Hemipenis Shape and Hindlimb Size Are Highly Correlated in Anolis Lizards

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Biological Journal of the Linnean Society, 2017, XX, 1–8. With 2 figures.

Hemipenis shape and hindlimb size are highly correlated in Anolis lizards

JULIA KLACZKO1,2*, CASEY A. GILMAN3,4 and DUNCAN J. IRSCHICK3

1Department of Physiological Sciences, Institute of Biological Sciences, University of Brasília, Asa Norte. Brasília - DF 70.910-900, Brazil 2Museum of Comparative Zoology and Department of Organismic and Evolutionary Biology, Harvard University, 26 Oxford Street, Cambridge, MA 02138, USA 3Department of Biology, University of Massachusetts, 221 Morrill Science Center III, 611 North Pleasant Street, Amherst, MA 01003-9297, USA 4Graduate Program in Organismic and Evolutionary Biology, University of Massachusetts Amherst, 230 Stockbridge Road , Amherst, MA 01003-9316, USA

Received 23 May 2017; revised 7 August 2017; accepted for publication 7 August 2017

The Squamata (lizards and snakes) is the only clade among vertebrates with hemipenes, a pair of male intromittent genital organs. Hemipenes are remarkably variable between species, and they evolve much faster than other organs. Therefore, they have been used extensively in systematics as a diagnostic character. However, few studies focus on the quantitative description of hemipenial variation, and few comparative evolutionary studies are available. Here we provide the first morphometric assessment of evolutionary hypotheses regarding male squamate genital evolution. We describe hemipenis shape in Anolis lizards from the Caribbean Islands Puerto Rico and Jamaica using elliptic Fourier analysis. To understand the forces driving hemipenis evolution, we tested the association between hemipenial shape and testis volume to assess sexual selection; we tested the association between hemipenial shape and hindlimb size to search for pleiotropic effects; and finally, we tested the association between hemipenis shape and ecomorphology. Our study results indicated a clear correlation between hemipenis shape and hindlimb size. However, we found no correlation between hemipenial morphology and testis volume, or ecomorphology. These results suggest that pleiotropy would be associated with hemipenial evolution, especially in the presence of hindlimbs, probably as a result of the physical, regulatory and genetic association between hindlimbs and genitalia development. This suggests that while sexual selection may be acting to drive hemipenis evolution, pleiotropy is somehow involved and may be slowing its evolution.

ADDITIONAL KEYWORDS: elliptic Fourier analysis – geometric morphometric – male genitalia – phylogenetic comparative analysis – pleiotropy – sexual selection.

INTRODUCTION 2004 for a thorough review), and most of the work has focused on describing which forms of sexual selection Male genital evolution in animals with internal fer- (cryptic female choice, sperm competition or sexual con- tilization is a recurrent topic in biological evolution in flict between the sexes) may be acting on genital diver- the last decades (Langerhans et al., 2016). Male geni- sification. Therefore, alternative explanations, such as talia are known to be extremely variable between spe- pleiotropy, have been relatively neglected (Reinhardt, cies, exhibit complex morphology and, when compared 2010). Showing that genital evolution has occurred as to other organs, evolve remarkably quickly (Arnqvist, a pleiotropic response to selection acting on another 1998; Hosken & Stockley, 2004; Böhme & Ziegler, 2009; trait is a challenging task. Nevertheless, few studies Eberhard, 2010; Klaczko, Ingram & Losos, 2015). A con- have reported indirect evidence of pleiotropic effects sensus exists that postmating sexual selection is the pri- on genital morphology, such as the clear correlation mary driver of genital evolution (see Hosken & Stockley, between genital and horn size in dung beetles (Parzer & Moczek, 2008), and the significant correlation between *Corresponding author. E-mail: [email protected] Drosophila’s genital and wing size (Andrade et al., 2009).

