Herpetologica, 58(4), 2002, 415–421 ᭧ 2002 by The Herpetologists’ League, Inc.

CONTINUOUS SPERMATOGENESIS IN THE SCELOPORUS BICANTHALIS (SAURIA: ) FROM HIGH ELEVATION HABITAT OF CENTRAL MEXICO

OSWALDO HERNA´ NDEZ-GALLEGOS1,FAUSTO ROBERTO ME´ NDEZ-DE LA CRUZ1, MARICELA VILLAGRA´ N-SANTA CRUZ2, AND ROBIN M. ANDREWS3,4 1Laboratorio de Herpetologı´a, Departamento de Zoologı´a, Instituto de Biologı´a, Universidad Nacional Auto´noma de Me´xico, A. P. 70-515, C. P. 04510, Me´xico, D. F. Me´xico 2Laboratorio de Biologı´a de la Reproduccio´n , Facultad de Ciencias, Universidad Nacional Auto´noma de Me´xico, A. P. 70-515, C. P. 04510, Me´xico, D. F. Me´xico 3Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA

ABSTRACT: Sceloporus bicanthalis is a viviparous lizard that inhabits high altitude temperate zone habitats in Me´xico. Our histological observations indicate that adult males exhibit spermato- genesis and spermiogenesis throughout the year; no seasonal differences were found in testes mass, height of epididymal epithelial cells, and number of layers of spermatogonia, primary and secondary spermatocytes, and spermatids. Seminiferous tubules exhibited slight, but statistically significant, seasonal variation in diameter. Continuous spermatogenesis and spermiogenesis of S. bicanthalis differ from the cyclical pattern exhibited by most species of and from lizard species sympatric with S. bicanthalis. Continuous reproductive activity of males of S. bicanthalis, and maturation at a relatively small size, is associated with a female reproductive activity in which vitellogenic or pregnant females are present in the population during all months of the year. As a consequence, males can encounter potential mates as soon as they mature. Key words: Reproductive cycle; Sceloporus bicanthalis; Spermatogenesis; Viviparity

REPRODUCTIVE activity of is ity of Sceloporus bicanthalis, a phrynoso- usually seasonal. In the temperate zone, matid lizard of high elevations of the cen- seasonal reproduction is associated with tral mountains of Me´xico. This species is the alternation of warm and cold seasons viviparous as are most other lizards that (Duvall et al., 1982; Fitch, 1970; Sher- live at comparable elevations. Based on brooke, 1975). In the lowland tropics, variation in the volume of testes, Guillette most reptiles also exhibit seasonal repro- (1981a, 1982) reported that males of this ductive cycles, but, in this case, they are species exhibit a seasonal reproductive cy- associated with the alternation of wet and cle. In contrast, our histological analysis dry seasons (Howland et al., 1990; Vitt and shows that males are actually spermato- Morato de Carvalho, 1992). Similarly, in genic year round. high elevation subtropical areas of Me´xico and Central America, most lizards also ex- MATERIALS AND METHODS hibit seasonal reproductive cycles (Guillet- We collected Sceloporus bicanthalis at te and Me´ndez-de la Cruz, 1993; Me´ndez- Parque Nacional Zoquiapan, State of Me´x- de la Cruz et al., 1998; Ramı´rez-Bautista ico (98Њ 37Ј 39Љ W, 19Њ 13Ј 10Љ N), at 3200 et al., 1998). Aseasonal reproduction is, m. The study area has a relatively cool cli- thus, an uncommon reproductive pattern for reptiles, regardless of habitat. At high mate with low temperature variation dur- elevations in Costa Rica, however, males of ing the year, and most rain falls during Barisia monticola exhibit continual sper- summer (Fig. 1). Vegetation is dominated matogenesis and spermiogenesis and ap- by pines, and the understory consists of pear to be reproductive throughout the various species of bunch-grasses, including year (Vial and Stewart, 1985). Festuca and Muhlenbergia (Rzedowsky, Our study examines the testicular activ- 1981). Sceloporus bicanthalis is terrestrial and is associated with the bunch-grass mi- crohabitat; individuals use the grasses for 4 CORRESPONDENCE: e-mail, [email protected] perches and for shelter. Collections were

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TABLE 1.—Monthly sample sizes for males of Scelo- porus bicanthalis and individuals selected for histo- logical analysis. J ϭ juvenile and Ad ϭ adult males.

