & Holland, 2015). Hence, other lizard species (Garcia, 2008; Garcia, Valterria divergence in head shape between morphs may pro- & Lister, 2010), and up to 30 species of insectivorous mote intrasexual resource partitioning that parallels birds that can reach densities of 60–70 individuals intersexual differences in diet in sexually dimorphic per hectare during the winter dry season when anoles and other lizard species (Schoener, 1967; Her- insect abundances are comparatively low (Hutto, rel et al., 2006). Larger heads and stronger bites are 1980). While small flocks of mainland birds occasion- also excellent predictors of the outcome of aggressive ally forage on San Agustın, the only resident breed- disputes between male lizards (Lailvaux & Irschick, ing bird species is the largely granivorous mourning 2007), and appear to enhance defence against preda- dove Zenaida macroura. tors as well (Leal, 1999; Runemark et al., 2015). Our comparisons of survivorship and predation Given these results, it appears likely that the rela- between island and mainland are in keeping with tively larger head size of mainland A. nebulosus is the prediction of life history theory that lower extrin- associated with utilization of larger arthropods that sic mortality on islands should result in the evolu- require greater bite force to capture and consume, tion of larger body sizes (Palkovacs, 2003). A more and possibly with predator defence, but further data complete understanding of the relative impacts of are needed to test these hypotheses. increased density, competition and relaxed predation Based on the results of our genomic analyses, it is on size awaits more extensive comparisons such as doubtful that gene flow from the nearby mainland, those conducted by Li et al. (2011). These authors or loss of genetic diversity through genetic drift, analysed an extensive data set on the body sizes, have constrained the shape divergence of the island resources, predators and life history parameters of population relative to the mainland source popula- rice frogs (Rana limnocharis) on 20 islands and two tion, and hence contributed to stasis (Calsbeek & mainland sites, and demonstrated convincingly that

© 2016 The Linnean Society of London, Biological Journal of the Linnean Society, 2016, 118, 852–872 866 H. H. SILICEO-CANTERO ET AL. reduced predation was the major force driving body populations, although private haplotypes were gigantism of the island frogs. observed in each group. The island population is less diverse than the mainland population in mtDNA Divergence in behaviour (h = 0.50 vs. 0.95), but not nucDNA (h = 0.38 vs. Increases in population density and intraspecific 0.32). Interestingly, our broad sampling of the competition on islands can promote greater competi- nuclear genome resulted in a clear distinction tion amongst males for mates as well as food. In the- between the island and mainland populations, indi- ory, increased sexual competition can also lead to cating a relative lack of gene flow. Taken together, selection for larger body (Stamps et al., 1997). In our results are likely to be capturing some evidence anoles and other lizards, larger size confers a num- of incomplete lineage sorting in the mtDNA, and ber of advantages. Larger males are socially domi- generally show evidence of a relaxation population nant, mate with more females and are more likely to genetic model, whereby the island population is win skirmishes over territories and females than evolving under the relative influences of drift and smaller males (Trivers, 1976; Losos, 2009). Our com- lack of migration. Senczuk et al. (2014) also geneti- parisons of A. nebulosus behaviour on San Agustın cally analysed San Agustın and mainland Ch-CBR and in the Ch-CBR forest revealed striking differ- A. nebulosus and found that the island population ences which suggest that intrasexual competition likewise exhibited reduced genetic variation relative among island males may be an important factor in to the mainland. That study, which utilized only the evolution of larger body size. In particular, the mtDNA from a smaller sample of individuals, also extraordinary amount of time and effort that island failed to detect haplotype sharing between island males expend in courtship and display is a likely and mainland populations. Those authors interpreted indicator of intense rivalry for access to females. this result as a single colonization of the island from Mainland A. nebulosus are amongst the most seden- the mainland, followed by minimal or non-existent tary lizards known, and Lister & Garcia (1992) inter- gene flow (relative isolation). Although not stated, preted their lack of movement and prolonged periods the reduced genetic diversity of the island population of hiding as adaptations that reduced exposure to pre- is, as in our study, probably due to either a historical dation. Release from mainland predators probably founder effect and/or a relaxation of allelic diversity enables the island