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 et al., 2016). could interfere with feeding, basking, and increased energy 3) Elevated androgens, associated with long-term sperm storage usage to avoid pursuit by males. Because of the potential costs in males, may be immunologically costly (Sacchi et al., 2016). associated with estrus (see Aldridge and Duvall, 2002), females should limit the time they are in estrus. The potential benefits and costs of prenuptial and methods (e.g., Uyeda et al., 2018). We also describe the postnuptial spermatogenic cycles are presented in Table 1. morphology of the efferent ducts of the testis and the Because polygyny is the dominant mating system in snakes seasonal patterns of sperm storage in snakes and lizards. (Rivas and Burghardt, 2005), species with prenuptial sper- Because many of the terms and descriptions were initially matogenesis are unlikely to deplete their sperm reserves focused on snakes, we approach each topic from a snake- during the mating season, whereas species with postnuptial oriented perspective. spermatogenesis may deplete their sperm stores during the We begin our review with an in-depth discussion of the spring mating season. literature on reproductive cycles in lepidosaurs before Here, we are interested in the evolutionary history of moving on to discussion of sperm storage, sperm storage spermatogenic cycles across squamates, and within snakes in structures, secondary sexual structures (sexual segment of the particular. It is well documented that many snake species kidney), and endocrinology of reproduction. In those have postnuptial spermatogenesis (Volsøe, 1944; Bons and sections, we provide our opinions on data conflict with Saint Girons, 1982; Saint Girons, 1982; Aldridge and Duvall, historical literature and a primarily subjective treatise on the 2002; Aldridge et al., 2009). The term ‘‘postnuptial spermato- evolution of reproductive cycles. Following, we assess the genesis’’ has also been used to describe the spermatogenic evolution of reproductive patterns in squamates using an pattern found in some lizards. One purpose of this study is to analytical approach with a phylogenetic backbone. determine if the patterns found in lizards are parallel to those found in snakes and to quantify the frequency of prenuptial MATERIALS AND METHODS and postnuptial spermatogenesis (as described by Volsøe, 1944) in lizards relative to snakes. We examine the evolution Taxa and terminology.—Data on reproductive cycles for of reproductive strategy using comparative phylogenetic individual squamate species were obtained from an exhaus- Aldridge et al.—Spermatogenesis and mating in squamates 233

Table 2. Classification of snake reproductive cycles based on the relationship of spermatogenesis to the female reproductive cycle. Female reproductive cycles are based on the population, male reproductive cycles on the individual. Number in parentheses is latitude of study, letter in parentheses: O ¼ oviparous; V ¼ viviparous. Taxa in bold in Viperidae have spermatogenesis and mating in the summer similar to the prenuptial pattern. Superscripts denote mating seasons or sperm storage for select taxa. A) Female reproductive cycle seasonal; male reproductive cycle seasonal with postnuptial spermatogenesis. All have spring mating season, those marked with superscript 1 also have summer/autumn mating season. Colubridae: (358N) Arizona elegans (O) Aldridge, 1979, 2001 (358N) Cemophora coccinea (O) Goldberg, 2016 (398N) Coluber constrictor (O) Fitch, 1963 (368N) Coronella girondica (O) Feriche, 1998 (588N) Coronella girondica (O) Bons and Saint Girons, 1982 (228N) Orthriophis taeniurus (O) Chiu and Wong, 1974 (318N) Lampropeltis pyromelana (O) Goldberg, 1997 (338N) Lampropeltis zonata (O) Goldberg, 1995a (368N) Macroprotodon brevis (O) Pleguezuelos and Feriche, 1998 (348N) Masticophis lateralis (O) Goldberg, 1975a (408N) Masticophis taeniatus (O) Goldberg and Parker, 1975 (358N) Opheodrys aestivus (O) Plummer, 1984; Aldridge et al., 1990 (408N) Pituophis catenifer (O) Goldberg and Parker, 1975 (368N) Rhinechis scalaris (O) Pleguezuelos and Feriche, 2006 (348N) Tantilla coronata (O) 1 Aldridge and Semlitsch, 1992 Dipsadidae: (398N) Carphophis amoenus (O) 1 Clark, 1970; Aldridge and Metter, 1973 (398N) Diadophis punctatus (O) 1 Wilkinson, 1962 (348N) Farancia abacura (O) Robinette and Trauth, 1992 (388N) Heterodon nasicus (O) 1 Platt, 1969 (388N) Heterodon platirhinos (O) 1 Platt, 1969 Lamprophiidae: (288S) Psammophylax rhombeatus (O) Flemming and Douglas, 1997

Natricidae: (368N) Natrix maura (O) Feriche, 1998 (498N) Natrix natrix (O) Petter-Rousseaux, 1953 (258N) Natrix piscator (O) Srivastava and Thapliyal, 1965 (318N) Natrix tessellata (O) Amer, 1976 (308N) Nerodia fasciata (V) Lorenz et al., 2011 (388N) Nerodia sipedon (V) Bauman and Metter, 1977 (368N) Nerodia taxispilota (V) White et al., 1982 (388N) Thamnophis elegans (V) 1 Fox, 1952 (428N) Thamnophis radix (V) 1 Cieslak, 1945 (518N) Thamnophis sirtalis (V) 1 Aleksiuk and Gregory, 1974 Elapidae: (308S) Austrelaps superbus (V) Shine, 1977 (308S) Hemiaspis signata (V) Shine, 1977 (258S) Micrurus corallinus (O) 5 Marques et al., 2013 (308N) Micrurus fulvius (O) 5 Quinn, 1979; Marques et al., 2013 (258S) Micrurus nigrocinctus (O) 5 Marques et al., 2013 (308S) Notechis scutatus (V) Shine, 1977 (308S) Parasuta dwyeri (V) 2, 5 Shine, 1977 Viperidae: (418N) Agkistrodon contortrix (V) 1 Aldridge and Duvall, 2002 (328N) Agkistrodon piscivorus (V) 1 Burkett, 1966 (328N) Crotalus atrox (V) 1, 5 Goldberg, 2007a (268N) Crotalus adamanteus (V) 1 Hoss et al., 2011; Fill et al., 2015 (348S) Crotalus cerastes (V) 1 Aldridge and Duvall, 2002 (418N) Crotalus concolor (V) Aldridge and Duvall, 2002 (268S) Crotalus durissus (V) 5 Almeida-Santos et al., 2004; Barros, 2011 (248N) Crotalus enyo (V) 5 Goldberg and Beaman, 2003 (388N) Crotalus horridus (V) 1, 5 Aldridge and Duvall, 2002 (398N) Crotalus lutosus (V) 5 Ashton, 2003; Glaudas et al., 2009 (338N) Crotalus mitchellii (V) Aldridge and Duvall, 2002 (338N) Crotalus molossus (V) 5 Goldberg, 1999a (358N) Crotalus oreganus (V) 1 Bromley, 1934; Fitch, 1949 234 Copeia 108, No. 2, 2020

