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Jul 25, 2015 - Abstract. The pattern of the testicular cycle and spermatogenic activity of the Neotropical yellow-striped snake Lygophis anomalus in Uru-.
South American Journal of Herpetology, 11(2), 2016, 114–118 © 2016 Brazilian Society of Herpetology

Male Reproductive Cycle of a Neotropical Snake, Lygophis anomalus (Dipsadidae), in a Temperate Geographic Distribution Jaim Sivan¹*, Alejandra Panzera², Raul Maneyro² ¹ Department of Life Sciences, Achva Academic College, 79800 M.P. Shikmim, Israel. ² Laboratorio de Sistemática e Historia Natural de Vertebrados, Facultad de Ciencias, Universidad de la República, Uruguay. * Corresponding author. Email: [email protected]

Abstract. The pattern of the testicular cycle and spermatogenic activity of the Neotropical yellow-striped snake Lygophis anomalus in Uruguay was investigated. We gathered data on testicular size, seminiferous tubules, and histological samples of preserved specimens. Testis volume and seminiferous tubule diameter did not differ significantly over the year, and individuals with sperm cells were found all year round. Nevertheless, histological observations show individuals in different stages of spermatogenetic cycle within a single month sample. Our results show that, despite their continuous sperm production, males of L. anomalus populations in Uruguay exhibit a seemingly seasonal cycle, as reflected by changes in testicular volume. Reproductive pattern, even if related to evolutionary lineage, is possibly regulated by environmental factors such as low winter temperatures. Keywords. Environmental factors; Natural history; Reproduction; Seasonality; Testicular cycle.

INTRODUCTION Snakes show a wide flexibility and variability in their reproductive tactics (Pizzatto et al. 2008). In most species of snakes that are distributed in tropical zones, spermatogenesis is continuous throughout the year, but seasonal cycles can also occur (Shine, 1977; Pizzatto and Marques, 2002; Salomão and Almeida-Santos, 2002). In contrast, in reptiles distributed within temperate zones, spermatogenesis is typically seasonal and follows one of two paths: post-nuptial path, in which spermatogenesis occurs after the mating season in preparation for the next reproductive season, and pre-nuptial path, in which spermatogenesis occurs before and is coupled to the current mating season (Seigel and Ford, 1987). Similarly, reptiles exhibit both dissociated and associated reproductive cycles in which the reproductive activity of males and females occur asynchronously or synchronously (Guillette and Méndez-de la Cruz, 1993; Méndez-de la Cruz et al., 1998). In reptiles distributed in temperate zones, pre- or post nuptial paths tend to occur with only one mating season early in the activity season. Different snake species along the world show a wide variety of reproductive paths (Fox, 1977), indicating that several factors influence the timing of reproductive activities, including environmental (e.g., precipitation, photoperiod, temperature) and phylogenetic factors (Shine, 1989; Cadle and Greene, 1993). There is a general trend in dipsadids toward continuous or extended reproductive cycles in tropical species and seasonal reproductive periods in temperate species (Vitt, 1983; Pizzatto and Marques, 2006; Pizzatto et al. 2008). Submitted: 25 July 2015 Accepted: 18 May 2016 

Populations of widely distributed species also seem to show a latitudinal gradient with broader reproductive cycles towards lower latitudes (e.g., Liophis miliaris, Pizzatto and Marques, 2006). Studies on the reproductive biology of South American species in temperate zones are scarce, especially considering the high species richness and diversity this region harbors (Lopez et  al., 2009). Further, studies that examine male reproductive cycles are underrepresented, probably because of the difficulties posed by histological preparation and analysis, and usually only variation in testes volume along the year is reported. The dipsadid genus Lygophis (Fitzinger 1843) comprises eight species (Uetz and Hošek, 2015) and is part of the subfamily Xenodontinae, whose monophyly has been corroborated by both morphological and molecular studies (Myers, 1986; Zaher, 1999; Vidal et al., 2000; Zaher et al., 2009). Lygophis species occur in moist environments from Panama to Uruguay and are mostly terrestrial (Dixon, 1985). The yellow-striped snake Lygophis anomalus is distributed in southern Brazil, northeastern Argentina, and Uruguay (Carreira et  al., 2005; Carreira and Maneyro, 2013). A recent study focused on female reproductive cycle from all the species distributional range found that even though gravid females occur between spring and summer, females with vitellogenetic follicles are present throughout the year (Panzera and Maneyro, 2013). Nevertheless, the authors observed seasonal variation in testis volume, with maximum sizes between August and November. However, they suggested that further analyses were necessary to asses the presence of sperm within

