applyparastyle "body/p[1]" parastyle "Text_First" 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 correla- tion 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). © 2017 The Linnean Society of London, Biological Journal of the Linnean Society, 2017, XX, 1–8 1 Downloaded from https://academic.oup.com/biolinnean/article-abstract/doi/10.1093/biolinnean/blx104/4162054/Hemipenis-shape-and-hindlimb-size-are-highly by [email protected] on 26 September 2017 2 J. KLACZKO ET AL. 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 subse- quent 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