population to adopt a much more (drift). In both studies, genetic data suggest reduced active life style including greater number of move- or non-existent gene flow from the mainland to the ments per hour, greater distances moved per hour and island populations and that island populations are prey capture rates that are 4- to 5-fold greater per evolving independently of mainland populations. hour (Supporting Information, Figs S7, S8). Similar behavioural shifts occur in populations of A. wattsi inhabiting small islands near Antigua where preda- 2. DOES THE MORPHOLOGY OF THE ISLAND AND tory Ameiva are missing (Kolbe, Colbert & Smith, MAINLAND POPULATIONS OF A. NEBULOSUS 2008). Other island–mainland comparisons have also CORRESPOND TO ANY OF THE SIX WEST INDIAN documented marked differences in activity, with ECOMORPHS? island anoles moving on average 4–5 times more fre- While the behaviour and ecology of mainland A. neb- quently per hour than mainland anoles (Losos, 2009). ulous most closely resemble West Indian trunk- Given intense male–male competition and the like- ground anoles (Lister & Garcia, 1992), the same pre- lihood of increased intersexual selection, we hypothe- dictable relationships between morphology and habi- size that island males should have evolved relatively tat use found in the West Indies do not apply to larger testes (Byrne, Roberts & Simmons, 2002), big- continental anoles, and the great majority of species ger dewlaps (Harrison & Poe, 2012; Harrison, 2014) that utilize habitats similar to West Indian anoles and greater signalling complexity (Ord, Blumstein & have not converged on similar morphologies (Irschick Evans, 2001) than their mainland counterparts. In et al., 1997; Pinto et al., 2008; however, see contrast, island females that spend the majority of Moreno-Arias & Calderon-Espinosa (2015) for recent their day avoiding male advances might be especially evidence indicating greater convergence between recalcitrant owing to their capacity for sperm storage mainland and Caribbean morphotypes than and long-term retention of fertilized eggs in the previously realized). From data presented by Schaad highly variable climate of Mexico’s west coast (Lister & Poe (2010), the probability that a mainland species & Garcia, 1992). has converged on any of the West Indian ecomorphs is 0.07–0.12 depending on the confidence level of the Genetic divergence assignment. The conditional probability of conver- Genetic analyses showed some evidence for mtDNA gence, given that the mainland species occupies a haplotype sharing between island and mainland trunk-ground microhabitat, is 0.0–0.016. Hence, our

© 2016 The Linnean Society of London, Biological Journal of the Linnean Society, 2016, 118, 852–872 DIMORPHISM/DIVERGENCE IN ISLAND AND MAINLAND ANOLES 867 initial expectation that the morphology of A. nebulo- in the West Indies, 34 of 40 resident male anoles sus would not correspond to any of the West Indian have SVLs between 55 and 75 mm, and, on 37 of the ecomorphs was confirmed. Like A. nebulosus, the 40 islands, female SVLs are between 40 and 60 mm shapes of the solitary, mainland-derived species (Schoener, 1970). The maximum sizes of the large- A. concolor and A. pinchoti do not fully match any of headed adult male and female morphs on San the West Indian ecomorphs (Calderon-Espinosa & Agustın lie within these ranges, as well as the males Barragan-Forero, 2011). To date, a comparative anal- and females of the solitary species A. concolor on ysis of shape in A. agassizi has not been carried out. Colombia’s San Andres Island (Calderon-Espinosa & Barragan-Forero, 2011). While the SVLs of female A. pinchoti, the other solitary species investigated by 3. IN THE ABSENCE OF MOST MAINLAND COMPETI- Calderon-Espinosa & Barragan-Forero (2011), are TORS, HAS THE ISLAND POPULATION OF A. NEBULO- within the West Indian size range, the mean and SUS UNDERGONE NICHE EXPANSION AND EVOLVED maximum SVLs of males are somewhat less than TOWARDS A GENERALIST PHENOTYPE? 55 mm (Corn & Dalby, 1973). Like A. ferreus in the Losos & de Queiroz (1997) tested the prediction that Lesser Antilles, A. agassizi is an outlier with respect anoles on one- and two-species islands in the Lesser to the sizes of most solitary anoles. The maximum Antilles and northern Caribbean should have evolved SVLs of the male morphs are 84 and 110 mm, while convergent, generalist morphologies. They defined females have a maximum SVL of 83 mm (Lopez-Vic- the generalist morphology as a hypothetical pheno- toria et al., 2011). Comparisons of A. nebulosus and type intermediate between the shapes of the six other island and mainland anoles with A. agassizi major Great Antillean ecomorphs. Only four West should be especially revealing given the latter spe- Indian species met this criterion. While the San cies’ large size, polymorphism, unique habitat and Agustın population of A. nebulosus has expanded the foraging behaviour, lack of territoriality and origins range of perch sites utilized relative to