Acta Herpetologica 9(2): 147-158, 2014 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.13128/Acta_Herpetol-13473 Comparative morphology of Liolaemus lizards precloacal glands Soledad Valdecantos1,*, Virginia Martínez1, Antonieta Labra2,3 1 IBIGEO (Instituto de Biología y Geología del NOA), Universidad Nacional de Salta-CONICET, Avenida Bolivia 5150, 4400, Salta, Argentina. *Corresponding author. E-mail: solevaldecantosp@gmail.com 2 Programa de Fisiología y Biofísica, Facultad de Medicina, Universidad de Chile, Casilla 70005, Correo 7, Santiago, Chile 3 University of Oslo, Department of Biosciences, Centre for Ecological and Evolutionary Synthesis, P.O. Box 1066 Blindern, N-0316 Oslo, Norway Submitted on 2013, 25th October; revised on 2014, 5th May; accepted on 2014, 4th June Editor: Giovanni Scillitani Abstract. Liolaemid lizards and amphisbaenids have precloacal pores in the anterior border of the cloaca, where epi- dermal glands drain and expel pheromonal secretions. Precloacal glands occur usually only in males, but in those few species where both sexes have precloacal glands, these are larger in males. Only the morphology and/or histol- ogy of precloacal glands of amphisbaenids have been described, and it is unknown whether in lizards these glands differ across ages, sexes and/or species, and if the lack of pores is associated with a lack of glands. We investigated for the first time the morphology and histology of lizard precloacal glands, by studying three Liolaemus species that differ in the presence of pores in their cloaca: L. irregularis, in which adults and juveniles of both sexes have pores; L. poecilochromus, in which only adult males have pores, and L. neuquensis, in which the adults of both sexes lack pores. Results show that the number of pores varies among species and sexes, but not between ages of a species. Adults, but not juveniles, of L. irregularis have sexual dimorphism in pore sizes; these are larger in males than in females. In addi- tion, pores are larger in adult males of L. irregularis than in L. poecilochromus. Glands are tubuloalveolar with holo- crine secretion, having similar structure across individuals, although adult males have larger glands than females and juveniles. Finally, the structure of Liolaemus precloacal glands is very similar to those of the amphisbaenid precloacal glands and the femoral glands of other lizard species. Keywords. Epidermal gland, histology, histochemistry, sexual dimorphism, ontogenetic variation. INTRODUCTION Many lizard species have epidermal glands with secretions, which are a complex mix of lipids and pro- teins, involved in intraspecific chemical communication (Mason, 1992; Louw et al., 2007; Weldon et al., 2008; Mar- tín and López, 2011; Louw et al., 2011). Externally, glands are recognized by patches of modified scales that allow the release of the secretions. One type, the generational glands, lacks a collecting portion and the associated scales have a glandular appearance through which secretions are expelled directly, without a pore (Van Wyk and Mouton, 1992). The other type of glands, the precloacal/femoral glands, have a unique and clear pore where the collect- ing portion of the gland drains secretions to be expelled (Cole, 1966a; Maderson and Chiu, 1970; Baig and Böhme, 1991; Van Wyk and Mouton, 1992; Jared et al., 1999). The precloacal pores are situated on the anterior border of the cloaca, while the femoral ones are located on the ventral surface of the thighs (Alberts et al., 1992; Imparato et al., 2007; Labra, 2008). Pores are usually present only in adult males (Chauhan, 1986; Dujsebayeva et al., 2009; Lobo et al., 2012). In species where both sexes have pores, these are larger in males (Cole, 1966b, Athavale et al., 1977, Fer- reira, 2007), and if juveniles have pores, these are larger in adults than in juveniles (Cole, 1966b; Alberts et al., 1992). In a few species, both sexes have secondarily lost these pores (Darevsky and Szczerbak, 1997; Lobo, 2005). 148 S. Valdecantos et alii The morphology of the femoral glands has received significant attention (e.g. Athavale et al., 1977; Chau- han, 1986; Ferreira, 2007; Imparato at al., 2007; Chamut et al., 2009), and they can be lobed (Antoniazzi et al., 1993; Imparato et al., 2007) or tubular, simple (Chiu and Maderson, 1975), branched (Chiu and Maderson, 1975; Ferreira, 2007) or acinar (Khannoon et al., 2013). In con- trast, the knowledge about the precloacal glands morphol- ogy is confused, because a variety of glands located in the cloacal area had been called precloacal glands, such as some generation glands and callose scales of several cor- dylid (e.g. Van Wyk and Mouton, 1992) and agamid liz- ards (e.g. Dujsebayeva et al., 2007). The other group of glands, those traditionally called precloacal glands (Ether- idge, 1995; Pinna et al., 2010), is located on the border of the cloaca, which have a recognizable secretory and col- lecting portion, and are found in amphisbaenids and liola- emid lizards. Focusing on these later precloacal glands, we can say that the knowledge on their morphology is