Males selected for Sample size histological analysis Month Ad J Ad J April 4 0 3 0 May 4 2 3 0 June 4 0 3 0 July 4 6 2 0 August 5 0 4 0 September 4 3 3 0 FIG. 1.—Mean temperature (solid circles) and October 4 1 3 0 rainfall (open circles) averaged over 3-mo intervals at November 3 0 3 0 the weather station (Rı´o Frı´o) located closest (about December 2 6 2 1 15 km) to the Zoquiapan study site (Garcı´a, 1973). January 5 0 3 0 February 5 0 2 0 March 4 1 2 1 made monthly (Table 1) but were analyzed seasonally: spring 1993 (April–June, n ϭ 14), summer 1993 (July–September, n ϭ mass and SVL as the covariate for other 22), fall 1993 (October–December, n ϭ measurements. In no case was the inter- 16), and winter 1994 (January–March, n ϭ action term between the class variable and 15). Morphological measurements taken the covariate significant at P Ͻ 0.05. from each animal were as follows: snout– vent length (SVL), body mass, and testic- RESULTS ular mass. The smallest sexually mature male was We performed conventional histological 35 mm SVL as judged by both gross mor- analyses (Estrada-Flores et al., 1990) of phology and histological analysis. Exter- testes from males exhibiting secondary nally, all individuals 35 mm in length or sexual characteristics (Table 1). An ocular more exhibited bright blue ventral color- micrometer was used to make the follow- ation and active femoral pores. Moreover, ing measurements on the left testis: mean all males 35 mm or more (n ϭ 33) had diameter of 25 representative seminiferous sperm in their seminiferous tubules and tubules and height of 25 epididymal epi- epididymal ducts. Adult males ranged in thelial cells. Germinal cells (spermatogo- size from 35–51 mm SVL (x¯ ϭ 42.6 mm, nia, primary and secondary spermatocytes, SE ϭ 0.61). Two males with SVL of 34 and spermatids) were classified according mm had traces of blue coloration and were to size and position in the seminiferous tu- selected for histological analysis. They did bules. Relative abundance of germinal not, however, have sperm in the seminif- cells was estimated by counting the num- erous tubules and epididymis duct and, ber of layers in 25 tubules. The presence therefore, were considered immature. of spermatozoa in the seminiferous tu- Testis mass of adult males did not vary bules and epididymis ducts was used to seasonally (F3,43 ϭ 0.8, P ϭ 0.51, ANCO- define the minimum size of sexual matu- VA), although testis mass was related to rity. We averaged the measurements for body mass (F1,43 ϭ 65.6, P Ͻ 0.0001). Anal- each individual; individuals were, thus, yses of histological data also indicated that represented only once in statistical analy- adult males of S. bicanthalis exhibit con- ses. tinuous testicular activity throughout the All statistical analyses were conducted year; no testicular recrudescence or re- with SAS software (SAS Institute, Inc., gression was observed. Mature sperm was Version 6.12, 1997). We used ANCOVA’s found in the seminiferous tubules (Fig. 2) to compare seasonal means of morpholog- and epididymal ducts in all specimens in ical measurements. In these analyses, we all seasons. The height of epididymal epi- used body mass as the covariate for testis thelial cells and the number of layers of December 2002] HERPETOLOGICA 417