the mainland in mainland Dactyloa rather than the Norops clade population, it has largely retained its ancestral phe- (Rand, Gorman & Rand, 1975; Losos, 2009). notype and has not evolved greater similarity to the The evolution of increased body size of anoles on putative generalist morphology compared with the San Agustın and other islands has important implica- mainland population (Fig. 6). tions for niche expansion and intraspecific competi- While these results suggest limited support for the tion. As maximum size increases, the range and generalist convergence hypothesis, we suggest that variance of prey sizes taken by a given population will this conclusion is unwarranted for three reasons. increase as well (Lister, 1976a). Moreover, as Bolnick First, the definition of the generalist phenotype et al. (2007) have shown, as the range of prey size assumes that anoles on small, biologically depauper- expands, the degree of prey size specialization among ate islands would somehow experience the net selec- individuals increases significantly, as predicted by the tion pressure resulting from the exploitation of the niche variation hypothesis (Van Valen, 1965). Greater same set of microhabitats utilized by all six eco- between-individual partitioning of prey size results in morphs. While this is theoretically possible, it is extre- reduced intraspecific competition and higher popula- mely doubtful that the diversity of vegetation on most tion densities. Mathematical analysis of growth in small islands would encompass the required range of continuously growing species indicates that as mean microhabitats. Even if such islands existed, the niches size increases, variance in size automatically of resident anoles would have to expand sufficiently to increases as well (Lister & McMurtrie, 1975). This encompass the entire range of vegetation occupied by effect is evident in the increased coefficient of varia- all of the more specialized ecomorphs. Second, the tion of SVLs on San Agustın (Supporting Information, effects of predation, intraspecific competition and Fig. S6). Such adaptive increases in size variation, changes in life history are all assumed to remain con- and concomitant increases in niche width and individ- stant on small islands. The third problem arises from ual specialization, are, in essence, emergent proper- defining the generalist phenotype with respect to ties of the evolution of larger size. shape in the first place. Below we offer an alternative As Schoener (1977) also discovered, solitary anoles definition that is consistent with known patterns in in the West Indies are convergent in their degree of the morphology of solitary anoles, and suggest that sexual dimorphism as well as size (Fig. 3). Like convergent evolution on a generalist phenotype should increases in overall size, greater sexual dimorphism be a common phenomenon on continental as well as increases both morphological and ecological diversity West Indian islands. within Anolis populations, as Figure 4 and the Sup- As Schoener (1970) originally observed, anoles on porting Information (Fig. S6) illustrate, and hence are one- and two-species islands are largely convergent important components in the evolution of niche width with respect to size. Among 40 monospecific islands (Butler & Losos, 2002; Butler et al., 2007). While

SSDs are available for only four solitary species with assistance with genotyping. The suggestions and com- mainland origins (Fig. 3), these initial data suggest a ments of three anonymous referees were greatly trend similar to West Indian counterparts. We predict appreciated and helped to improve the article in many that additional samples from continental island popu- ways. We also acknowledge the support of the gradu- lations should cluster around a modal SSD of approxi- ate programme in the biological sciences at the Insti- mately 1.3. Finally, it follows that the behavioural tuto de Biologia (IB) Universidad Nacional Autonoma release from predation exhibited on San Agustın de Mexico (UNAM) and the Estacion de Biologıa Cha- should also occur on most small islands lacking main- mela from IB-UNAM. Brad Lister was funded by land predators. We suggest that solitary anoles National Science Foundation grant 1038013. The should converge on a repertoire of behaviours similar authors declare no conflict of interests through any to insular A. nebulosus, behaviours that drive selec- commercial or other relationships. tion for larger male body size, and possibly propor- tional increases in testes and dewlap size as well as similar endocrine functioning and display complexity. REFERENCES Multiple variables, including climatic variation, distance to source of colonists, prey size distribution and Andrews RM. 1979. Evolution of life histories: a comparison vegetation, will undoubtedly impact the evolution of of Anolis lizards from matched island and mainland habi- shape, size, SSD and behaviour on any given island. tats. Breviora 454: 1–51. However, the primary forces affecting size in solitary Andrews RM, Nichols JD. 1990. 