Table 2. Continued. (318N) Crotalus pricei (V) 5 Goldberg, 2000; Prival et al., 2002 (338N) Crotalus ruber (V) Aldridge and Duvall, 2002 (338N) Crotalus scutulatus (V) 1, 5 Schuett et al., 2002 (338N) Crotalus tigris (V) 5 Goldberg, 1999b (358N) Crotalus viridis (V) 5 Aldridge, 1979, 1993 (318N) Crotalus willardi (V) 5 Holycross and Goldberg, 2001 (408N) Sistrurus catenatus (V) 1 Reinert, 1981 (OK) B) Female reproductive cycle seasonal; male reproductive cycle seasonal with prenuptial spermatogenesis. Acrochordidae: (128S) Acrochordus arafurae (V) Shine, 1986 (108N) Acrochordus granulatus (V) Gorman et al., 1981 Colubridae: (118N) Boiga cyanea (O) Saint Girons and Pfeffer, 1972 (338N) Chilomeniscus cinctus (O) 4 Goldberg, 1995b (338N) Chionactis occipitalis (O) 4 Goldberg and Rosen, 1999 (338N) Chionactis palarostris (O) 4 Goldberg and Rosen, 1999 (378N) Hemorrhois hippocrepis (O) 4 Pleguezuelos and Feriche, 1999 (328N) Hemorrhois hippocrepis (O) 4 Bons and Saint Girons, 1982 (338N) Phyllorhynchus browni (O) 4 Goldberg, 1996 (338N) Phyllorhynchus decurtatus (O) 4 Goldberg, 1996 (298N) Spalerosophis diadema (O) 4 Amer and Elshabka, 1978 (338N) Trimorphodon biscutatus (O) 3 Goldberg, 1995c Dipsadidae: (308S) Liophis semiaureus (O) 4 Lopez´ et al., 2009 (238S) Sibynomorphus mikanii (O) 2 Rojas et al., 2013 Elapidae: (28N) Enhydrina schistosa (V) Voris and Jayne, 1979 (258S) Micrurus altirostris (O) 5 Marques et al., 2013 (258S) Micrurus decoratus (O) 5 Marques et al., 2013 (258S) Micrurus frontalis (O) 5 Marques et al., 2013 (258S) Micrurus lemniscatus (O) 5 Marques et al., 2013 (248N) Naja naja (O) Lofts et al., 1966 (308S) Pseudechis porphyriacus (V) 5 Shine, 1977 (308S) Pseudonaja nuchalis (O) Shine, 1977 (308S) Pseudonaja textilis (O) Shine, 1977 Homalopsidae: (118N) Enhydris bocourti (V) Saint Girons and Pfeffer, 1972 (118N) Enhydris enhydris (V) Saint Girons and Pfeffer, 1972 (118N) Homalopsis buccata (V) 4 Saint Girons and Pfeffer, 1972 Lamprophiidae: (368N) Malpolon monspessulatus (O) 4 Feriche et al., 2008 (328N) Malpolon monspessulatus (O) 4 Cheylan et al., 1981 (08N) Psammophis phillipsi (O) Butler, 1993 (268N) Psammophis schokari (O) 4 Cottone and Bauer, 2009 (298N) Psammophis sibilans (O) 3 Amer and Elshabka, 1978 Natricidae: (188N) Nerodia rhombifer (V) Aldridge et al., 1995 Pythonidae: (338S) Morelia spilota (O) Slip and Shine, 1988 Typhlopidae: (338S) Anilios nigrescens (O) 3 Shea, 2001 Viperidae: (288S) Bitis gabonica (V) Bodbijl, 1997 (98N) Bothrops asper (V) Solorzano´ and Cerdas, 1989 (mating March) (238S) Bothrops jararaca (V) 2, 5 Janeiro-Cinquini et al., 1993a, 1993b (188N) Bothrops leucurus (V) 2, 5 Barros et al., 2014 (308N) Cerastes cerastes (V) 4 Saint Girons, 1962 (318N) Cerastes vipera (V) 3 Sivan et al., 2012 (478N) Vipera ammodytes (V) 3 Saint Girons, 1972, 1976 (478N) Vipera aspis (V) 3, 5 Saint Girons, 1976 (558N) Vipera berus (V) 3, 5 Volsøe, 1944 (588N) Vipera berus (V) 3 Nilson, 1980 Aldridge et al.—Spermatogenesis and mating in squamates 235

Table 2. Continued. (478N) Vipera latastei (V) 3, 5 Saint Girons, 1976 (478N) Vipera seoanei (V) 3, 5 Saint Girons, 1976 (478N) Vipera ursinii (V) 3 Saint Girons, 1976 C) Female reproductive cycle seasonal; male spermatogenesis continuous. Colubridae: (108N) Drymobius margaritiferus (O) Goldberg, 2003 Homalopsidae: (108N) Cerberus rhynchops (V) Gorman et al., 1981 Dipsadidae: (108N) Erythrolamprus bizona (O) Goldberg, 2004 (218S) Sibynomorphus mikanii (O) Pizzatto et al., 2008 (228S) Sibynomorphus neuwiedi (O) Pizzatto et al., 2008 (298S) Sibynomorphus ventrimaculatus (O) Pizzatto et al., 2008 D) Female reproductive cycle continuous (or extended); male spermatogenesis continuous. Seasonal changes in testis size may occur in some species. Colubridae: (118N) Boiga irregularis (O) Mathies et al., 2010 (208S) Chironius flavolineatus (O) Pinto et al., 2010 (208S) Chironius quadricarinatus (O) Pinto et al., 2010 (118N) Dendrelaphis pictus (O) Saint Girons and Pfeffer, 1972 (118N) Oligodon taeniatus (O) Saint Girons and Pfeffer, 1972 Dipsadidae: (168S) Dipsas catesbyi (O) Alves et al., 2005 (168S) Dipsas neivai (O) Alves et al., 2005 (168S) Leptodeira annulata (O) Pizzatto et al., 2008 (158S) Liophis miliaris (O) Pizzatto and Marques, 2006 (228S) Oxyrhopus guibei (O) Pizzatto and Marques, 2002 (168S) Pseudoboa nigra (O) de Paula Orofino et al., 2010 Elapidae: (108N) Laticauda colubrina (O) Gorman et al., 1981 Homalopsidae: (38N) Homalopsis buccata (V) Berry and Lim, 1967 1 Both summer and spring mating season. 2 Summer spermatogenesis and mating. 3 Mixed cycle, summer spermatogenesis, spermiation occurs during mating season. 4 Vernal spermatogenesis and mating. 5 Long-term sperm storage in ductus deferens. tive literature search. Only species in which both male and Continuous spermatogenesis: a taxon in which spermatogen- female cycles have been described are included. Species esis occurs throughout the year (Saint Girons, 1982). included on Tables 2 and 3 were chosen to represent as many reproductive strategies, families, and latitudes as possible. The list of species is not exhaustive. However, all lizard Some latitudes presented in the tables were estimated based studies in which the author states or implies that the on broad occurrence data (Uetz et al., 2018). spermatogenic cycle is postnuptial are included. We discuss Because reproductive cycles of lizards differed substantially sperm storage in males (in efferent ducts) and in females (in from snakes, we focused on the temporal relationship oviducts) as ‘‘short-term’’ sperm storage if sperm are stored between spermatogenesis and mating. Reproductive cycles for days to weeks for a single clutch or successive clutches were classified based on the terminology of Volsøe (1944): during the breeding season, as ‘‘long-term’’ sperm storage for sperm that are stored over winter for spring mating, and as Postnuptial spermatogenesis: a taxon in which spermatogen- ‘‘very long-term’’ sperm storage for sperm stored for succes- esis occurs after the mating season, and sperm are stored in sive breeding seasons. The terms oviparity and viviparity are the efferent ducts of the testis for an extended period used to denote egg laying versus live birth. following testicular regression. If a species has two mating The majority of the discussions will focus on the seasons (summer/autumn and spring) and sperm are stored reproductive cycles of temperate species, whether due to in the efferent ducts following testicular regression, sper- latitude or altitude, because the presence of a ‘‘winter’’ matogenesis is considered postnuptial (i.e., sperm storage in requires long-term sperm storage if mating occurs in the males is required). summer and fertilization in the spring. In squamates, long- term sperm storage occurs in females of some snakes and Prenuptial spermatogenesis: a taxon in which spermatogen- lizards; however, long-term sperm storage in males, which is esis occurs immediately prior to mating. This definition is the key component of postnuptial spermatogenesis, has only consistent with Licht (1984). been described in snakes. 236 Copeia 108, No. 2, 2020

Table 3. Classification of lizard reproductive cycles based on the authors’ interpretation. We contend that the taxa in bold actually represent prenuptial spermatogenesis, because the studies fail to show long-term sperm storage, required for postnuptial spermatogenesis. Female reproductive cycles are based on the population; male reproductive cycles on the individual. Number in parentheses is latitude of study, letter in parentheses: O ¼ oviparous; V ¼ viviparous. Superscripts denote mating seasons for select taxa. A) Female reproductive cycle seasonal; male reproductive cycle seasonal. Author implies/states, or it appears that spermatogenesis is postnuptial. Anguidae: (328N) Elgaria kingi (O) 1 Goldberg, 1975b (238N) Elgaria paucicarinata (O) 1 Goldberg and Beaman, 2004 Cordylidae: (338S) Chamaesura anguinus (V) 2, 3 Du Toit et al., 2003 (288S) Smaug giganteus (V) 2, 3 Van Wyk, 1995 (288S) Pseudocordylus melanotus (V) 1, 3 Flemming, 1993 Gekkonidae: (348S) Christinus marmoratus (O) 1 King, 1977 Lacertidae: (398S) Podarcis muralis (O) 4 Kwiat and Gist, 1987 (468S) Podarcis muralis (O) 4 Saint Girons and Duguy, 1970 Liolaemidae: (398S) Phymaturus zapalensis (V) 4 Boretto et al., 2012 Scincidae: (298N) Plestiodon egregius (O) 1 Mount, 1963 (338S) Hemiergis decresiensis (V) 1, 3 Murphy et al., 2006 (338S) Niveoscincus coventryi (V) 1, 3 Murphy et al., 2006 (428S) Niveoscincus metallicus (V) 1, 2 Swain and Jones, 1994 (428S) Niveoscincus ocellatus (V) 1, 2 Jones et al., 1997 B) Female reproductive cycle seasonal; male reproductive cycle seasonal with prenuptial spermatogenesis. Agamidae: (228S) Agama aculeata (O) Heideman, 1994, 2002 (288S) Agama atra (O) Van Wyk and Ruddock, 2000