lim- ited to some studies on amphisbaenas (e.g. Antoniazzi et al., 1993). However, because the structure of the glands of liolaemid lizards is unknown, the understanding of the extent of morphological precloacal gland variation as compared to the femoral gland, it is not possible. The lack of information regarding the morphol- ogy of precloacal glands and pores in Liolaemus lizards, the most diverse Liolaemidae genus (Lobo et al., 2010a), with more than 240 species (Uetz and Hošek, 2014), con- trasts with the extensive use of precloacal pores as a taxo- nomic character, as well as for sex discrimination (Lobo, 2005). Gland secretions are involved in social recognition (Labra, 2008), and their lipid compositions differ among species and populations (Escobar et al., 2001; 2003). The glands apparently share a function across Liolaemus, but the variation in morphology among species, sexes or age classes (i.e., juveniles vs. adults) is largely unknown, and it is unclear if the lack of external pores is associated with a complete absence of these epidermal glands. To shed some light on these questions, we studied the external, anterior ventral border of the cloaca in three Liolaemus species that differ in the occurrence of precloa- cal pores: 1- L. irregularis: adults and juveniles of males and females have pores (Valdecantos and Lobo, 2007); 2- L. poecilochromus: only adult males have pores (Laurent, 1986); and, 3- L. neuquensis: adults of both sexes lack pores (Scolaro et al., 2007). MATERIALS AND METHODS Animals We processed individuals of the herpetological collection of the Museo de Ciencias Naturales of Universidad Nacional de Salta (Argentina), in order to have an adequate sample size without affecting negatively the natural populations. Specimens were fixed in 10% buffered formalin solution and preserved in 70% alcohol, and were collected in consecutive summers (December-February of 2007, 2008, 2009), not in their mat- ing season (Ramírez-Pinilla, 1991). We used 83 individuals of L. irregularis, 38 of L. poecilochromus and two of L. neuquen- sis. The description of the color of the secretions, however, was based on field observations of wild lizards. Histology We removed the external, anterior ventral border of the cloaca of 31 L. irregularis (♂: 7 juveniles, 8 adults; ♀: 9 juve- niles, 7 adults), 22 L. poecilochromus (♂: 5 juveniles, 8 adults; ♀: 2 juveniles, 7 adults) and two L. neuquensis adults (1♂, 1♀), and dehydrated in graded series of ethanol solution, cleared in xylene and embedded in paraffin. Thereafter, paraffin sagittal serial sections (5-7 µm) were obtained with a rotary microtome (Reichter, Germany) and were stained with hematoxylin-eosin (H & E). Based on previous studies on sexual maturity (Valde- cantos and Lobo, 2007), we considered juveniles of L. irregula- ris and L. poecilochromus individuals which snout-vent lengths ranged from 32.76 to 48.63 mm, without including newborns since they were not available in the collection. Adults of L. irregularis had sizes from 70.4 to 99.1 mm, and L. poecilochro- mus, from 62.14 to 75.03 mm. No age classes were studied in L. neuquensis, and the adult male measured 60.44 mm, while the adult female measured 59.25 mm. Gland descriptions and their measurements were done on the three best central mid-sagittal sections (i.e., not on their borders) of the best-sectioned gland from each specimen. Descriptions follow the nomenclature of Cole (1966b), Geneser (2000), and Gartner and Hiatt (1994). Histochemistry To identify neutral and acid mucosubstances, paraffin sections were treated separately with periodic acid Schiff ’s rea- gent (PAS) and alcian blue (AB) pH 2.5 (Martoja and Martoja- Pierson, 1970). Separately, we stained new slides with Bromo- phenol blue to identify proteins (Imparato et al., 2007). We did not search for lipids, because they solubilize in alcohol, which was used to preserve the lizards. Slides were observed under an Olympus BX40 microscope (Tokyo, Japan) and photographed with a digital camera Olympus DP25 (Tokyo, Japan). Scanning electron microscopy We processed the external, anterior ventral cloacal border of four L. irregularis, one of each sex and age (juvenile, adult), and one adult male of L. poecilochromus. The tissues were dehydrated by critical point, dried with liquid carbon dioxide, sputtered with gold, and examined under a scanning electron microscopy (JEOL JSM-6480, Tokyo, Japan). 