Handling Editor: Carlos Arturo Navas Iannini doi: 10.2994/SAJH-D-15-00021.1 

South American Journal of Herpetology, 11(2), 2016, 114–118

the testis is necessary in order to confirm a seasonal testicular cycle in males. A comprehensive analysis of male reproductive cycles in this species could shed some light on which factors (e.g., environmental, phylogenetic) shape the reproductive cycles in temperate xenodontines. Hence, the aim of this study is to determine the annual testicular cycle and spermatogenic activity of the temperate Uruguayan populations of Lygophis anomalus. MATERIALS AND METHODS This work is based on material deposited in the scientific collection of vertebrates housed at Facultad de Ciencias (Universidad de la República, Uruguay), where the specimens were fixed and stored in 10% formalin for several years. Data on dates and localities of specimens were taken from collection records. Only adults were selected for this study. Snout–vent length (SVL) was measured in each specimen to the nearest 1  mm using a plastic ruler. Length and width of testes were measured with a digital caliper to the nearest 0.1 mm and testicular volume was calculated using the ellipsoid volume formula (Dunham, 1983). Testicular volume was then related to SVL (Relative Testicular Volume = RTV) and a bimonthly average (n = 66) was calculated to better express the seasonal changes. Seminiferous tubule diameters were measured using a microscope and ocular micrometer. The average of 15 measurement of different tubules of the same testis was considered to be the representative diameter of the individual. As in testicular volume, an average of a bimonthly value was found to express the seasonal changes. The significance of bimonthly variation was tested using the non-parametric Kruskall-Wallis test. We compare both testicular volume and seminiferous tubules diameter for populations collected north of Rio Negro (n = 30) to populations collected in southern regions (n = 36). All analyses were performed using Statistica 6.0 (StatSoft, 2004). For histological observations, samples of Lygophis anomalus testes (n = 33) were: (1) washed in clean buffer, (2) post-osmicated for 1 h in 1% OsO4 in the same buffer, (3) dehydrated in a series of increasing concentrations of ethanol, and (4) embedded in an epoxy resin. Semi-thick sections ( 0. 05.

three individuals displayed significant spermatogenetic activity in the germinative layers (Fig. 2H). One of those individuals showed sperm without spermatogenetic activity. In contrast, some individuals (n = 3) contained no sperm in the lumen but spermatogenetic activity in germinative layers, and some (n = 2) presented no spermatogenetic activity and no sperm (Fig. 2G). In summer (December–February; n  =  5), some individuals (n = 3) exhibited sperm in the tubule lumen and no spermatogenetic activity (Fig.  2B), one individual showed spermatogenetic activity but no sperms in the lumen, while another displayed no sperm and no spermatogenetic activity. In fall (March–May; n = 12), some individual (n = 8) contained sperm in the tubule lumen (Fig. 2A, D) and 3 of them also showed spermatogenetic activity (Fig.  2E). In contrast, other specimens (n = 3) showed no sperm and no spermatogenetic activity, as found in summer sample (Fig. 2C, F). One specimen showed spermatogenetic activity but lacked sperm in the lumen. In winter (June; n = 2), both individual show sperm in tubule lumen and spermatogenetic activity. In specimens with no spermatogenetic activity unidentified cells were observed in the lumen, but in one case, a small number of cell layers were found in the tubule wall (Fig. 2C, F), and in another there were more cells and organized layers (Fig. 2G). Seminiferous tubule diameter We found no significant differences in the seminiferous tubule diameter between seasons (Fig. 3). Nevertheless, a decrease of seminiferous tubule diameter is apparent in the colder months of the year, from the end of fall (May) until the end of spring (October).

The Uruguayan populations of Lygophis anomalus, which are distributed in a temperate region, present a reproductive pattern that differs from those seen in many species of the family with tropical distribution (Pizzatto and Marques, 2006). Although individuals with mature sperm occur throughout the year, mature females with vitellogenic follicles and egg laying occur predominantly between September–March (Panzera and Maneyro, 2013), suggesting a seasonal reproductive cycle. Males with sperm were found throughout the year, thereby constituting potentially reproductive individuals. Specimens with high spermatogenic activity and sperm in the lumen were found in every season; however, within the same monthly or bimonthly samples we found individuals in different stages of the spermatogenetic cycle and lacking sperm in the correspondent lumen. Some testis samples presented collapsed seminiferous tubules in April, May, and June. Those collapsed tubules may be evidence of copulation prior to the observation, so there is no concordance with the vitellogenic activity reported for female follicles (Panzera and Maneyro, 2013). Moreover, the mean seminiferous tubule diameter did not change over the year. The absence of spermatogenetic activity or sperm in the lumen may reflect a cleaning process after copulation, a reorganization stage with rebuilding of generative tissue in the testes toward a new reproductive cycle in each individual, as observed in other species (Sivan et al., 2012). If those collapsed tubules can be considered evidence of mating before examination, in April–May, and there is no evidence of mature follicles in females, then a delay in fertilization of oocytes could be hypothesized, as well as storage and nutrition of sperm in the female reproductive system. Long- term sperm storage is a way to profit from mature follicles and is necessary when asynchronic cycles are displayed by the genders (Almeida-Santos and Salomao, 1997). Our results suggest that Lygophis anomalus in temperate Uruguay undergoes a tropical spermatogenesis cycle but that maturation of gonads is strongly influenced by environmental factors. This conclusion is reinforced by the fact that neonates appear synchronously (AP, unpublished data). It seems, that the process of sperm production is as follows: (a)  spermatogenesis with a high level of activity in the germinative layers of the tubule walls, (b)  temporary sperm storage in the tubule lumen and break in the spermatogenetic activity and reduction in germinative cells density, (c) clearing of lumen by mating or a parallel activity, (d) collapse of the germinative layers and filling of the lumen with those cells, and (e) reorganization of the germinative layers and re-initiation of the spermatogenetic process.