(228S) Agama planiceps (O) Heideman, 1994, 2002 (88S) Acanthocercus cyanogaster (O) Robertson et al., 1965 (138N) Calotes nemoricola (O) Subba Rao and Rajabai, 1972 (158N) Calotes versicolor (O) Gouder and Nadkarni, 1979 (128N) Calotes versicolor (O) Sarkar and Shivanandappa, 1989 (128N) Eutropis carinata (O) Sarkar and Shivanandappa, 1989 (348N) Phrynocephalus vlangalii (V) 5 Wu et al., 2015 (128N) Psammophilus dorsalis (O) Sarkar and Shivanandappa, 1989 (348N) Stellagama stellio (O) Childress, 1970 (138N) Sitana ponticerinata (O) Subba Rao and Rajabai, 1972 (208N) Uromastyx acanthinura (O) Kehl, 1944 Amphisbaenidae: (378S) Amphisbaena kingii (O) Vega, 2001 Anguidae: (468N) Anguis fragilis (V) see Saint Girons, 1963 (488N) Elgaria coerulea (V) Vitt, 1973 (338N) Elgaria multicarinatus (O) Goldberg, 1972 Anniellidae: (228N) Anniella pulchra (V) Goldberg and Miller, 1985 Blanidae: (328S) Blanus cinereus (O) Bons and Saint Girons, 1963 Cordylidae: (328S) Ouroborus cataphractus (V) Flemming and Mouton, 2002 (288S) Karusasaurus polyzonus (V) Van Wyk, 1990 (278S) Platysaurus capensis (O) Van Wyk and Mouton, 1996 (238S) Platysaurus minor (O) Van Wyk and Mouton, 1996 (328S) Hemicordylus capensis (V) Van Wyk and Mouton, 1998 Crotaphytidae: (358N) Crotaphytus collaris (O) Trauth, 1979 (338N) Crotaphytus vestigium (O) Goldberg and Mahrdt, 2010 Dactyloidae: (308N) Anolis carolinensis (O) Fox, 1958 Aldridge et al.—Spermatogenesis and mating in squamates 237

Table 3. Continued. Gekkonidae: (358N) Gekko japonicus (O) Ikeuchi, 2004 (158S) Hemidactylus brookii (O) Shanbhag et al., 2000 (298S) Hemidactylus flaviviridis (O) Sanyal and Prasad, 1967 (198S) Heteronotia binoei (O) Clerke and Alford, 1993 (358N) Christinus marmoratus (O) King, 1977 Helodermatidae: (358N) Heloderma suspectum (O) Goldberg and Lowe, 1997 Iguanidae: (348N) Sauromalus obesus (O) Prieto and Sorenson, 1977; Abts, 1988 Lacertidae: (538N) Lacerta agilis (O) Saveliev et al., 2006 (408N) Lacerta lepida (O) Castilla and Bauwens, 1990 (238S) Meroles cuneirostris (O) Goldberg and Robinson, 1979 (468S) Podarcis muralis (O) Saint Girons and Duguy, 1970 (408N) Podarcis sicula (O) Angelini and Picariello, 1975 (408S) Psammodromus algirus (O) Diaz et al., 1994 Leiocephalidae: (268N) Leiocephalus carinatus (O) Meshaka et al., 2006 (218N) Leiocephalus psammodromus (O) Smith and Iverson, 1993 Liolaemidae: (408S) Liolaemus elongatus (V) Ibarguengoyt¨ ´ıa and Cussac, 1998 (518S) Liolaemus magellanicus (V) Ferna´ndez et al., 2017 (518S) Liolaemus sarmientoi (V) Ferna´ndez et al., 2017 (398S) Phymaturus zapalensis (V) Boretto and Ibarguengoyt¨ ´ıa, 2009 (288S) Phymaturus punae (V) 1 Boretto et al., 2007, 2014 Phrynosomatidae: (338N) Phrynosoma coronatum (O) Goldberg, 1983 (278N) Sceloporus cyanogenys (V) 1 Crisp, 1964 (198N) Sceloporus grammicus (V) 1 Jimenez-Cruz´ et al., 2005 (198N) Sceloporus grammicus (V) 1 Guillette and Casas-Andreu, 1980 (188N) Sceloporus horridus (O) Valdez-Gonza´ ´lez and Ram´ırez-Bautista, 2002 (348N) Sceloporus jarrovii (V) 1 Goldberg, 1971; Ballinger, 1973 (108N) Sceloporus malachitus (V) Marion and Sexton 1971 (198N) Sceloporus mucronatus (V) Estrada-Flores et al., 1990 (348N) Sceloporus occidentalis (O) Wilhoft and Quay, 1961; Goldberg, 1974 (348N) Sceloporus orcutti (O) Mayhew, 1963 (318N) Sceloporus poinsettii (V) 1 Ballinger, 1973 (348N) Sceloporus uniformis (O) 1 Goldberg, 2012 (188N) Sceloporus spinosus (O) Valdez-Gonza´ ´lez and Ram´ırez-Bautista, 2002 (198N) Sceloporus torquatus (V) Guillette and Mendez-de la Cruz, 1993 (328N) Sceloporus virgatus (O) Ballinger and Ketels, 1983 (368N) Sceloporus undulatus (O) Altland, 1941 (258N) Uma exsul (O) Gadsden et al., 2006 (348N) Uma notata (O) Mayhew, 1966 (348N) Uma scoparia (O) Mayhew, 1966 (198N) Urosaurus bicarinatus (O) Ram´ırez-Bautista and Vitt, 1998 (338N) Urosaurus graciosus (O) Vitt et al., 1978 (338N) Urosaurus ornatus (O) Van Loben Sels and Vitt, 1984 (318N) Uta stansburiana (O) Hahn, 1964 Phyllodactylidae: (198N) Phyllodactylus lanei (O) Ram´ırez-Sandoval et al., 2006 Polychrotidae: (78N) Polychrus acutirostris (O) Vitt and Lacher, 1981 Scincidae: (308S) Anepischetosia maccoyi (O) Robertson, 1976, 1981 (148S) Carlia amax (O) James and Shine, 1985 (158S) Carlia fusca (O) Wilhoft and Reiter, 1965 (148S) Carlia gracilis (O) James and Shine, 1985 (198S) Carlia pectoralis (O) Clerke and Alford, 1993 (308S) Carlia tetradactyla (O) James and Shine, 1985 (208S) Carlia triacantha (O) James and Shine, 1985 238 Copeia 108, No. 2, 2020