149Precloacal glands of Liolaemus lizards Morphometry of precloacal pores and glands We determined the number and diameter (mm) of the precloacal pores of L. irregularis and L. poecilochromus (see Table 1 for sample size). To obtain the pore diameters, we measured the length of the minor and major axes of each pore, using a binocular Arcano magnifying glass (HG 272931, Shangai, China) with a graduated ocular lens. Each individual was character- ized by the average value of the diameters of all its pores. Using a microscope Olympus magnifying glass (Tokyo, Japan) with a graduated ocular lens, and following Cole (1966b), we measured the maximum length of the glands from the anterior end to the posterior end of the basal layer of germinative epithelial cells. The maximum width was measured in the transverse axis of the precloacal gland (see Table 2 for sample size and Fig. 5 for indi- cations of how these measurements were taken). To remove the effect of body size in the statistical anal- yses of pores and glands sizes, we measured the snout-vent length of these lizards (see below). Statistics The numbers of precloacal pores were not normally dis- tributed, and comparisons were made with non-parametric Mann-Whitney tests. The log10 transformed pore diameters and the log10 length and width of the precloacal glands were nor- mally distributed, and were compared using analysis of covari- ance (ANCOVA) with snout-vent length as covariate, followed by a posteriori Tukey tests (Zar, 1999). RESULTS Gross morphology and morphometry Juveniles and adults of both sexes of L. irregula- ris (Figs. 1A, B, C, D), and all males of L. poecilochromus (Fig. 1E), had a line of precloacal pores, and each pore was located in a single modified scale. Two adult males of L. poecilochromus had, immediately posterior to the line of precloacal pores, a row of “secondary pores” (Fig. 1F), consisting of three to four pores, smaller than the pri- mary ones. Because these “secondary pores” were uncom- mon, they were not considered in the final analyses of pore morphology. Three out of the seven adult females of L. poecilochromus had one or two precloacal pores (Fig. 1G), and one of these females had a gland that opened inside the cloaca, rather than with a pore in a scale. The remain- ing females did not have any trace of pores (Fig. 2F). Final- ly, three out of five juvenile males of L. poecilochromus had in the scales of the cloacal border, deep invaginations with- out openings, i.e. clefts (Fig. 1H). The inspection of the two L. neuquensis individuals did not reveal pores or clefts. The secretions of living animals had an orange color (Fig. 1A). In adult males of L. irregularis and L. poecilochromus secretions were always visible as a solid and compact pack of dead squamous cells and secre- tory cells (Fig. 2A), which in most adult males emerged through the pores as long and rigid cylinders, resembling wax crayons (Figs. 1A, E). In contrast, in females and juveniles little of the glandular secretions emerged out of the pore (Figs. 1B, C, D). The morphometry of precloacal pores is in Table 1. Males (adults and juveniles) of L. irregularis had more precloacal pores than females (adults: W = 338.50, P = 0.0001; juveniles: W = 25.00, P = 0.004). In addi- tion, adult males had larger pores than adult and juve- nile females (Figs. 2A, C), but there were no differences between juveniles of both sexes (Figs. 2B, D). Differences in pore size between ages were observed in females, but not in males (F = 26.51, P<0.0001). Finally, L. irregula- ris had more (W = 122.00, P<0.0001, Figs, 1A, E, F) and larger precloacal pores (F = 9.14, P = 0.0054, Figs. 2A, E) than L. poecilochromus. Histology Each pore was associated to a tubuloalveolar gland, compound in adult (Figs. 3A, E), simple in juveniles (Figs. 4A, C). Glands always had secretion in the lumen, independent of the age and sex of the individuals. The morphology, but not the size of these glands was simi- lar between sexes and ages (Table 2; length: F = 9.16, P = 0.0004; width: F = 21.06, P<0.0001). Adult males of L. irregularis had longer glands than adult females and juveniles, but there were no differences between juve- niles of both sexes. The width of the glands was larger in adult males than in females, but no differences were found between age classes by sex. Finally, adult males of L. irregularis had longer (F = 5.93, P = 0.0331), but not wider (F = 0.0001, P = 0.9910), precloacal glands than L. poecilochromus. The size of the glands increased with the body size in adult males of L. irregularis (Figs. 5A, B), which are large enough to touch each other (Fig. 3A). The following description corresponds to the glands of adult males of L. irregularis (Fig. 3A) and L. poecilochromus (Fig. 3E). The gland is surrounded by an envelope of a thin layer of connective tissue (Figs. 3B, E), which extends into the partitions that divide the gland into several elongated lobules (Figs. 3B, C, E). Each lobule has several tubules and alveoli that converge in a single and short central duct that collects the secretions, which finally reach the pore (Figs. 3A, E). Tubules and alveoli are internally coated by a layer of basal cells, the germinative epithelium (Figs. 3D, F). This layer consists of small, flat to cubic cells that have oval to spherical nuclei, with conspicuous nucleoli, which occupy the larg- 150 S. Valdecantos et alii Fig. 1. Ventral view of adult and juvenile of Liolaemus lizards showing the cloacal area. Liolaemus irregularis: (A) adult male, (B) adult female, (C) juvenile male, (D) juvenile female. L. poecilochromus: (E) and (F) adult males, (G) adult female, (H) juvenile male. The white arrowhead from (A) to (G) shows the precloacal pores with secretions and in (H), the clefts. The white circle in (F) indicates a “secondary pore”. Figures (A), (B) and (E) were taken in wild lizards in the field, showing the normal condition of these secretions. The other pictures were taken from specimens from the herpetological collection. 