FIG. 2.—Seminiferous tubules of Sceloporus bicanthalis from Zoquiapan, State of Me´xico: (a) spring, (b) summer, (c) fall, and (d) winter. Note the presence of spermatogonia (SG), primary spermatocytes (PS), secondary spermatocytes (SS), spermatids (SP), and spermatozoa (S) in all seasons. Scale bar ϭ 30 ␮m. 418 HERPETOLOGICA [Vol. 58, No. 4

sexual segment of the kidney, are known to increase in height and secretory activity in association with reproduction (Wilhoft, 1963). While we did not study the sexual segment of the kidney, the lack of seasonal variation in the size of the cells in the ep- ithelial lining of the epididymis duct indi- cates that reproduction by males was pos- sible year round. The bright blue ventral coloration and active femoral pores throughout the year further suggest con- tinual reproductive activity; these features presumably reflect high androgen levels throughout the year (Cooper, 1995; Hews and Moore, 1995). Our conclusion about the reproductive cycle of males of S. bicanthalis differs from that of Guillette (1981a, 1982). A possible reason for this difference is that observations by Guillette and by us were made on different populations and for dif- ferent years. Geographic variation in re- productive cycles is known for several spe- cies (Me´ndez-de la Cruz et al., 1994; Vil- lagra´n-Santa Cruz et al., 1994). More like- ly explanations, however, relate to methodology. GuilletteЈs study is based on FIG. 3.—Morphological measurements of testis estimates of testis volume whereas our mass and histological structures expressed as means conclusions are based on histological anal- and their standard errors. When standard error bars are not shown, they were too small to be distin- ysis. The problem with the use of testis guished from the means. Values with different super- size to assess reproductive status is that scripts are significantly different (P Ͻ 0.05). variation in testis size may reflect the de- gree of hydration (Vitt, 1986) or variation in intensity of sperm production (Garcı´a- spermatogonia, primary and secondary Collazo et al., 1993). Moreover, Guillette spermatocytes, and spermatids did not did not correct estimates of testis size for vary seasonally or as a function of SVL (P the size of the lizard, and testis size is values 0.11–0.95 for overall models). The strongly correlated with body size in our only feature that varied seasonally was the investigation. diameter of seminiferous tubules (F3,28 ϭ Our results call into question male re- 4.0, P ϭ 0.018, ANCOVA) (Fig. 3A,B). productive cycles assessed only by macro- scopic measures, particularly when fe- DISCUSSION males show noticeably asynchronous re- Reproductive Seasonality production. For example, Sceloporus for- Our observations indicate that adult mosus (Guillette and Sullivan, 1985) and males of S. bicanthalis exhibit continuous Eumeces copei (Ramı´rez-Bautista et al., spermatogenesis and spermiogenesis 1996) are classified as lizards with fall re- throughout the year. The relative amount productive activity, but vitellogenic fe- of proliferation from one germinal cell lay- males are found during most of the year, er to another within the testes did not vary which suggests that male reproductive ac- seasonally nor did the height of epithelial tivity could be similarly protracted. Only cells in the epididymis duct. The epithelial histological studies of testes will clarify the lining of the epididymis duct, as well as the reproductive cycles for males of these spe- December 2002] HERPETOLOGICA 419 cies, because the variation in reproductive sonal reproduction as well. Continuous re- activity through histological examination productive activity of S. bicanthalis, thus, provides confirmation of the degree of cor- is not necessarily related to climate or mi- respondence between macroscopic chang- crohabitat features. es in gonadal cycles and gametogenic ac- Continuous spermatogenesis by males tivity (Estrada-Flores et al., 1990). of S. bicanthalis is associated with two un- The only morphological measurement usual features of the reproductive biology that varied seasonally was the diameter of of this species. The SVL of males of S. the seminiferous tubules, which was great- bicanthalis at sexual