Table 3. Continued. (198S) Cryptoblepharus virgatus (O) Clerke and Alford, 1993 (158S) Ctenotus essingtonii (O) James and Shine, 1985 (308S) Ctenotus robustus (O) Way, 1979 (318S) Ctenotus taeniolatus (O) Taylor, 1985 (128N) Eutropis carinata (O) Sarkar and Shivanandappa, 1989 (368S) Hemiergis decresiensis (V) 1 Robertson, 1981 (348S) Hemiergis peronii (V) 1 Smyth and Smith, 1968 (178S) Lygisaurus foliorum (O) James and Shine, 1985 (348S) Menetia greyii (O) Smyth and Smith, 1974 (348S) Morethia boulengeri (O) Smyth and Smith, 1974 (358S) Oligosoma suteri (O) Towns, 1975 (358N) Plestiodon anthracinus (O) Trauth, 1994 (198N) Plestiodon copei (O) Guillette, 1983 (358N) Plestiodon fasciatus (O) Trauth, 1994 (328N) Plestiodon laticeps (O) Vitt and Cooper, 1985 (338S) Pseudemoia entrecasteauxii (V) Murphy et al., 2006 (258S) Trachylepis capensis (V) Flemming, 1994 (78S) Trachylepis heathi (O) Vitt and Blackburn, 1983 (258S) Trachylepis sparsa (V) Goldberg, 2007b (288S) Trachylepis spilogaster (O) Goldberg, 2006a Teiidae: (358N) Aspidoscelis inornatus (O) Christiansen, 1971; Stevens, 1983 (358N) Aspidoscelis sexlineatus (O) Johnson and Jacob, 1984 (358N) Aspidoscelis sexlineatus (O) Brackin, 1979 (348N) Aspidoscelis tigris (O) Goldberg, 1976 Trogonophiidae: (328S) Trogonophis wiegmanni (V) Bons and Saint Girons, 1963 Tropiduridae: (238S) Tropidurus itambere (O) Ferreira et al., 2009 Varanidae: (268S) Varanus griseus (O) Kehl, 1944 (148S) Varanus glauerti (O) 2 James et al., 1992 (148S) Varanus glebopalma (O) 2 James et al., 1992 (158S) Varanus storri (O) 2 James et al., 1992 (148S) Varanus gilleni (O) 2 James et al., 1992 (168N) Varanus olivaceus (O) Auffenberg, 1988 Xantusiidae: (328N) Xantusia vigilis (V) 4 Miller, 1948 (338N) Xantusia riversiana (V) 4 Goldberg and Bezy, 1974 C) Female reproductive cycle seasonal; male spermatogenesis continuous. Agamidae: (08S) Agama agama (O) Marshall and Hook, 1960 (128N) Calotes versicolor (O) Kasinathan and Basu, 1973 Dactyloidae: (98N) Anolis auratus (O) Sexton et al., 1971 (98N) Anolis limifrons (O) Sexton et al., 1971 (98N) Anolis tropidogaster (O) Sexton et al., 1971 Gekkonidae: (218N) Dixonius siamensis (O) Goldberg, 2008 Scincidae: (98S) Carlia bicarinata (O) Zug et al., 1982 (188S) Carlia rhomboidalis (O) Wilhoft, 1963 (198S) Carlia pectoralis (O) Clerke and Alford, 1993 (268S) Trachylepis variegata (O) Goldberg, 2006a D) Female reproductive cycle continuous (or extended); male spermatogenesis continuous. Seasonal changes in testis size may occur in some species. Agamidae: (78S) Agama agama (O) Ejere and Adegoke, 2005 (18N) Draco melanopogon (O) Inger and Greenberg, 1966 (18N) Draco quinquefasciatus (O) Inger and Greenberg, 1966 Anguidae: (98N) Mesaspis monticola (V) Vial and Stewart, 1985 Aldridge et al.—Spermatogenesis and mating in squamates 239

Table 3. Continued. Gekkonidae: (68S) Cosymbotus platyurus (O) Church, 1962 (18N) Cyrtodactylus malayanus (O) Inger and Greenberg, 1966 (18N) Cyrtodactylus pubisulcus (O) Inger and Greenberg, 1966 (68S) Hemidactylus frenatus (O) Church, 1962 (218S) Lygodactylus verticillatus (O) Vences et al., 2004 (68S) Gehyra mutilata (O) Church, 1962 Gymnophthalmidae: (98S) Neusticurus ecpleopus (O) Sherbrooke, 1975 Lacertidae: (238S) Aporosaura anchietae (O) Goldberg and Robinson, 1979 (268S) Meroles suborbitalis (O) Goldberg, 2006b Phrynosomatidae: (198N) Sceloporus bicanthalis (V) Herna´ndez-Gallegos et al., 2002 (188N) Sceloporus variabilis (O) Ram´ırez-Bautista et al., 2006 Scincidae: (158S) Cryptoblepharus plagiocephalus (O) James and Shine, 1985 (198S) Cryptoblepharus virgatus (O) Clerke and Alford, 1993 Teiidae: (78S) Ameiva ameiva (O) Vitt, 1982 (68N) Aspidoscelis lemniscatus (O) Mojica et al., 2003 (78S) Aspidoscelis ocellifer (O) Vitt, 1983 Tropiduridae: (78S) Tropidurus torquatus (O) Vitt and Goldberg, 1983; Vieira et al., 2001 (78S) Tropidurus semitaeniatus (O) Vitt and Goldberg, 1983 Varanidae: (118N) Varanus indicus (O) Wikramanayake and Dryden, 1988 1 Mating in summer/autumn (winter in mild climates). 2

Mating season unknown. 3 Spring mating season speculated. 4 Spring mating season. 5 Mixed mating season.

Single Sample Chi Square Test with an a ¼ 0.05 was used to approach these issues cautiously, using methods that attempt assess the frequency of reproductive parameters. to account for model uncertainty and decreased power for estimating parameters at internal nodes. All of our analyses Ecological and physiological data.—From the literature, we were conducted in the R package ‘phytools’ (Revell, 2012), gathered information on the geographic distribution (trop- with associated models therein. ical/temperate), parity mode (oviparous/viviparous), and First, we estimated ancestral states and transitions using sperm-production strategy (prenuptial/postnuptial/continu- stochastic mapping (Bollback, 2006), which samples poten- ous) of a number of species spanning the diversity of the tial character histories from their posterior distribution Squamate Tree of Life. From a total of 261 species, we were jointly across nodes. We did this to ensure that our estimates able to match 254 to a recent time-calibrated phylogenetic of the evolutionary trajectories of previously analyzed traits analysis (Tonini et al., 2016) for the purposes of phylogenetic (parity mode and climate) were congruent with existing comparative analyses. Of these 254 species, we were also able reconstructions. For our new mating-strategy data, we wished to gather the range-wide mean of Mean Annual Temperature to estimate the first origin and subsequent transition- (BIO1 from the BIOCLIM dataset; Hijmans et al., 2005) from sequence of postnuptial spermatogenesis. All lizards exhibit the dataset of Meiri et al. (2013) for 102 lizard species. For either prenuptial or continuous spermatogenesis, while 104 snakes, S. Meiri and A. Feldman (unpubl. data) provided snakes exhibit all three modes. We sampled 100 independent us with equivalent mean-temperature estimates drawn from character histories using the ARD (all-rates-different) model, the snake range maps published by Roll et al. (2017). These from a random tree from Tonini et al. (2016). While the trees data allow us to perform a comprehensive analysis of the relationship between climatic preference and mating strategy of Tonini et al. (2016) were intended to be used as a in squamates, with a particular focus on transitions to novel distribution representing phylogenetic uncertainty for Squa- sperm-production strategies in snakes. mata, 238 of the 254 species were represented in the fixed backbone including all major lineages and character states, Hypothesis testing.—Characters such as parity (Pyron and and thus little uncertainty was present. Burbrink, 2014), climate (Pyron, 2014), temperature (Meiri et Second, we used the binary phylogenetic correlation al., 2013), and reproductive strategies (Shine, 2003) have model of Pagel (1994) to test for evolutionary correlations been studied widely in squamate reptiles, but their ancestral between: parity mode/climate, parity mode/mating strategy states and evolutionary correlations are still subject to debate (snakes), climate/mating strategy (snakes), parity mode/ (Wright et al., 2015; Harrington and Reeder, 2017). Thus, we mating strategy (lizards), and climate/mating strategy (liz- 240 Copeia 108, No. 2, 2020