151Precloacal glands of Liolaemus lizards est part of the cells (Figs. 3E, F). Mitoses are observed in the germinative stratum (Fig. 3F), indicating a continu- ous proliferation of cells that replenish the dead ones. As cells proliferate, they move toward the periphery, and during this migration, they enlarge, increasing their cyto- plasmic content with granules (Figs. 3D, G), which show a weak PAS positive reaction (i.e., pink tones; Fig. 6A). Towards the center of the tubules and alveoli, most free/ luminal cells are degenerated as evidenced by the gradual pyknosis of their nuclei or because they are enucleated, with a disaggregated cytoplasm (Figs. 3D, G). Inside the central duct, the structural integrity of the cells is not rec- ognizable any more (Fig. 3G). The final secretion appears as a mixture of secretory granules and cell debris, corre- sponding to a holocrine secretion (Figs. 3G, 4B, D). Three out of seven adult females of L. irregularis had precloacal glands with similar structure as those of adult males. The glands of the remaining adult females (n=4), those of the adult females of L. poecilochromus (n=3) with precloacal pores, and those of juveniles of both sexes of L. irregularis had fewer or non-existent partitions and alveoli, although they had secretions (Figs. 4A, B, C, D). Fig. 2. Scanning electron microscopy of the precloacal pores of Liolaemus irregularis (A to D) and L. poecilochromus (E and F). (A) adult male, (B) juvenile male, (C) adult female, (D) juvenile female, (E) adult male, (F) adult female. : pack of dead squamous cells and secre- tory cells. 152 S. Valdecantos et alii Table 1. Descriptive statistics (mean ± SD) of snout-vent length (SVL, mm) of the individuals of L. irregularis and L. poecilochromus meas- ured to characterize the precloacal pores, considering separately both sexes and age classes. %IP: percentage of individuals with precloacal pores from the total number on individuals examined. The characteristics of the precloacal pores of these two Liolaemus species are: N°P: number and PD: diameter (mm) of pores. In parentheses, the minimum and maximum values. n: sample size. L. irregularis L. poecilochromus Juveniles Adults Adults Females n = 9 Males n = 8 Females n = 16 Males n = 15 Males n = 15 SVL 36.6 ± 3.4 (31.7–43.9) 38.9 ± 2.6 (36.7–45.0) 73.5 ± 6.1 (62.0–85.0) 82.0 ± 11.25 (66.3–97.6) 68.2 ± 5.3 (58.3–76.1) % IP 67 100 100 100 100 N°P 4.1 ± 3.2 (0–8) 8.4 ± 1.5 (7–11) 5.9 ± 1.9 (1–8) 8.4 ± 1.1 (7–10) 4.0 ± 1.2 (3–7) PD 0.08 ± 0.06 (0.09–0.15) 0.15 ± 0.04 (0.12–0.23) 0.22 ± 0.05 (0.16–0.33) 0.54 ± 0.19 (0.25–0.81) 0.49 ± 0.14 (0.18–0.75) Fig. 3. Histological sections of the anterior cloacal borders of an adult male of Liolaemus irregularis (A to D and G) and an adult male of L. poecilochromus (E and F). (A) General view of the precloacal glands draining, each one, in a single scale. (B) Magnification of the box in (A), showing details of the enve- lope surrounding the gland, a thin layer of connective tissue, and the division of glands in lobules. (C) Magnification of the box in (B), showing details of the partitions that divide the gland into several elongated lob- ules and the germinative epithelium that coated tubules and alveoli, which consists of small, flat to cubic cells, with oval to spheri- cal nuclei. (D) Magnification of the box in (C), with details of the different cell types. (E) General view of precloacal glands. (F) Details with immersion of the box shown in (E), showing germinative cells with spheri- cal nuclei and conspicuous nucleoli and mitoses in the germinative stratum. (G) Details with immersion of a basal portion of alveoli of precloacal gland where the dif- ferent cell types and degenerated cells can be appreciated. Degenerated cells are enucle- ated with a disaggregated cytoplasm where cytoplasmatic membranes cannot be recog- nized anymore. Abbreviations: CD: central duct, CGrC: cell with granular cytoplasm, CPyN: cell with pyknotic nucleus, E: epider- mis, EC: enucleated cells, En: envelope, GC: germinative cells, L: lobule, M: mitosis, N: nucleus, Nu: nucleolus, P: pore, Pa: partition, PG: precloacal gland, PrG: Proctodeal gland, S: secretion, Sc: scale, SPTA: secretory por- tion tubule alveolar, T: tubule, UA: unstained area; : enucleated cells with a disaggre- gated cytoplasm and degenerated cells. Stain: Hemalum-Eosin. 