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Lizards and snakes are the only vertebrates to pos- high rates of diversification and extensive morphologi- sess a pair of intromittent male genitalia called hemi- cal differentiation (Losos, 2009). Ecologically similar penes. The hemipenes are retracted into the base of the species on different islands in the Greater Antilles tail when not in use and are everted during copulation. (Cuba, Hispaniola, Jamaica and Puerto Rico) have Hemipenes show considerable variation in morphology convergently evolved a suite of morphological traits, across species, and therefore have long been used in tax- including limb length. At the extreme, species using onomic studies (Arnold, 1986b). However, little is known broad surfaces such as tree trunks or the ground have about the factors underlying hemipenial evolution in evolved very long hindlimbs, whereas twig species have squamates. As with other taxa, some studies have sug- converged on short limbs (Williams, 1972; Losos, 2009; gested that sexual selection may be driving hemipenial Mahler et al., 2013). Studies on morphological evolu- morphological variation; nevertheless, these studies do tion within Anolis show that their hemipenes evolve not discard the possibility of pleiotropy, or even the lock- six times faster than other morphological non-genital and-key mechanism (Arnold, 1986a; Sales Nunes et al., traits (Klaczko et al., 2015). Anolis hemipenial mor- 2014; Klaczko et al., 2015; Klaczko & Stuart, 2015). phology ranges from short, squat, unilobed (cylindri- Recent studies have discussed the development cal) structures to long, tendril-like bilobed (y-shaped) of amniote male genitalia (Holmes & Wade, 2005; structures, with every mix of shapes in-between. Gredler et al., 2014; Tschopp et al., 2014; Gredler, Some Anolis species can be morphologically similar Sanger & Cohn, 2015; Infante et al., 2015; Sanger, externally and only differ in their genital morphology Gredler & Cohn, 2015). In squamates the hemipe- (Köhler & Sunyer, 2008; Köhler & Vesely, 2010; Köhler, nial swelling initiations are located at the base of Dehling & Köhler, 2010). the hindlimbs buds, and they share the same devel- Here we examined the male genitalia of 14 Anolis opmental route with, and several regulatory genes species from the Caribbean islands Puerto Rico (eight (Gredler et al., 2014; Tschopp et al., 2014; Infante et species) and Jamaica (six species). We analysed on al., 2015). Such regulatory topologies could favour a average five specimens per species, with a range of correlation between hindlimbs and hemipenes (Lonfat 1–30 (Supporting Information, Table S1). One of the et al., 2014). Therefore, if there is a pleiotropic effect hemipenes was dissected from each specimen and on limbs and hemipenes, hemipenial morphology and prepared following Klaczko et al. (2015). We obtained limb traits should be correlated. digital images of hemipenes on the side containing the Despite the great interest in hemipenis morphol- sulcus spermaticus (the sperm channel) using a JVC ogy and evolution, most studies focus on qualitative camera KYF75U attached to a stereomicroscope with descriptions of hemipenial variation, rarely analysing it the aid of Auto-Montage Pro, 5.02. in a comparative perspective (Sales Nunes et al., 2014; Klaczko et al., 2015; Klaczko & Stuart, 2015). Here we describe hemipenial shape variation of Anolis lizards Morphological measurements using geometric morphometric techniques, which are Using digital calipers, we obtained the following meas- well known and commonly used in studies of genital urements from each male: snout–vent length (SVL) as evolution on invertebrates, but have never been used a proxy of body size, length of the thigh, length from to analyse hemipenial variation (Andrade et al., 2009; knee to ankle (shank) and testis volume. Thigh length Macagno et al., 2011; Oneal & Knowles, 2013). We assess was measured ventrally from the insertion point of whether pleiotropy or sexual selection (or both) are the head of the thigh to the knee. Shank length was involved in hemipenial evolution by testing the correla- measured dorsally. For testis volume, we measured tion between hemipenial shape and hindlimb size. We testis linear dimensions to the nearest 0.01 mm and also test for a correlation between hemipenial shape and calculated the volume using the formula for spheroid testis volume, using testis volume as a proxy for mating volume: vol = (4/3) π × length/2 × width/2 × height/2. system because males showing larger testes often mate We measured all traits twice and used the mean of with more females (Gage & Freckleton, 2003; Lüpold the measurements. For each trait, we performed a log- et al., 2009; Immler et al., 2011). Finally, we tested if log regression of species mean trait measurements on hemipenial shape and ecomorphology are correlated. species mean SVL. The residuals from these regres- sions were used as size-corrected measures in subsequent statistical analyses. MATERIAL AND METHODS