ards). These divisions are appropriate given the single forms (n ¼ 2, x2 ¼ 10.32, P ¼ 0.001). In one species with evolutionary transition to postnuptial spermatogenesis that continuous spermatogenesis, Homalopsis buccata, spermato- occurs in the ancestor of all snakes (see Results and genesis is cyclic but not seasonal. Discussion section). For snakes, we recoded mating strategy One major difficulty in determining spermatogenic cycles as a binary character of postnuptial/(prenuptial þ continu- in tropical snakes is the lack of histological data. In his review ous). As this involved at least five different potentially non- of tropical reproductive cycles, Mathies (2011) included 31 independent comparisons, we used an adjusted significance species. Of these, only three included histological measure- level of 0.05/5 ¼ 0.01 for discussion, though all significant ments. In our analysis, we initially listed Sibynomorphus results yielded P values drastically lower than 0.01 (see mikanii (Pizzatto et al., 2008) as having continuous sper- Results and Discussion section). matogenesis, based on gross measurements. A more recent Third, we estimated the evolutionary relationship between study, based on histological analysis (Rojas et al., 2013), temperature and mating strategy separately in lizards and revealed that the species was cyclic and had prenuptial snakes, using Felsenstein’s (2012) threshold model. We used spermatogenesis. the same strategy described above, where lizards exhibit only In snakes from temperate areas (.23.58 latitude) the two states, and snakes were recoded as having a trinary majority of the species have postnuptial spermatogenesis. mating strategy. This model samples an unobserved correla- We hypothesize that spermatogenesis in temperate regions tion between a continuous variable and an underlying was derived by: 1) emigration of snakes from lower latitudes, genetic liability that causes a change in state over a certain 2) passive development of prenuptial spermatogenesis due to threshold. It thus acts to estimate the correlation between a mild climate, and 3) independent evolution of prenuptial continuous independent variable (temperature, in this case) spermatogenesis. The independent evolution of prenuptial and a binary or ordered multi-state dependent variable spermatogenesis suggests that there may be a significant cost (mating strategy). We used one chain of 1,000,000 genera- to the postnuptial spermatogenic pattern (see Table 1). tions for each, which was sufficient to produce visual Rationale of our hypothesis is as follows: stationarity for both runs and ESS . 100. 1. Emigration of snakes from lower latitudes: Emigration of species from more mild climates may explain the RESULTS AND DISCUSSION occurrence of prenuptial spermatogenesis in colubrids Snake reproductive cycles.—In species in which the female of southern Europe, a pythonid and several elapids of reproductive cycle is seasonal, the frequency of prenuptial Australia. In the Iberian Peninsula, Pleguezuelos and and postnuptial spermatogenesis in snakes from all latitudes Feriche (1999) reported that Hemorrhois hippocrepis and (Table 4) is not significantly different by number of families Malpolon monspessulanus are the only Palearctic colubrid (x2 ¼ 0.64, P ¼ 0.42), genera (x2 ¼ 0.02, P ¼ 0.89), or species (x2 species with prenuptial spermatogenesis. These species ¼ 1.4, P ¼ 0.24). The frequency of oviparous and viviparous are sympatric with several other species (Colubridae: forms is not significantly different in prenuptial (x2 ¼ 0.08, P Coronella girondica, Elaphe scalaris, Macroprotodon cucul- ¼ 0.78) or postnuptial (x2 ¼ 0.16, P ¼ 0.69) spermatogenic latus;Natricidae:Natrix maura), all of which have patterns. postnuptial spermatogenesis (Pleguezuelos and Feriche, When examined by latitude, however, the frequencies of 1999). These authors suggested that the difference in spermatogenic patterns vary significantly at several levels. In these cycles may be due to phylogeny. Both H. hippo- snakes from tropical and subtropical latitudes (23.58 crepis and M. monspessulanus have ranges that extend latitude), the pattern at the family level (x2 ¼ 2.28, P ¼ from North Africa to the Iberian Peninsula. Bons and 0.13) was not significantly different; however, at the genus Saint Girons (1982) reported that the North African (x2 ¼ 0.03, P ¼ 0.03, 90%) and species (x2 ¼ 8.64, P ¼ 0.003, populations of M. monspessulanus have prenuptial 93%) levels, the majority were prenuptial. The sole exception spermatogenesis. According to Carranza et al. (2006), to tropical/subtropical species having prenuptial spermato- Hemorrhois hippocrepis and Malpolon monspessulanus genesis is Orthriophis taeniurus (Colubridae; 228N latitude). In originated in the Maghreb region of North Africa and tropical snakes with prenuptial spermatogenesis, the fre- migrated to the Iberian Peninsula, perhaps during the quency of oviparous forms (n ¼ 3) is not significantly Pleistocene, by ‘‘hopping’’ across the Strait of Gibraltar different from viviparous forms (n ¼ 10, x2 ¼ 2.76, P ¼ 0.10). (Carranza et al., 2006). Thus, the presence of prenuptial In temperate snakes (.23.58 latitude), in which the female spermatogenesis in these species may be the result of cycle is seasonal, the frequency of occurrence of prenuptial their recent migration from northwest Africa (Carranza and postnuptial spermatogenic patterns is not significantly et al., 2006). different at the family level (x2 ¼ 0.00, P ¼ 1.00); however, the 2. Passive development of prenuptial spermatogenesis frequency is different at the genus (x2 ¼ 8.76, P ¼ 0.003) and because of mild climate: Several species of Australian species (x2 ¼ 6.48, P ¼ 0.01) levels. In temperate species, the snakes exhibit prenuptial spermatogenesis. In the frequency of oviparous species and viviparous species is not python Morelia spilota (338S), spermatogenesis occurs significantly different in prenuptial (x2 ¼ 0.11, P ¼ 0.11) or during the winter and continues in the spring during postnuptial species (x2 ¼ 0.28, P ¼ 0.60). the mating season (Slip and Shine, 1988). Following the Males with continuous spermatogenesis occur in species in spring mating season, the testes regress. The range of which the female reproductive cycle is seasonal (n ¼ 6) or Morelia spilota is extensive, covering much of the continuous (n ¼ 13). All of the species are from subtropical/ tropical area in Australia, but also extending across the tropical climates except Sibynomorphus ventrimaculatus (Dip- temperate regions of southern Australia. This species is sadidae) from 298S latitude. The frequency of oviparous (n ¼ described as mostly nocturnal and crepuscular (Cogger 17) species was significantly greater than the viviparous et al., 1983); however, basking is frequently observed Aldridge et al.—Spermatogenesis and mating in squamates 241

Table 4. Number of families, genera, and species of snakes and lizards with prenuptial and postnuptial spermatogenesis by latitude; number of oviparous and viviparous species in parentheses (A ¼ snakes, B ¼ lizards). C ¼ number of families, genera, and species of snakes and lizards with continuous spermatogenesis by latitude; number of oviparous and viviparous species in parentheses. All latitudes Tropical 23.58 latitude Temperate .23.58 latitude

(A) Prenuptial Postnuptial Prenuptial Postnuptial Prenuptial Postnuptial

Families 9 5 6 1* 5 5 Genera 26 28 9 1* 9 28 Species 45 (24:21) 58 (27:31) 13 (3:10) 1* (1:0) 32 (21:11) 57 (26:31) (B) Prenuptial Postnuptial Prenuptial Postnuptial Prenuptial Postnuptial

Families 20 0 20 0 18 0 Genera 63 0 26 0 49 0 Species 121 (88:33) 0 43 (38:5) 0 78 (50:28) 0 (C) Snakes Lizards Snakes Lizards Snakes Lizards

Families 5 11 5 11 1 0 Genera 15 20 15 20 1 0 Species 19 (2:17) 32 (2:30) 18 (1:16) 32 (2:30) 1 (1:0) 0 * Elaphe taeniura

(Slip and Shine, 1988). The presence of prenuptial January 10.78C, February 12.48C, March 14.88C, April spermatogenesis may be the result of retention of the 18.88C, May 23.38C; Climate-Zone.com: https://www. tropical reproductive pattern due to its basking behavior climate-zone.com/climate/united-states/arizona/ (Pearson et al., 2005) and mild winter climate, described tucson/index_centigrade.htm). These warm tempera- as mesothermal (Cogger et al., 1983). Our phylogenetic tures, with nearly continuous sunshine, may stimulate reconstruction (see below) indicates that Acrochordus, spermatogenesis prior to females beginning vitellogen- Anilios nigrescens, and Morelia spilota appear to have esis (for dates of spermatogenesis and vitellogenesis, see