153Precloacal glands of Liolaemus lizards The clefts of the juvenile males of L. poecilochromus are invaginations of the epidermis on the cloacal border (Fig. 4E), without secretions, which are located in the same position where the large pores of adults are observed. The “secondary pores” found in two adult males of L. poecilochromus were morphologically different from the precloacal glands, as they are a sac filled with a material whose nature probably is secretion (Fig. 4F). No traces of precloacal pores or clefts were observed in either sex of L. neuquensis. Fig. 4. Histological section of the cloa- cal border of L. irregularis (A to D) and L. poecilochromus (E and F). (A) Precloacal gland of a juvenile female showing the pre- cloacal gland. (B) Magnification of the box in (A), with details of the different cell types. (C) Precloacal gland of a juvenile male with a single partition. (D) Magnification of the box in (C), showing details of partition, germina- tive cells and secretion. (E) Cleft of a juvenile male. (F): “Secondary pore” of an adult male. Abbreviations: C: cleft, CPyN: cell with pyk- notic nucleus, E: epidermis, EC: enucleated cells, GC: germinative cells, Pa: partition, PG: precloacal gland, S: secretion, SP: secondary pore. Stain: Hemalum-Eosin. Table 2. Descriptive statistics (mean ± SD) of the length (LG; mm) and width (WG; mm) of precloacal glands of L. irregularis and L. poecilochromus, considering separately both sexes and age classes. n: sample size. L. irregularis L. poecilochromus Juveniles Adults Adults Females n = 9 Males n = 5 Females n = 7 Males n = 7 Females n = 3 Males n = 7 LG 0.13 ± 0.04 0.21 ± 0.04 0.26 ± 0.06 0.90 ± 0.41 0.16 ± 0.05 0.43 ± 0.19 WG 0.12 ± 0.03 0.19 ± 0.05 0.23 ± 0.06 0.83 ± 0.22 0.20 ± 0.08 0.58 ± 0.20 154 S. Valdecantos et alii Histochemical Observations The secretion in the glandular lumen showed posi- tive reaction to PAS (i.e. pink tones, Fig. 6A) and AB (i.e., light blue tones, Fig. 6B). Intracytoplasmatic granules and secretions were positive to Bromophenol blue (Fig. 6C). The cytoplasms of many cells showed unstained areas, independent of the stain used (Figs. 3D, G). DISCUSSION Each precloacal pore in Liolaemus is located in a unique scale, without modification, such as those observed in teiids, where the scales resemble a rosette (e.g. Imparato et al., 2007). Each pore has an associated epider- mal gland, and if glands are present, they are always func- tional, i.e. they have secretions in the lumen, independent of the sex and age of the individual. In L. irregularis, there is sexual dimorphism in the size and number of these pores, biased towards males. In the case of L. poecilochro- mus, we found “secondary pores” in some adult males, and juvenile males had clefts, which would represent the origin of the adult precloacal pores, as what was report- ed for the femoral pores of Crotaphytus (Cole, 1966b). To our knowledge, we are describing for the first time in Liolaemus these clefts, likely because they are not eas- ily detected by the naked eye. The “secondary pores” had been described in some other Liolaemus species, which are used as a taxonomic character (e.g. Lobo et al., 2010b), Fig. 5. Length (A) and width (B) of precloacal glands in relation to the snout- vent length of L. irregularis and L. poecilochromus. Each point represents an individual. The regression values were obtained including all the individuals measured in this study, independent of the species. Each graph has an insert showing how glands were measured. Fig. 6. Histological section of precloacal glands of adult males with different stains. (A) Details of a partition and cells with granular cytoplasm of L. poecilochromus, stained with PAS. The pink cyto- plasms indicate a weak positive reaction. (B) Details of the secre- tion and glands of L. irregularis stained with AB. The light blue color indicates a weak positive reaction. (C) Details of a partition and cells with granular cytoplasm and secretion of L. poecilochro- mus, stained with Bromophenol blue. The intense blue tone indi- cates a strong positive reaction. Abbreviations: CGrC: cell with granular cytoplasm, L: lobule, Pa: partition, PG: precloacal gland, S: secretion. 155Precloacal glands of Liolaemus lizards although their function is unknown, and so, if their prod- uct might be involved in chemical communication. Individuals of L. irregularis had functional pre- cloacal glands from early in the ontogeny (i.e. juve- niles), which experience a marked ontogenetic change in males; the number of tubules, alveoli and the size of glands increase from juveniles to adults. Even though we did not include newborns of L. irregularis (indi- viduals with ~ 27 mm of snout-vent length), the fact that all juveniles have functional secreting glands, sug- gests that individuals areborn with precloacal glands. The observed ontogenetic variation of the L. irregularis glands is similar to what was reported for the femoral glands; in Crotaphytus collaris there is an increase in the size and complexity of glands after birth in males, but not in females (Cole, 1966b). In Cordylus polyzonus polyzonus, the variation of the males’ gland size is corre- lated with spermatogenic activity (Van Wyk, 1990), and in Iguana iguana, the activity of the glands varies across the reproductive cycle, and they are always larger in males than in females (Ferreira, 2007). Unlike L. irreg- ularis, the precloacal glands of L. poecilochromus are absent in juveniles, which only have invaginations of the epidermis, in the same position where pores are located in adults, which suggests that glands