Copeia 108, No. 2, 2020, 231–264 REVIEWS Seasonal Timing of Spermatogenesis and Mating in Squamates: A Reinterpretation Robert D. Aldridge1, Dustin S. Siegel2, Stephen R. Goldberg3, and R. Alexander Pyron4 The squamates occur in a variety of climates from tropical to Arctic regions. Being poikilotherms, snakes and lizards in temperate regions, and high elevation tropical environments, must adjust their reproductive biology to reproduce at a time that optimizes offspring survival. The two major components of the reproductive cycle in both males and females are gametogenesis and mating. The reproductive cycle of males is the focus of this study. In snakes in temperate climates, sperm production (spermatogenesis) may occur immediately prior to mating (prenuptial spermatogenesis) or following mating (postnuptial spermatogenesis). In postnuptial spermatogenesis, sperm are produced following the mating season and stored in the efferent testicular ducts (primarily the ductus deferens) until the following spring mating season. Given that most recent phylogenetic reconstructions resolve snakes as a monophyletic group of highly specialized lizards, it is generally assumed that lizards have spermatogenic cycles similar to snakes. Lizard spermatogenic cycles are often described as prenuptial or postnuptial. We propose that the major difference between snake and lizard spermatogenic cycles is the presence of postnuptial spermatogenesis in snakes and the absence of true postnuptial spermatogenesis in lizards. Our interpretation of lizard spermatogenic cycles suggests that all lizards have prenuptial spermatogenesis (i.e., sperm are produced immediately prior to mating). If fertilization occurs months after mating, the female, and not the male, stores the sperm until spring ovulation and fertilization. Using a variety of analytical tools, we analyzed the reproductive strategies of snakes and lizards, and we have concluded that they differ in fundamental ways. Most notably, prenuptial spermatogenesis is the ancestral condition for Squamata with continuous spermatogenesis evolving multiple times independently within lizards and snakes. We also found that postnuptial spermatogenesis evolved early in the evolutionary history of snakes but, we argue, has never evolved in lizards. We suggest that the evolutionary origin of snakes may account for the differences observed in snake versus lizard reproductive cycles, and we present a scenario for the evolution of snake reproductive cycles. INCE snakes are a monophyletic group of highly tial species, the seminiferous tubules are generally regressed specialized lizards (Camp, 1923; Estes et al., 1988; at the time of mating, while the epididymis and secondary S Eckstut et al., 2009; Pyron et al., 2013), it is generally sex characters, such as the sexual segment of the kidney assumed that lizards and snakes have similar spermatogenic (SSK), are hypertrophied. One major difference between cycles. In fact, the majority of studies of snakes and lizards prenuptial and postnuptial spermatogenesis is the age of the use the same terms to describe the relationships between sperm at mating. In snakes with prenuptial spermatogenesis, mating and spermatogenesis, suggesting that there are few or the sperm may be stored for less than a month in the ductus no differences between lizard and snake reproductive cycles. deferens prior to mating, whereas in snakes with postnuptial Early in the study of squamate reproductive cycles, spermatogenesis, the sperm are stored in the ductus deferens investigators realized that gametogenesis in males and for six months or more prior to mating. Another difference females could occur at different times of the year. Volsøe between postnuptial (many species of snakes) and prenuptial (1944) described the temporal relationship between sper- spermatogenesis (many species of snakes and almost all matogenesis and mating in Vipera berus and compared this lizards) is that, in order to coevolve to a new (female) mating cycle to that of other reptiles. He introduced the terms ‘‘pre- season, species with prenuptial spermatogenesis need to nuptial spermatogenesis’’ to describe species in which adjust the seasonal timing of spermatogenesis, whereas spermatogenesis occurred immediately prior to mating and species with postnuptial spermatogenesis, and long-term ‘‘post-nuptial spermatogenesis’’ to describe species in which sperm storage, do not need to adjust the timing of spermatogenesis occurred in the summer preceding the spermatogenesis. spring mating season. In species with postnuptial spermato- Volsøe (1944) realized that the terms ‘‘pre-nuptial’’ and genesis, sperm are produced in the summer and stored in the ‘‘post-nuptial’’ represented the extremes, and that interme- ductus deferens until the spring mating season. In postnup- diates would likely occur. He also noted that the terminology 1 Department of Biology, Saint Louis University (Emeritus), St. Louis, Missouri 63103; Email: [email protected]. Send reprint requests to this address. 