retained the prenuptial state of their lizard ancestors; Aldridge et al., 2009). however, caution should be taken with this interpreta- 3. Independent evolution of prenuptial spermatogenesis: tion because of the lack of reproductive cycle data on The third mechanism for the evolution of prenuptial basal snakes. spermatogenesis is found in some Vipera from Europe. In a study of elapids in temperate New South Wales, In these species, vitellogenesis occurs in the spring Australia (308S), both prenuptial and postnuptial sper- (Saint Girons, 1982). Saint Girons (1976) examined the matogenic cycles occurred (Shine, 1977). Of the seven reproductive biology of six species and nine subspecies species for which there were sufficient data, three of Vipera (see Viperidae in Table 2) and found that these species (Pseudechis porphyriacus, Pseudonaja nuchalis, species have cycles that resemble both prenuptial and and P. textilis) exhibited prenuptial and four (Austrelaps postnuptial spermatogenic patterns. He termed these superbus, Hemiaspis signata, Notechis scutatus, and Un- cycles ‘‘mixed’’ and noted that there were two distinct echis gouldii) had postnuptial spermatogenesis. Of the types of mixed cycles that corresponded to whether the three prenuptial species, one was oviparous and the snakes had a single spring mating season (V. berus)or other two viviparous. There was no single factor that two mating seasons, one in the summer/autumn and accounted for the occurrence of the prenuptial species, another in the spring (V. aspis). In the cycle found in V. and too few species were examined to detect phyloge- berus, spermatogenesis began in the late spring and netic trends (Shine, 1977). The three species with continued through the autumn. Following hibernation, prenuptial spermatogenesis were the largest of the spermiogenesis began and sperm were produced for the seven examined, were diurnal species that basked, and spring mating season, typical of prenuptial spermato- had ranges that extended in latitude to less than 168S. genesis. Because sperm were not present in the ductus The temperatures at the study sites are mild with deferens during the winter, all of the sperm were temperatures at the Quambone study site of 348C produced in the spring. Nilson (1980) described the (summer) and 16.68C (winter) and at Armidale site cycle found in V. berus as having postnuptial sperma- 278C (summer) and 12.28C (winter). tocytogenesis and a prenuptial spermiogenesis. Passive development of prenuptial spermatogenesis The pattern in V. aspis differs from the pattern in V. may occur in some temperate Nearctic species (Chilo- berus in that spermiogenesis occurs in the summer and meniscus cinctus, Goldberg, 1995b; Chionactis occipitalis the following spring. In species with the pattern found and C. palarostris, Goldberg and Rosen, 1999) simply in V. berus, mating may occur in the summer, but the because the climate warms early in the spring to permit primary mating season is in the spring. Sperm produced spermatogenesis. In the small to medium-sized snakes in the summer are stored in the ductus deferens. The in the deserts of the southwest United States (US), the pattern in V. aspis differs from the typical postnuptial winters are relatively mild and warm temperatures and pattern in that spermiogenesis resumes in the spring. persistent sunshine occurs early in the year (Tucson, AZ; Because sperm are produced in the spring, prior to the 242 Copeia 108, No. 2, 2020

spring mating season, both patterns qualify as prenup- tial. Both of these cycles may have been derived from an ancestral postnuptial spermatogenesis cycle according to our phylogenetic reconstruction (see below). Saint Girons (1992) also suggested that the spermato- genic cycles in Vipera could reflect phylogenetic relationships. This does not appear to be true. Saint Girons (1992) reported that V. aspis, V. latastei, and V. seoanei had two mating seasons and V. ammodytes, V. berus, and V. ursinii had a single spring mating season. An analysis of the relationships among the Vipera using molecular data (Garrigues et al., 2005) indicated that V. berus, V. seoanei, and V. ursinii form a single clade. In this group, V. berus and V. ursinii had a single mating season and V. seoanei had two mating seasons. Thus, the timing of mating seasons evolved independently of phylogeny. The evolution of prenuptial spermatogenesis in Vipera could be the result of adaptations in both females and males. Because we do not know the ancestral mating season(s) of the genus, several scenarios are possible. Focusing on the most studied taxa, V. aspis and V. berus, we propose that female V. aspis either retained two matings seasons while V. berus lost the summer Fig. 1. Representations of the major patterns of spermatogenic and mating season, or, if a single summer mating season is vitellogenic cycles of snakes in temperate climates. (A) Typical the ancestral pattern, both species added a spring reproductive pattern of North American Crotalidae. (B) Typical reproductive pattern of North American Colubridae, Xenodontidae, mating season and V. berus lost the summer mating and others. Bold solid line represents spermatogenesis, the dashed line season. The addition of a spring mating season is represents vitellogenesis, and horizontal boxes represent mating common in several families of snakes (e.g., Colubridae periods. Individual species differ in that they may have only mating and Crotalidae). season 1, or 2, or both (see Aldridge and Duvall, 2002; Aldridge et al.,

In males, assuming summer spermatogenesis is the 2009 for mating seasons of individual species). primitive state, the major change is the continuation of spermatogenesis/spermiogenesis into the spring. It and spring matings, may shed light on the selection of appears that the date of initiation of spermatogenesis mating season in Vipera. in Vipera is similar to many species of snakes. What makes Vipera defined as having prenuptial spermato- The diversity of snake reproductive cycles in temperate genesis is that spermatogenesis/spermiogenesis is ex- climates makes illustration of cycles difficult. Saint Girons tended into the spring. In V. aspis, spermiogenesis (1982) illustrated reproductive cycles from Europe. To begins in the summer, is quiescent over the winter, and illustrate how males adapt to changes in estrus cycles, we resumes during the spring mating season. In V. berus, present the reproductive cycles of two North American snake spermatogenesis begins in the summer, is quiescent in families, Crotalidae (Aldridge and Duvall, 2002) and Colu- the winter, then resumes in the spring. The extension of bridae (Aldridge et al., 2009), in Figure 1. In these species, spermiogenesis into the spring prevents one of the because sperm are stored for extended periods of time, a major disadvantages of postnuptial spermatogenesis, change from summer to spring mating or vice versa, or the i.e., the possibility of depleting sperm reserves during addition of another mating season, does not necessarily the mating season (Table 1). This hypothesis could be affect the seasonal timing of spermatogenesis. tested by analysis of sperm storage in the ductus The widespread occurrence of postnuptial spermatogenesis deferens through the mating seasons. in several families of North American snakes supports the In V. berus, which mates primarily in the spring, primitive nature of this pattern. In several families of North Hoggren¨ and Tegelstrom¨ (2002) found that the first America snakes (Colubridae, n ¼ 20; Natricidae, n ¼ 28; male that mates with the female fathers the majority of Xenodontidae, n ¼ 6), Aldridge et al. (2009) reported that all the offspring (in 8 litters, male 1 ¼ 75%, male 2 ¼ 18%, but two species had a spring mating season. More recent male 3 ¼ 7%). They did not examine paternity from studies have documented spring matings in the two species summer matings. Hoggren¨ and Tegelstrom¨ (2002) added missing a spring mating season (Drymarchon couperi, Hyslop that in V. berus, summer/autumn matings are rare and et al., 2009; and Thamnophis sauritus, Langford et al., 2011). there is no evidence that these matings result in Thus, all species of North American colubrids breed in the offspring. In V. aspis, females routinely mate in the spring, and of these, 18 (33%) also had a summer/autumn summer and spring. The sperm of V. aspis from mating season. The uniform presence of the spring mating summer/autumn matings are stored in the posterior season suggests that this is the ancestral pattern, and that the oviduct during the winter and move anteriorly to addition of a summer/autumn breeding season is the derived infundibular sperm storage tubules at least several weeks condition. prior to ovulation (Saint Girons, 1957, 1959). Exami- In viperids of North America (Agkistrodon, Crotalus, and nation of paternity in V. aspis, which include summer Sistrurus), Aldridge and Duvall (2002) reported that of the 15 Aldridge et al.—Spermatogenesis and mating in squamates 243 species examined, one had a spring-only mating season synchronous and asynchronous reproductive patterns in (confirmed by Dugan et al., 2008), seven had summer-only the Cordylidae. They concluded that the basal pattern for mating seasons, and seven had summer and spring mating cordylids was synchronized breeding (gametogenesis in both seasons. In Table 2, we have included studies completed males and females), with oviparity and spring spermatogen- following Aldridge and Duvall (2002). Species with a spring- esis. They added that the evolution of asynchronized only and spring and summer mating seasons are clearly breeding (autumn spermatogenesis and breeding with spring postnuptial (require long-term sperm storage). We contend ovulation) occurred simultaneously with the evolution of that species with a summer/autumn mating season (in bold viviparity. Following the change to asynchronous breeding, type in Table 2) also have postnuptial spermatogenesis. some western species (see Mouton et al., 2012) reverted back We base this conclusion on these observations: 1) all the to synchronous breeding. Mouton et al. (2012) also discussed species studied thus far in Crotalus (including Crotalus climatic factors that may have influenced the changes in durissus from South America; Almeida-Santos et al., 2004; reproductive patterns. We contend that the conversion from Barros, 2011) have summer spermatogenic cycles; 2) in the synchronous (spring spermatogenesis) to asynchronous eight species that have been studied for sperm storage, all (summer spermatogenesis) and back again, resulting from have long-term sperm storage in males; 3) that within species the evolution and loss of viviparity, suggests that the female groups of Crotalus in North America, four of the six species determines the mating period and consequently, because of groups had species with different mating seasons, suggesting the lack of long-term sperm storage in male lizards, that the loss or gain of a mating season is a highly variable spermatogenesis. trait; and 4) Aldridge and Duvall (2002) proposed that the Crews (1984) introduced the terms ‘‘associated’’ and primitive pitviper mating pattern for temperate species ‘‘dissociated,’’ which originally applied to the relationship consisted of a summer and spring mating season. They between steroid hormones and gamete maturation within a added that spring-only and summer-only patterns were sex. In the associated pattern, gamete maturation and derived due to a loss of one of the mating periods. Thus, it maximal sex steroid hormone secretion immediately precede appears that presence of a summer-only mating period is not or coincide with mating behavior, and in the dissociated the result of spermatogenesis being moved in response to a pattern, mating occurs when the gonads are not producing change in the mating season, but rather the loss of a spring gametes and blood levels of sex steroids are basal (see Crews, mating season. 1999). In a recent review, Van Dyke (2015) used these terms to describe the temporal relationship between mating and Lizard reproductive cycles.—Through an in-depth literature spermatogenesis. In the associated pattern, species mate review, it superficially appears that lizards have species with