start to develop when males are juveniles, reaching maturity in adults. All together, observations suggest that gland develop- ment would be associated with the individual’s sexual maturity, as was reported for other Liolaemus species (e.g. Valdecantos and Lobo, 2007). The Liolaemus precloacal secretions contain acid mucosubstance, which contrast to the neutral mucosub- stances found in other species of lizards and amphis- baenids (Antoniazzi et al., 1993; Imparato et al., 2007). Unfortunately, there are no studies exploring the role played by these mucosubstances. The precloacal secre- tions also contain proteins, as has been reported for the femoral/precloacal gland secretions of other species (Antoniazzi et al., 1993; Imparato et al., 2007). It was pro- posed that proteins of the femoral secretions of Iguana iguana might act as a barrier to reduce lipid evaporation (Alberts, 1991). In addition, the observed intra and inter- specific variability in the protein profiles may provide significant information about the identity of the owner (Alberts et al., 1993). On the other hand, it is well known that femoral/precloacal secretions contain lipids (Impar- ato et al., 2007; Martín and López, 2011), also described in the precloacal secretions of Liolaemus species (Escobar et al., 2001; 2003). In this study, we could not explore the presence of lipids because specimens were preserved in alcohol, but the unstained areas observed in the glands suggest that the content would be of lipidic nature. The secretions are expelled as rigid cylinders, in opposition to the flexible condition of femoral secretions (e.g. “paste-like secretions”) reported by Chauhan (1986). Probably precloacal secretions are delivered passively in thin layers while lizards perform their daily activi- ties. In fact, we never observed pieces of these cylinders on lizard perches in the field, nor individuals with bro- ken cylinders of secretion. A passive delivery is facilitated by the position of these pores, as has been suggested for Amphisbaena alba (Jared et al., 1999). Moreover, secre- tions may be adhered to faeces during defecation, con- tributing to their pheromonal properties (Labra, 2008). This does not exclude, however, the possibility that secre- tions may be more actively delivered by dragging the clo- aca in the substrate (Labra et al., 2002). The precloacal glands of these Liolaemus species have a general histological structure similar to the precloacal glands of amphisbaenas (Antoniazzi et al., 1993; Jared et al., 1999), and to the femoral glands of lizards from dif- ferent families (Cole, 1966b; Imparato et al., 2007). Thus, it may be possible to consider that the femoral and pre- cloacal glands are homologous; both are holocrine with pheromonal secretions (Martín and López, 2011) and have the same embryological origin (ectoderm). It is intriguing however, what determines variation in the area where glands appear (cloaca vs. thighs). In Liolaemus, there is a significant intra and inter- specific variation in the number of precloacal pores, and moreover, some few species have secondarily lost them (Etheridge, 1995; Lobo, 2005). In most species (> 90%) pores are only present in males, and it is unclear which factors determine the loss (e.g. in some species) or gain (e.g. in females) of these glands, or their occurrence early in the ontogeny (e.g. L. irregularis). In Cordylidae, the presence of generational glands correlated with the envi- ronment temperature; females, and in few cases males, of species that inhabit at high altitude lack these glands (Cordes et al., 1995). In contrast, Escobar et al. (2001) found that Liolaemus species that live at higher altitude have more pores than those from lower altitude, which was explained as a need to produce more secretions due to a faster degradation of these at high altitude. As for the species studied here, altitude/environment temperature cannot explain the difference in the number of precloa- cal pores, as both species with pores, L. irregularis and L. poecilochromus, inhabit high elevations and the presence of precloacal pores in females differed between these two species. Moreover, L. neuquensis is one of the few species of the subgenus Liolaemus (Lobo et al., 2010a) that lacks precloacal pores although it lives at low elevations (< 500 masl), while other species in this Liolaemus subgenus that inhabit at high elevations, have precloacal pores (e.g. 156 S. Valdecantos et alii Martínez Oliver and Lobo, 2002). Potentially, phyloge- netic constraints may explain the interspecific difference between L. irregularis and L. poecilochromus that belong to distinct subclades of Eulaemus (Schulte et al., 2000; Pincheira-Donoso et al., 2008; Lobo et al., 2010a). Further than phylogenetic constraints upon the occurrence of the precloacal pores, their evolution may also be tightly related to selective forces upon chemical communication, considering that precloacal secretions are involved in social communication (Labra, 2008). If so, the lack of precloacal glands (i.e. L. neuquensis) may be related to a reduction in the use of