Study group Hemipenial shape Caribbean Anolis lizards have become a model system We described hemipenial shape using geometric mor- for the study of biological evolution, as they exhibit phometrics. Given that the hemipenis is soft tissue

and lacks defined homologous landmarks, we used least squares (D-PGLS). D-PGLS is a phylogenetic elliptic Fourier analysis (EFA) to quantify morpho- comparative method that uses a permutation proce- logical variation of the hemipenes. EFA is an outline dure to assess statistical hypotheses describing pat- analysis method, useful for describing shape variation terns of shape variation and covariation, taking into on landmark-poor shapes (Crampton, 1995). account the phylogenetic proximity of the analysed The outline is summarized as a series of harmoni- species could induce non-independence in the distribu- cally related sine and cosine equations termed har- tion of phenotypic traits (Adams, 2014). monics. This data set is then corrected for the effect In an attempt to visually compare the amount of of rotation, position and size by standardizing ellipti- variation on hemipenis morphology between related cal Fourier descriptors by the first harmonic of each species of limbed and limbless squamates, we selected specimen. Afterwards, it uses multivariate statisti- five species of Anolis and five Colubridae snakes spe- cal analyses in the same way that landmark-based cies, from two clades that diverged around 26.5 Mya. morphometric data sets are used (Crampton, 1995; We trimmed the phylogenic trees from the squamate Claude, 2008). Outline shapes were digitized using phylogeny (Zheng & Wiens, 2016). tps software. The EFAs were performed in the R package Momocs (Bonhomme et al., 2014). We used the Fourier power equation to remove superfluous vari- RESULTS ables (Crampton, 1995; Claude, 2008) and determined that ten harmonics were required to reconstruct 99% Hemipenial shape within Caribbean Anolis species of the detail of the digitized outline; thus, the first ten can be summarized in the first two PCAs that account harmonics were used for subsequent analysis of shape for 82.6% of explained shape variation in our overall variation. We calculated species mean shapes and used data set; each of the subsequent PCs accounts for less those in all further analyses. than 3% of the variation. Figure 1I is the biplot of the first and second principal (PC1 × PC2) axes, showing hemipenial shape variation. Shape differences along Comparative analyses PC1 are mainly associated with the relative propor- For comparative analyses that account for species phy- tion of hemipenis lobe and body. This axis strongly dis- logenetic relationships, we used the maximum clade criminates Anolis cooki, Anolis reconditus and Anolis credibility phylogenetic tree from a Bayesian analysis krugi, showing values smaller than zero, and Anolis of mitochondrial DNA of 187 Anolis species (Mahler valencienni, on the opposite side of the graphic, with et al., 2010). We pruned it to include only the 14 species positive values. PC2 describes changes associated with analysed for this study. relative length of the hemipenis, showing A. krugi, All of our statistical analyses were performed in R Anolis poncensis and A. valencienni separated from all (v3.2.3) using the R package Geomorph (v. 3.0) (Adams other species at the bottom side of the figure. & Otárola-Castillo, 2013; Adams, Collyer & Sherratt, All traits show Blomberg’s K values not significantly 2016). We used principal component analysis (PCA) to greater than zero: hemipenial shape K = 0.69 and examine the variation in hemipenial shape across the P = 0.08; testis volume K = 0.67 and P = 0.23; femur/thigh 14 Anolis species. K = 0.69 and P = 0.19; and metatarsus/shank K = 0.61 We estimated phylogenetic signal of hemipenial and P = 0.48. In accordance with this pattern, the phy- shape, hindlimb measurement and testis volume using lomorphospace shows that sister-species were separated Blomberg’s K statistic (Blomberg, Garland & Ives, in shape space, and consequently, morphologically simi- 2003). The K statistic can indicate little or no phyloge- lar hemipenis are often distantly related (Fig. 1II). netic signal (K << 1) or greater than the degree of phy- Testis volume and ecomorphology do not predict logenetic signal expected under a Brownian motion hemipenial shape when phylogeny is incorporated