immediately following spermatogenesis (i.e., prenuptial both prenuptial and postnuptial spermatogenesis. Lizards spermatogenesis), whereas in the dissociated pattern, sper- have either a summer/autumn (or early winter in mild matogenesis occurs months prior to the mating season, and climates) or spring mating season, not both. In addition, the the sperm are stored (in the epididymis) for the following lizard mating season is always immediately preceded by mating period (i.e., postnuptial). As examples, Van Dyke spermiogenesis, making spermatogenesis prenuptial. (2015) cites Jones et al. (1997) and Murphy et al. (2006). In some species of lizards described as having postnuptial In lizards, postnuptial spermatogenesis is described or spermatogenesis (Table 3), spermatogenesis and mating occur superficially apparent in six families, nine genera, and 13 in the summer and investigators suggest that sperm are species (Table 3). A close inspection of the components of the stored in the efferent ducts of the male for a spring mating reproductive cycles, however, suggests that all of these lizards season. However, long-term sperm storage in the male and actually have prenuptial spermatogenesis. In each of these the occurrence of a second mating season in the spring have species, males are spermatogenic immediately prior to the not been definitively shown to occur in lizards. In general, if mating season (Fig. 1). They appear to be postnuptial because mating and vitellogenesis occur in different seasons in the female’s breeding season occurs only (or primarily) in the lizards, long-term sperm storage occurs in the oviduct rather summer/autumn, preceding spring ovulation and fertiliza- than in the efferent ducts of the testis. We contend that the tion. The reproductive cycles of lizards from these six families use of the term postnuptial to describe spermatogenesis in are discussed below. lizards is inaccurate. Guillette and Mendez-de´ la Cruz (1993) introduced the 1. Anguidae: In Anguidae (Elgaria kingii, Goldberg, 1975b; terms synchronous and asynchronous to describe variations E. paucicarinata, Goldberg and Beaman, 2004), spermio- in the timing of male and female reproductive cycles in genesis begins in the spring (May) and spermiation lizards (reviewed by Mendez-de´ la Cruz et al., 2014). In occurs in the summer/autumn (August–November). synchronous cycles, spermatogenesis begins immediately Vitellogenesis begins in the summer/autumn (October) prior to mating (i.e., prenuptial), whereas in asynchronous at which time mating occurs. The authors speculate that cycles, spermatogenesis begins several months prior to females store sperm until spring ovulation. This sper- mating, and mating occurs at maximum spermatogenesis matogenic cycle is clearly prenuptial. or during the early regressive stage of spermatogenesis 2. Cordylidae: Three species of cordylids have been (Mendez-de´ la Cruz et al., 2014). Using our definition of described as having postnuptial spermatogenesis. In the timing of spermatogenesis, asynchronous is prenuptial Smaug giganteus, van Wyk (1995) reported that sper- spermatogenesis. Mendez-de´ la Cruz et al. (2014) add that in matogenesis begins in the spring (December) and both synchronous and asynchronous species, sperm are spermiogenesis occurs from summer to autumn (Febru- stored in the oviduct until fertilization. ary–April). The reproductive cycle of Smaug giganteus is Mouton et al. (2012) studied the role of phylogeny and graphically presented in Figure 2, along with the parity mode (oviparity and viviparity) in evolution of reproductive cycle of Ouroborus cataphractus, a cordylid 244 Copeia 108, No. 2, 2020

phy of the SSK, and 6) male–female interactions observed in autumn. In late winter and spring, the SSK and epididymis are nonsecretory, and the maximal epididymal epithelial height (12 lm) and the maximal ductus deferens epithelial height (6 lm) are much less that the autumnal levels. If the autumnal mating season is confirmed, this spermatogenic cycle is prenuptial. Du Toit et al. (2003) examined the reproductive cycle of Chamaesaura anguina in the Western Cape region of South Africa. They reported preovulatory females were present throughout the year; however, the male reproductive cycle appeared to be seasonal and post- nuptial. Males were spermatogenic from spring to autumn, and then the testes were regressed in late autumn through the winter. During the summer (January–February, n ¼ 9), the testes were spermatogenic and all males had sperm in the epididymis. By June (n ¼ 8), only half of the males have epididymal sperm and by September and October (n ¼ 10), one-third had sperm. The authors suggest a spring mating season, which would suggest that spermatogenesis is postnuptial. The authors, however, present no data on the mating season Fig. 2. Representations of the reproductive cycle of cordylid lizards in or on sperm storage in females. temperate climates. Lines/boxes as in Figure 1. (A) Reproductive cycle In Pseudocordylus melanotus, Flemming (1993) report- of Ouroborus cataphractus (Flemming and Mouton, 2002), described ed that spermatogenesis begins in the spring (Novem- as having a prenuptial spermatogenesis. (B) Reproductive cycle of ber), and peak spermiogenesis occurs from summer to Smaug giganteus (van Wyk, 1995) described as having postnuptial autumn (February–April). The testes regress in the spermatogenesis. In Ouroborus cataphractus, vitellogenesis, spermato- autumn and winter (May–July). Sperm are present in genesis, and the mating period occur in the spring, typical of prenuptial the epididymis from summer (February) into the spring spermatogenesis. In Smaug giganteus, vitellogenesis occurs in the (October). The epididymis was empty in late spring spring, similar to Ouroborus cataphractus; however, spermatogenesis occurs in the summer. Based on limited data, van Wyk (1995) states (December–January). Vitellogenesis begins in summer that mating occurs in the spring (mating box 1). However, other data (March; Flemming, 1993), continues through the winter suggest the mating season occurs in the summer (mating box 2, see (August), and culminates with ovulation in the spring text), and the sperm would be stored over winter in the oviduct. If (October). Mouton and van Wyk (1993) noted the mating is restricted to the summer, this is prenuptial spermatogenesis, existence (unpublished) of mating occurring in the fall. whereas, if mating occurs in the spring (with or without the summer However, they also cited Mackay (1993), who examined mating period), this pattern would be postnuptial spermatogenesis, the oviducts of this species and found evidence of because sperm would be stored in the efferent ducts of the male over sperm storage. Flemming (1993) suggested that the winter. extended epididymal sperm storage insured fertilization in the spring. with prenuptial spermatogenesis. The testes regress in If this species has primarily a spring mating season, the late autumn and winter (June–July). Sperm are spermatogenesis would be postnuptial. More recent present in the testes, epididymis, and ductus deferens data, however, suggest a summer/autumn mating from summer (March) into the winter (August). The SSK season. Moon (2001) observed copulation in the wild is hypertrophied during spermiogenesis. In other on 26 April and male–male aggression, which is months (May–January), the SSK cytoplasm is nonsecre- associated with mate guarding and territoriality, was tory. Vitellogenesis begins in summer (January–Febru- observed on 2 June. Based on the presence of mating in ary), continues through the winter (August), and the summer/autumn, this species has prenuptial sper- culminates with ovulation in the spring (October– matogenesis. This species has extended sperm storage November; van Wyk, 1994). The mating season has over winter, but it may not be used in mating. More not been documented; however, based on plasma data are needed to confirm mating seasons. testosterone levels and other factors, van Wyk (1995) 3. Gekkonidae: In the gekkonid Christinus marmoratus, suggested that this species has a spring mating season. If vitellogenesis begins and spermatogenesis and mating S. giganteus has a spring-only mating season, this species occur in the summer (King, 1977). Sperm are stored in has postnuptial spermatogenesis. Van Wyk (1995) vaginal lamellae of the posterior vagina throughout the added that the possibility of an autumnal mating winter. Ovulation occurs in the spring. We argue that season cannot be excluded. We suggest that the because spermatogenesis occurs immediately prior to following reproductive data presented by van Wyk the mating season, this spermatogenic cycle is prenup- (1995) are more consistent with an autumnal mating tial. season: 1) the high plasma testosterone level, 2) 4. Lacertidae: In the lacertid Podarcis muralis, vitellogenesis maximal sperm stores in the epididymis, 3) maximal and mating occur in the spring. In males, spermato- epididymal epithelial height (40 lm), 4) maximal genesis begins in the fall and spermiogenesis is ductus deferens epithelial height (15 lm), 5) hypertro- completed in the spring (Saint Girons and Duguy, Aldridge et al.—Spermatogenesis and mating in squamates 245