chemical communi- cation, unless other pheromonal sources fulfill the func- tion of these glands. In contrast, L. irregularis, the spe- cies in which pores are present in all individuals, may use significantly more chemical communication, and precloacal secretions may encode important informa- tion for different aspects of their social communication, while secretions in L. poecilochromus would play only a key role in the reproductive success of adult males. It is unclear, however, why some females of L. poecilochromus develop these glands. It seems intuitive that this species is experiencing selective forces to use more the precloa- cal secretions in chemical communication, which also may explain the occurrence of “secondary pores” in some males, and precloacal glands in some females. In summary, this first morphological study on liz- ard precloacal glands shows that in Liolaemus their pres- ence varies across sexes and species, although, they are always functional, producing secretions. Future studies will unravel the selective forces that determine gains and losses of these glands across species, sexes, and ages. ACKNOWLEDGEMENTS Authors thank Fernando Lobo, Donald Griffin and two anonymous reviewers for their significant contribu- tion to an early version of this manuscript and especially to Hervé Seligmann, who also fixed the language. We are indebted with Oscar Leone for his invaluable help with the laboratory work and morphological descriptions. SV thanks CONICET for a visitor fellowship that contributed to develop part of this study. Funds come from CIUNSA Nº 1918 (Universidad Nacional de Salta) to SV and VM, and from FONDECYT 1120181 to AL. REFERENCES Alberts, A.C. (1991): Phylogenetic and adaptive varia- tion in lizard femoral gland secretions. Copeia 1991: 69-79. Alberts, A.C., Sharp, T.R., Werner, D.I., Weldon, P.J. (1992): Seasonal variation of lipids in femoral gland secretions of male green iguanas. J. Chem. Ecol. 18: 703-712. Alberts, C.A., Phillips, J.A., Werner, D.I. (1993): Sources of intraspecific variability in the protein composition of lizard femoral gland secretions. Copeia 1993: 775-781. Antoniazzi, M.M., Jared, C., Pellegrini, C.M.R., Macha, N. (1993): Epidermal glands in Squamata: morphol- ogy and histochemistry of the pre-cloacal glands in Amphisbaena alba (Amphisbaenia). Zoomorphology 113: 199-203. Athavale, N.V., Asnani, M.V., Pilo, B., Shah, R.V. (1977): Histo-morphology of the femoral glands in the Aga- mid lizard, Uromastix hardwickii (Gray). J. Anim. Morphol. Physiol. 24: 51-55. Baig, K.J., Böhme, W. (1991): Callous scalation in female agamid lizards (Stellio group of Agama) and its func- tional implications. Bonn. Zool. Beitr. 42: 275-281. Chamut, S., Valdez, V.G., Manes, M.E. (2009): Function- al morphology of femoral glands in the Tegu lizard, Tupinambis merianae. Zool. Sc. 26: 289-293. Chauhan, N.B. (1986): Histological and structural obser- vations on pre-anal glands of the gekkonid lizards, Hemidactylus flaviviridis. J. Anat. 144: 93-98. Chiu, K.W., Maderson, P.F.A. (1975): The microscopic anatomy of epidermal glands in two species of gek- konine lizards, with some observations on testicular activity. J. Morphol. 147: 23-40. Cole, C.J. (1966a): Femoral glands in lizards: A review. Herpetologica 22: 199-206. Cole, C.J. (1966b): Femoral glands of the lizard, Crota- phytus collaris. J. Morphol. 118: 119-135. Cordes, I.G., Mouton, P.L.F.N., van Wyk, J.H. (1995): Sexual dimorphism in two girdled lizard species, Cor- dylus niger and Cordylus cordylus. S. Afr. J. Zool. 30: 187-196. Darevsky, I.S., Szczerbak, N.N. (1997): A new gecko of the genus Gonydactylus (Sauria: Gekkonidae) with a key to the species from Vietnam. Asian Herpetol. Res. 7: 19-22. Dujsebayeva, T., Ananjeva, N., Böhme, W., Wagner, P. (2007): Studies on specialized epidermal derivatives in iguanian lizards. I. Gross morphology, topography and histology of callose scales in the Asian Rock Aga- ma, Laudakia himalayana (Steindachner, 1869) (Squa- mata: Agamidae). Amphibia-Reptilia 28: 537-546. Dujsebayeva, T., Ananjeva, N., Böhme, W., Wagner, P. (2009): Studies on specialized epidermal derivatives in iguanian lizards: II. New data on the scalation of the Malagasy iguanas of the genus Oplurus (Sauria: Iguanidae). Amphibia-Reptilia 30: 89-97. 157Precloacal glands of Liolaemus lizards Escobar, C.A., Labra, A., Niemeyer, H.M. (2001): Chemi- cal composition of precloacal secretions of Liolaemus lizard. J. Chem. Ecol. 27: 1677-1690. Escobar, C.M., Escobar, C.A., Labra, A., Niemeyer, H.M. (2003): Chemical composition of precloacal secretions of two  Liolaemus fabiani  populations: Are they differ- ent? J. Chem. Ecol. 29: 629-638. Etheridge, R. (1995): Redescription of Ctenoblepharys adspersa Tschudi, 1845, and the taxonomy of Liola- eminae (Reptilia: Squamata: Tropiduridae). Am. Mus. Novit. 314: 1-34. Ferreira, A. (2007): Morphology of the femoral glands of the lizard Iguana iguana (Linnaeus, 1758) (Reptilia, Iguanidae). Bioikos 21: 97-103. Gartner, L.P., Hiatt, J.L. (1994): Color Atlas of Histology. 2nd ed. Williams & Wilkins, Baltimore. Geneser, F. (2000): Histología. 