random-walk model of evolution (K >> 1). To test if the in the analyses (testis volume: D-PGLS, F1,12 = 1.3,

data contain a signal of phylogeny (i.e. K > 0), we ran- P = 0.418 and ecomorph: D-PGLS, F1,12 = 1.66, domly permuted the order of species on the tree 1000 P = 0.135), but the length of the hindlimbs was sig- times and recalculated K values for each permutation. nificantly correlated with hemipenial shape (thigh:

We then compared the observed K value to this null D-PGLS, F1,12 = 6.49, P = 0.005 and shank: D-PGLS,

distribution to assess significance. To visualize the F1,12 = 4.38, P = 0.012). evolutionary history of hemipenis shape, we projected the phylogenetic tree used into the PCA morphospace. We examined the degree to which hemipenial shape DISCUSSION variation among species is evolutionarily associated with testis volume, hindlimb size and ecomorphology Previous studies have shown that male genitalia have using a Procrustes distance phylogenetic generalized great morphological diversity, show faster evolutionary

evolution. However, few studies have refuted other hypotheses concerning genital evolution. Our results indicate that, on the one hand, a clear correlation exists between hemipenial shape and hindlimb length and, on the other hand, that no correlation occurs between hemipenial morphology and testis volume or ecomorphology. The close physical association between the hindlimb buds and hemipenis swellings, the share of signalling pathways and gene expression similarities could be indicative of a developmental and evolutionary link between limb and genitalia (Lonfat et al., 2014; Tschopp et al., 2014). The outgrowth of limbs and genitalia during embryonic development depends on common genes, such as Hox genes; as showed by Kondo et al. (1997) that removed the posterior Hox gene function and it resulted in a concomitant loss of digits and genital bud derivatives. Additionally, disor- ders such as the human hand-foot-genital syndrome are the result of mutations in these developmental regulators (Mortlock & Innis, 1997). Moreover, it was revealed that these loci have similar regulatory topologies in developing limb buds and genital tubercle, and some limb enhancers could also function in developing genitalia (Lonfat et al., 2014). Such regulatory topologies may have favoured the evolution of pleiotropic developmental loci among vertebrates (Lonfat et al., 2014). Therefore, the strong correlation between hemipenis shape and hindlimb length could be a direct effect of pleiotropy. If hemipenes are under pleiotropic effect, we could expect that the rate and direction of evolution would be constrained, as pleiotropic genes act as stabilizing factors that impose a pressure on how far genital morphology could change (Lande, 1980; Cheverud, 1996; Otto, 2004). Pleiotropy driving male genital evolution was first suggested by Mayr (1963), that proposed that the variation in genital morphology is selectively neutral, and an exclusively indirect result of evolution of genetically correlated characters. Figure 1. The morphospace of Anolis hemipenial shape. I, Infante et al. (2015) showed that lizards’ limbs biplot of principal components 1 and 2 (PC1 vs. PC2) show- and genitalia transcriptional networks share cis- ing the hemipenial mean shape for the analysed species regulatory elements. Moreover, the analysis of snakes of Anolis. II, projection of the phylogenetic tree into mor- revealed that they retained specific enhancers that phometric space. Species letters are defined as follows: A, regulate activities between developing limbs and Anolis cooki; B, A. reconditus; C, A. krugi; D, A. lineatopus; genitalia, even in the complete absence of limbs. The E, A. grahami; F, A. cristatellus; G, A. gundlachi; H, A. ever- analysis by Infante et al. (2015) showed that HLEB manni; I, A. opalinus; J, A. stratulus; K, A. pulchellus; L, A. orthologs in snakes do not drive limb expression, but garmani; M, A. poncensis; N, A. valencienni. have retained activity within subdomains of the external genitalia. Finally, engineering of an HLB deletion rates than other morphological traits and usually in mice leads to defects in the development of both are more diverse in promiscuous species (House & limbs and genitalia, demonstrating pleiotropic roles Simmons, 2003; Böhme & Ziegler, 2009; Eberhard, of this enhancer. 2010; Rowe & Arnqvist, 2012; Klaczko et al., 2015). Therefore, it is possible that hemipenes evolution In the last few years, sexual selection frequently has may be less constrained in the absence of limbs. In been pointed to as the principal force driving genital snakes for example, there is an astounding diversity of