Male Reproductive Cycle of a Neotropical Snake, Lygophis anomalus (Dipsadidae), in a Temperate Geographic Distribution Jaim Sivan, Alejandra Panzera, Raul Maneyro

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In contrast with snakes in temperate and subtropical zones, some tropical snake species are reproductive over long periods of the year and some may even reproduce year-round (Saint Girons, 1982). As mentioned above, in every sample over the year, we found individuals in different stages of the spermatogenetic cycle. This fact may lead to the conclusion that at individual level, an din contrast to the population level, Lygophis anomalus show a discontinuous cyclical pattern, where gonads or accessory glands become quiescent for some period during the year (Mathies, 2011). As such, at the individual level, L. anomalus males undergo aseasonal cycles. In conclusion, males of Lygophis anomalus are potentially ready to copulate throughout the year there, as occurs in tropical species. Since, as mentioned above, mature females with vitellogenic follicles and egg laying occur predominantly between September and March (Panzera and Maneyro, 2013), and neonates appear synchronously (AP, unpublished data), we conclude that copulation time is semi-synchronous and depends mainly on female condition. REFERENCES Almeida-Santos S.M., Salomão O.M.G. 1997. Long-term sperm storage in the neotropical rattlesnake Crotalus durissus terrificus (Viperidae: Crotaline). Japanese Journal of Herpetology 17:46–52. Cadle J.E., Greene H.W. 1993. Phylogenetic patterns, biogeography, and the ecological structure of Neotropical snake assemblages. Pp. 281–293, in Ricklefs R.E., Schluter D. (Eds.) Species Diversity in Ecological Communities: Historical and Geographical Perspectives. University of Chicago Press, Chicago. Carreira S., Maneyro R. 2013. Guía de Reptiles del Uruguay. Ediciones de la Fuga, Montevideo. Carreira S., Meneghel M., Achaval F. 2005. Reptiles del Uruguay. Universidad de la República, Facultad de Ciencias. Montevideo. Dixon J.R. 1985. A review of Liophis anomalus and Liophis elengantissimus, and the description of a new species (Serpentes: Colubridae). Copeia 1985:565–573. Dunham A.E. 1983. Realized niche overlap, resource abundance and intensity of interspecific competition. Pp.  261–280, in Huey R.D., Pianka E.R., Schoener T.W. (Eds.) Lizard Ecology. Harvard University Press, London. Fitzinger L. 1843. Systema Reptilium. Fasciculus primus, Amblyglossae. Braumüller et Seidel. Vienna. doi:10.5962/bhl.title.4694 Fox H. 1977. The urogenital system of reptiles. Pp. 1–157, in Gans E.C., Parsons T.S. (Eds.), Biology of the Reptilia. Volume 6. Morphology. Academic Press, London. Guillette L.J. Jr, Méndez-de la Cruz F.R. 1993. The reproductive cycle of the viviparous Mexican lizard Sceloporus torquatus. Journal of Herpetology 27:168–174. doi:10.2307/1564933 Lopez S.M, Giraudo A.R, Arzamendia V., Chiaraviglio M. 2009. Reproductive biology of the semi-aquatic snake Liophis semiaureus (Serpentes, Colubridae) in the north-east of Argentina [in Spanish]. Revista Chilena de Historia Natural 82:233–244. doi:10.4067/ S0716-078X2009000200006 Mathies T. 2011. Reproductive cycles of tropical snakes. Pp. 511–550, in Sever D.M., Aldridge R.D. (Eds.), Reproductive Biology and Phylogeny of Snakes. CRC Press. Boca Ratón.

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Male Reproductive Cycle of a Neotropical Snake, Lygophis anomalus (Dipsadidae), in a Temperate Geographic Distribution Jaim Sivan, Alejandra Panzera, Raul Maneyro