3th ed. Editorial Médica Panamericana S. A., Madrid. Imparato, B.A., Antoniazzi, M.M., Rodrigues, M.T., Jared, C. (2007): Morphology of the femoral glands in the liz- ard Ameiva ameiva (Teiidae) and their possible role in semiochemical dispersion. J. Morphol. 268: 636-648. Jared, C., Antoniazzi, M.M., Silva, J.R.M.C., Freymüller, E. (1999): Epidermal glands in Squamata: Microscopi- cal examination of precloacal glands in Amphisbaena alba (Amphisbaenia, Amphisbaenidae). J. Morphol. 241: 197-206. Khannoon, E., Breithaupt, T., El-Gendy, A., Hardege, J.D. (2013): Comparative study of the pheromone-manu- facturing femoral glands in two sympatric species of lacertid lizards (Acanthodactylus). Zool. Sci. 30: 110- 117. Labra, A. (2008): Sistemas de comunicación en reptiles. In: Herpetología de Chile, pp. 547-577. Vidal, M.A., Labra, A., Eds, Science Verlag, Santiago, Chile. Labra, A., Escobar, C.A., Aguilar, P.M., Niemeyer, H.M. (2002): Sources of pheromones in the lizard Liolaemus tenuis. Rev. Chil. Hist. Nat. 75: 141-147. Laurent, R.F. (1986): Descripciones de nuevos Iguanidae del género Liolaemus. Acta Zool. Lilloana 38: 87-105. Lobo, F. (2005): Las relaciones filogenéticas en el grupo chiliensis de Liolaemus (Iguania: Liolaemidae). Sumando nuevos caracteres y taxa. Acta Zool. Lilloa- na 49: 67-89. Lobo, F., Espinoza, R., Quinteros, S. (2010a): A critical review and systematic discussion of recent classifica- tion proposals for liolaemid lizards. Zootaxa 2549: 1-30. Lobo, F., Slodki, D., Valdecantos, S. (2010b): Two new species of lizards of the Liolaemus montanus group (Iguania: Liolaemidae) from the Northwestern uplands of Argentina. J. Herpetol. 44: 279-293. Lobo, F., Abdala, C., Valdecantos, S. (2012): Morphologi- cal diversity and phylogenetic relationships within a South-American clade of iguanian lizards (Liolaemi- dae: Phymaturus). Zootaxa 3315: 1-41. Louw, S., Burger, B.V., Le Roux, M., Van Wyk, J.H. (2007): Lizard epidermal gland secretions. I: Chemical characterization of the femoral gland secretion of the sungazer, Cordylus giganteus. J. Chem. Ecol. 33: 1806- 1818. Louw, S., Burger, B.V., Le Roux, M., Van Wyk, J.H. (2011): Lizard epidermal gland secretions. II. Chemi- cal characterization of the generation gland secretion of the sungazer, Cordylus giganteus. J. Nat. Prod. 74: 1364-1369. Maderson, P.F.A., Chiu, K.W. (1970): Epidermal glands in gekkonid lizards: Evolution and phylogeny. Herpeto- logica 26: 233-238. Martín, J., López, P. (2011): Pheromones and reproduc- tion in reptiles. In: Hormones and Reproduction in Vertebrates, pp. 141-167. Norris, D.O., López, K.H. Eds, Academic Press, London. Martínez Oliver, I., Lobo, F. (2002): Una nueva especie de Liolaemus del grupo alticolor (Igunia: Liolaemidae) de la Puna Salteña. Cuad. Herpetol. 16: 47-64. Martoja, R., Martoja-Pierson, M. (1970): Técnicas de His- tología Animal. Toray-Masson, S. A., Barcelona. Mason, R.T. (1992): Reptilian pheromones. In: Hor- mones, Brain and Behavior. Biology of Reptilia, pp. 114-228. Gans, C., Crews, D. Eds, The University Chi- cago Press, Chicago, USA. Pinna, P.H., Mendoça, A.F., Bocchiglieri, A., Fernandes, D.S. (2010): A new two-pored Amphisbaena Linnaeus from the endangered Brazilian Cerrado biome (Squa- mata: Amphisbaenidae). Zootaxa 2569: 44-54. Pincheira-Donoso, D., Hodgson, D.J., Tregenza, T. (2008): Comparative evidence for strong phylogentic inertia in precloacal signalling glands in a species-rich lizard clade. Evol. Ecol. Res.10: 11-28. Ramírez-Pinilla, M.P. (1991): Estudio histológico de los tractos reproductivos y actividad cíclica anual repro- ductiva de machos y hembras de dos especies del género Liolaemus (Reptilia: Sauria: Igunidae). Unpub- lished doctoral dissertation. Universidad Nacional de Tucumán, Tucumán. Schulte, J.A., Macey, J.R., Espinoza, R.E., Larson, A. (2000): Phylogenetic relationship in the iguanid lizard genus Liolaemus: Multiple origins of viviparous repro- duction and evidence for recurring Andean vicariance and dispersal. Biol. J. Linnean Soc. 69: 75-102. Scolaro, A., Videla, F., Puig, S., Marcus, A. (2007): Difer- encias morfológicas y status taxonómico de las espe- cies simpátricas Liolaemus coeruleus y Liolaemus neu- 158 S. Valdecantos et alii quensis (Reptilia: Iguania: Liolaemida). Multequina 16: 53-63. Uetz, P., Hošek, J., eds. The Reptile Database, http://www. reptile-database.org, accessed April 20, 2014. Valdecantos, M.S., Lobo, F. (2007): Dimorfismo sexual en Liolaemus multicolor y L. irregularis (Iguania: Liolemi- dae). Rev. Esp. Herp. 21: 55-69. Van Wyk, J.H. (1990): Seasonal testicular activity and morphometric variation in the femoral glands of the lizard Cordylus polyzonus polyzonus (Sauria: Cordyli- dae). J. Herpetol. 24: 405-409. Van Wyk, J.H., Mouton, P.L.F.N. (1992): Glandular epi- dermal structures of cordylid lizards. Amphibia-Rep- tilia 12: 1-12. Weldon, P.J., Flachsbarth, B., Schulz, S. (2008): Natural products from the integument of nonavian reptiles. Nat. Prod. Rep. 25: 1-39. Zar, J. (1999): Biostatistical Analysis. 4th ed. Prentice Hall, New Jersey, USA.Figures