hemipenial shape, as well as ornamentation, such as testis volume and mating system has never been spines, papilla and calyces. Snakes have more diverse tested in Anolis species, so the underlying assumption and more complex hemipenes than any limbed liz- of this hypothesis remains to be tested in anoles. ards that we have examined (Fig. 2). This hypothesis Finally, we tested if hemipenial shape were corre- remains to be tested in a broad study of hemipenis mor- lated to ecomorphology, since the association between phology diversity and evolutionary rate across limbed hemipenes and hindlimbs could be an indirect result of and limbless squamate species – we predict that when natural selection acting on limbs, or, moreover, hemi- such studies are conducted, they will show greater penis and ecomorphology could be correlated as an rates of genital evolution in limbless squamates. indirect result of the association between mating sys- To test if the hemipenes of Anolis lizards are under tem and habitat use (Johnson, Revell & Losos, 2010). sexual selection, we used testis volume as a proxy of However, our analyses fail to recover that association. the mating system because it has been found that ani- Likely, genital evolution is the result of a set of fac- mal species with promiscuous mating systems have tors with different degrees of influence. Our results comparatively larger testis volumes and more variable do not rule out the hypothesis of sexual selection genitalia (Gomendio & Roldan, 1991; Harcourt, Purvis because both forces, sexual selection and pleiotropy, & Liles, 1995; Arnqvist, 1998; Hosken & Ward, 2001; could be acting simultaneously on hemipenial evolu- Byrne, Roberts & Simmons, 2002). If hemipenes are tion. Sexual selection may act on genital evolution in under sexual selection, we should find a close asso- a number of ways, including the three most commonly ciation between testis volume and hemipenial shape. studied: cryptic female choice, sperm competition and Yet we failed to find it. These results do not explain sexual conflict between the sexes (Hosken & Stockley, why Anolis hemipenes evolve six times faster than 2004). Genital shape may evolve to stimulate females other morphological non-genital traits, as described and act as signals of good genes, or females may by Klaczko et al. (2015). Also, the correlation between ‘choose’ these males because of the fitness benefits

Figure 2. Comparative morphology of hemipenes between a snake clade (Colubridae, Serpents) and an Anolis clade, both being diverged around 26.5 Mya. The phylogenic trees were trimmed from the squamate phylogeny (Zheng & Wiens, 2016). Species letters are defined as follows: A, sulcate and assulcate views of Mastigodryas bifossatus hemipenis; B, sulcate and assulcate views of Spilotes pullatus hemipenis; C, sulcate and assulcate views of Drymarchon corais hemipenis; D, sulcate and assulcate views of Chironius grandisquamis hemipenis; E, sulcate and assulcate views of C. exoletus hemipenis; F, sulcate view of Anolis stratulus hemipenis; G, sulcate and assulcate views of A. pulchellus hemipenis; H, sulcate view of A. gundlachi hemipenis; I, sulcate and assulcate views of A. poncensis hemipenis; J, sulcate and assulcate views of A. cristatellus hemipenis.

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Table S1. List of analysed specimens of Anolis species.

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