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SUPPORTING INFORMATION Additional Supporting Information may be found online in the supporting information tab for this article: Figure S1. Histograms of adult male A. nebulosus snout and forearm lengths for the island (A, B) and mainland (C, D) populations. For both locations, the light grey histobars correspond to the long-armed, small- headed morph, and the dark grey histobars to the short-armed, large-headed morph. Figure S2. Cumulative survival and hazard functions for A. nebulosus individuals tethered at 2 m height to tree trunks on San Agustın (blue) and the Ch-CBR forest (red) during the wet and dry seasons. The overall mean survival time on the island was 7.4 days vs. 2.8 days on the mainland. During both seasons, survival rates were significantly higher on the island (Mantel-Cox log rank test, P < 0.0001). Figure S3. Morphological groupings of the four mainland and four island morphs in two-dimensional discriminant space. Polygons encompassing the outermost scores for each morph are shown. Sixty-seven per cent of individual cases were correctly classified with respect to the eight morph-groups. The large-headed, short- limbed and longer SVL morphs are on the right of the diagram, and the small-headed, long-limbed and shorter SVL morphs are grouped on the left. Figure S4. Vectors of the loadings of the labelled variables on the two PC axes shown in Figure 5. A, male morphs. For males, the two PC axes accounted for 65% of the total variation in the size-adjusted variables. B, female morphs. For females, the two PC axes accounted for 55% of the total variation in the size-adjusted variables. Note the negative loadings along PC1, the axis associated with relative size differences, for snout length and mandibular width, and the positive loadings for forelimb and hind limb length. Figure S5. A, plot of ln height vs. ln diameter of woody plants measured along transects through the island (triangles) and mainland (circles) study areas. Island filled triangle: x̅ln height = 5.68 cm; x̅ln diameter = 1.30 cm. Mainland filled circle: x̅ln height = 5.74 cm; x̅ln diameter = 1.10 cm. B, plot of ln perch height vs. ln perch diameter for adult male and female A. nebulosus morphs combined from the island (triangles) and mainland (circles) study areas. Island filled triangle: x̅ ln perch height = 4.5 cm; x̅ ln perch diameter = 1.63 cm. Mainland filled circle: x̅ln perch height = 3.9 cm; x̅ln perch diameter = 1.63 cm.

Figure S6. Distribution of snout–vent length measurements for all morphs combined from the island (A) and mainland (B) populations. Both the coefficient of variation (CV) and the Shannon diversity measure (H) were significantly higher for the island population. Figure S7. Number of moves per hour (A) and total distance moved per hour (B) for male and female A. nebulosus observed in the mainland and island study areas during the wet and dry seasons. Mainland males dark red; mainland females light red; island males dark blue; island females light blue. Figure S8. Number of prey captures per hour for adult male A. nebulosus observed in the mainland (red) and island (blue) study areas as a function of the number of arthropods captured in sticky traps during the time period of observation. Mainland males red; island males blue. Best fit linear regression lines have been drawn through the island and mainland data. Table S1. Descriptive statistics for male and female A. nebulosus morphs. Table S2. Sexual size dimorphism (SSD) indices calculated by taking the ratios of mean and maximum SVLs of male morphs to the mean and maximum SVLs of the corresponding female morphs. Table S3. The morphospace areas calculated from Figure 4 for the island and mainland male and female morphs. Table S4. Loadings of the size-adjusted predictor variables on the first two discriminant axes in the Support- ing Information (Fig. S3). Table S5. Results of the one-way MANOVA with Morph Group as the independent variable and the eight size-adjusted morphological variables as the dependent variables. Table S6. Results of Tukey’s post-hoc test for differences between size-adjusted snout lengths (SL) and mand- ible widths (MW) of equivalent mainland and island morphs. Table S7. Euclidean distances between the centroids in Figure 5 for the island and mainland male (IslM and MLM) and island and mainland female (IslF and MLF) morphs. Table S8. Euclidean distances between the centroids of insular and mainland A. nebulosus males, the centroids of the West Indian male ecomorphs, and the putative generalist morphology, in Figure 6. Table S9. Mean trunk heights and diameters in the island and mainland study areas, mean perch diameters and heights utilized by adult males and females on the island and mainland, and niche widths (Simpson measure) of perch site utilization. Table S10. Mitochondrial DNA summary statistics for island and mainland populations of A. nebulosus.