2 Department of Biology, Southeast Missouri State University, Cape Girardeau, Missouri 63701; Email: [email protected]. 3 Department of Biology, Whittier College, Whittier, California 90608; Email: [email protected]. 4 Department of Biological Sciences, The George Washington University, Washington, D.C. 20052; Email: [email protected]. Submitted: 6 May 2019. Accepted: 24 December 2019. Associate Editor: W. L. Smith. Ó 2020 by the American Society of Ichthyologists and Herpetologists DOI: 10.1643/CH-19-230 Published online: 4 May 2020 232 Copeia 108, No. 2, 2020 is further complicated because, in some taxa (i.e., the turtle Table 1. Potential benefits and costs of prenuptial and postnuptial Sternotherus odoratus, Risley 1938), females may also mate in spermatogenesis. the summer (prior to hibernation) and store the sperm in the Prenuptial spermatogenesis oviduct. We now know that mating occurs many months Benefits: before ovulation (and vitellogenesis) in many snakes (Al- 1) Males produce sperm continuously during mating season; dridge et al., 2009). individual males unlikely to deplete their sperm reserves during Bons and Saint Girons (1982) and Saint Girons (1982) the mating season. extended the discussion on the evolution of spermatogenic 2) In the non-mating season, energy is not spent on storing cycles in reptiles. Based on the fact that most postnuptial sperm in the efferent ducts, and (perhaps as a side effect) species have spermatogenesis in the summer/autumn, and maintaining the development of sexual segment of the kidney. most prenuptial species have spermatogenesis in the spring, Costs (real and potential): they used the term ‘‘aestival’’ to describe postnuptial 1) If individual females come into estrus early in the spring spermatogenesis and ‘‘vernal’’ to describe prenuptial sper- mating season, some males may not have produced enough matogenesis. To describe those species in which spermato- sperm to fertilize these females. genesis is initiated in the summer/autumn (postnuptial) but 2) Cool temperatures may slow the production of sperm in spring not completed until the following spring, both authors used thus limiting the male’s ability to fertilize an entire clutch. the term ‘‘mixed’’ spermatogenesis. 3) In heliothermic species, males may be exposed during We believe the focus on the season in which spermato- prolonged thermoregulation (Saint Girons, 1976). genesis occurs, rather than the relationship between 4) The energy required for the production of sperm may affect spermatogenesis and mating, obscures differences in repro- the amount of energy available for the development of the ductive strategies, especially in species in which the female sexual segment of the kidney and male courtship and territorial has a single mating period, many months prior to behaviors. vitellogenesis and ovulation. Thus, we will use the terms Postnuptial spermatogenesis prenuptial and postnuptial to describe spermatogenic Benefits: cycles. We also use the terminology of Aldridge and Duvall 1) Cool temperatures in the early spring do not affect the (2002) and define ‘‘estrus’’ as the period when an individual numbers of sperm present in this mating season. female is receptive to male courtship, and the term ‘‘mating 2) With adequate numbers of sperm stored in the ductus season’’ as the collective time during which females are in deferens, males can use their energy developing the sexual estrus. segment of the kidney and in scramble mating strategies, finding females, or in species with male combat, spend the Some authors have suggested that the mating season is a energy in growth and fighting potential male rivals. compromise between males and females (Gibson and Falls, 3) For those species that also have a summer/autumn mating 1975). However, we will contend that females determine the season, males are continuing to produce sperm and are not seasonal timing of the mating season and males evolve likely to deplete the sperm reserves in the summer/autumn. hormonally and behaviorally to be prepared to mate when Costs (real and potential): at least some females enter estrus. Some potential costs to 1) During the spring mating season, males risk using up all the females that may be associated with estrus include: advertising stored sperm. costs (pheromone production), advertising movements (in- 2) Elevated androgens, associated with long-term sperm storage crease in exposure to predators), and harassment by males that in males, may be energetically costly (Sacchi