maturity is the small- est in summer. Seasonal change in tubule est among species of Sceloporus (Fitch, diameter is inexplicable because the 1978; Tinkle et al., 1970). Small size at ma- change in diameter was not associated turity may be advantageous for males be- with variation in the number of cells (the cause receptive females are available number of germinal cells did not vary sea- throughout the year and mating could be sonally). Moreover, because testis mass did possible whenever males mature. More- not vary seasonally, the increase in the di- over, the ratio of male SVL to female SVL ameter of the seminiferous tubules was (0.87) is the lowest in the genus (Fitch, probably not associated with greater hy- 1978). In the bunch-grass microhabitat of dration of the tubules. Given these obser- S. bicanthalis, larger males may not have vations, and the relatively small variation an advantage over smaller males. Low vis- in the diameter of the tubules, we consider ibility precludes the effective territorial that the seasonal change in tubule diam- monitoring and long-range territorial dis- eter is not likely to be significant biologi- plays of species that occupy open micro- cally. habitats. In this latter situation, growth to Females of S. bicanthalis at Zoquiapan relatively large body sizes is beneficial to also exhibit continuous reproduction; vi- males (Fitch, 1978). In contrast, males of tellogenic and pregnant females are pre- S. bicanthalis may employ strategies of sent in the population year round (Man- courtship that are effective at small as well rı´quez-Mora´n, 1995; Me´ndez-de la Cruz as large body sizes. et al., 1998). The presence of vitellogenic females during most months is associated Evolution of the Reproductive Cycle with the continuous reproductive activity Sceloporus bicanthalis is the only mem- in males; potential mates are always avail- ber of the genus Sceloporus known to ex- able. Males and females of S. bicanthalis hibit continuous spermatogenesis and at other sites, Nopalillo, Hidalgo (2900 m), spermiogenesis. We postulate that the and Nevado de Toluca (4100 m), State of largely aseasonal reproduction by both Me´xico, also exhibit continuous reproduc- males and females of S. bicanthalis is re- tion (F. R. Me´ndez-de la Cruz, unpub- lated to the recent origin of viviparity of lished data). this species. Sceloporus bicanthalis is the For S. bicanthalis, continuous testicular sister-species of the oviparous Sceloporus activity could be associated with low sea- aeneus, and these species are genetically sonal variation in temperature (Duvall et very similar (Benabib et al., 1997; Guillet- al., 1982; Vial and Stewart, 1985). This ex- te, 1982; Mink and Sites, 1996). In Scelo- planation is unlikely, as males of the sym- porus, the evolution of viviparity is asso- patric Sceloporus mucronatus (Guillette, ciated with a change in reproductive phe- 1981b) and Sceloporus grammicus (Guil- nology from spring mating and fertilization lette and Casas-Andreu, 1980) exhibit sea- in oviparous species to spring and summer sonal reproduction. Moreover, two species mating and fall fertilization in viviparous from different genera that share the bunch species (asynchronous reproduction). This grass microhabitat with S. bicanthalis (E. change in phenology is associated with the copei: Guillette, 1983; Ramı´rez-Bautista et invasion of cool climates at high elevations al., 1996; and B. imbricata: Guillette and (Me´ndez-de la Cruz et al., 1998). The re- Casas-Andreu, 1987) appear to exhibit sea- productive cycle of S. bicanthalis suggests 420 HERPETOLOGICA [Vol. 58, No. 4 that an initial prolongation of the repro- ANDREU. 1990. Gonadal changes throughout the ductive season may represent a transition reproductive cycle of the viviparous lizard Scelo- porus mucronatus (Sauria: Iguanidae). Herpetolo- between the reproductive cycles of ovipa- gica 46:43–50. rous and viviparous species (Herna´ndez- FITCH, H. S. 1970. Reproductive cycles in lizards and Gallegos, 1995; Me´ndez-de la Cruz et al., snakes. University of Kansas Museum of Natural 1998). History, Miscellaneous Publication 52:1–247. . 1978. Sexual size differences in the genus RESUMEN Sceloporus. 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