1970; Kwiat and Gist, 1987) during the mating season bite marks on females. Wapstra et al. (1999) report that (Gribbins and Gist, 2003). Saint Girons and Duguy in the spring, the testes are at their smallest and some, (1970) presented additional evidence of a spring-only but not all, males had eversible hemipenes, milky mating season in its native range. They reported that epididymides, and ejaculated motile sperm. Following the SSK, which is hypertrophied during the mating the spring mating period, the epididymis appeared season, was not developed in the summer/autumn (SSK empty and remained this way until spermiation in the tubular diameter 49 lm) but highly developed in the summer. In both of the studies on N. ocellatus, testis spring (SSK tubular diameter 120 lm; Saint Girons and mass, not histological examination, was used to assess Duguy, 1970). Thus, this species has prenuptial sper- testicular activity (Jones et al., 1997; Wapstra et al., matogenesis. Carretero (2006) stated that male and 1999). The presence of motile sperm in the ductus female cycles in lacertids are linked. He added that this epididymis and the presence of bite marks on some link means that neither sperm needs to wait for ovarian females in the spring suggests that long-term sperm follicles to mature nor ovarian follicles become ‘‘frozen’’ storage (postnuptial spermatogenesis) may occur in waiting for spermiogenesis, and concluded that no some males in this species. Both studies, however, long-term sperm storage occurs in the whole family. expressed some hesitation in the importance of spring 5. Liolaemidae: The spermatogenic cycle of the liolaemid mating, with Jones et al. (1997) using the term Phymaturus zapalensis has been described as postnuptial ‘‘circumstantial’’ to describe spring mating and Wapstra (Boretto et al., 2012). This classification is based on et al. (1999) describing stored sperm in the spring as males beginning spermatogenesis in the summer prior ‘‘residual.’’ to the spring mating season. However, the authors state We believe the descriptions provided by Jones et al. that the spermatogenesis initiated in the summer is not (1997) and Wapstra et al. (1999) are consistent with our completed prior to hibernation and they do not find definition of postnuptial spermatogenesis; however, the sperm stored in the epididymis over winter. In the relatively low proportion of snakes displaying this trait spring, spermatogenesis resumes and mating occurs. (30–40%; Wapstra et al., 1999) suggests that this may be This cycle is best described as prenuptial spermatogen- incipient postnuptial spermatogenesis. If selection esis. favors long-term sperm storage and spring mating, this 6. Scincidae: Five species of scincids have also been trait may become fixed in these populations. Successful reported to have postnuptial spermatogenesis. In Ples- spring matings could lead to the loss of the autumn tiodon egregius (Mount, 1963), spermatogenesis begins mating season. We speculate that this scenario may be several months prior to the mating season, and

one possible mechanism leading to the evolution of following mating, the sperm are stored in the oviduct postnuptial spermatogenesis within the Squamata. over the winter. In this species, vitellogenesis and Further work on spring testis development and obser- ovulation occur in the spring. We argue that because vations on spring mating are necessary to confirm these spermatogenesis occurs prior to the mating season, observations. these spermatogenic cycles are prenuptial. Murphy et al. (2006) reported that Hemiergis decre- In Niveoscincus metallicus, Swain and Jones (1994) siensis and Niveoscincus coventryi displayed autumn speculate that there may also be a spring mating season. spermatogenesis with epididymal sperm storage over They base this possibility on the presence of elevated winter and spring vitellogenesis, mating, and ovulation. testosterone levels (in 2 of 14 males in September) and However, the data supporting these conclusions are the presence of bite scars on females that are associated lacking (limited data on the number of specimens and with mating. More data are needed to confirm the times of the year specimens were examined), and spring mating season. Without the spring mating evidence of the timing of the mating season is season, spermatogenesis in this species is prenuptial. speculative. Murphy et al. (2006) recognized the Males of the viviparous lizard Niveoscincus ocellatus shortcomings of the data and recommended that have primarily a prenuptial spermatogenic cycle; how- further work on these species is necessary to support ever, some males also store sperm over winter and may the existence of long-term epididymal sperm storage. mate again in the spring (Jones et al., 1997; Wapstra et Adding to the uncertainty of the mating season are al., 1999). In this species, males undergo spermatogen- studies by Pengilley (1972, cited by Murphy et al., esisinthesummerfollowedimmediatelybythe 2006), who found sperm in oviducts over winter for N. primary mating season in the autumn (Fig. 2B, with coventryi in southeast Australia, and by Robertson mating period 2). Jones et al. (1997) stated that all (1981), who reported a fall mating season in H. sexually mature females mate in the autumn and store decresiensis from 368S latitude. sperm in their oviducts over winter. Females begin vitellogenesis in the autumn, corresponding to the We conclude that the majority of, if not all, lizards have summer mating season, and store sperm in the oviduct prenuptial spermatogenesis. The only family that has species until the following spring. Vitellogenesis slows during with extended sperm storage in males belongs to the the winter, and then resumes in late winter–spring. Scincidae. If long-term sperm storage in males occurs, it Jones et al. (1997) also reported that during copulation, would represent an independent evolution of postnuptial the males bite the female near the shoulders, leaving U- spermatogenesis. However, further studies are needed to shaped marks. As vitellogenesis resumes in the spring, confirm long-term sperm storage in males. Wapstra et al. (1999) observed that 30–40% of the females appear to have mated again (Fig. 2B, with Reproductive cycle of Sphenodon.—The reproductive biology mating periods 1 and 2), based on the presence of fresh of Sphenodon punctatus from Stephens Island (408S) was 246 Copeia 108, No. 2, 2020 described by Saint Girons and Newman (1987). The sper- epithelial height of the ductus epididymis are difficult to matogenic cycle is prenuptial, with spermatogenesis begin- detect. He reported that the epithelial height varied from a ning in the spring (October) and reaching a peak in summer low of 22 lm in May to a high of 34 lm in August; however, (February). Mating occurs in the summer (January and there was considerable variation in the monthly samples. Fox February). The seminiferous tubules do not regress complete- (1952) added that he could only detect faint traces of ly in the winter; however, spermiogenesis is halted. The granules in the epididymis. authors suggest that the spermatogenic cycle of Sphenodon, Sever (2010) examined the efferent ducts in Seminatrix which resembles lizards, may be due to similar adaptations to pygaea and reported that the epididymis had a basophilic climate, rather than by phylogenetic relatedness with lizards. cytoplasm that stains for neutral carbohydrates and for Stephens Island has a maritime climate in which the summer proteins. He added that the epididymis does not undergo (16.28C) and winter (9.28C) temperatures differ by only 78C. marked seasonal variation. Previous investigators suggested Thus, spermatogenesis occurs throughout the year but at a that the epididymis of snakes is non-secretory (Volsøe, 1944; much-reduced rate in the winter. Fox, 1952; Dufaure and Saint Girons, 1984), though Sever The seasonal changes in the epididymis parallel changes (2010) confirmed the presence of secretory material in the observed in snakes and lizards. The epithelial height epididymis. Sever (2010) added that S. pygaea possesses a increases from a low in winter of 40 lm (August) to a high constitutive secretory pathway in which the secretions are of 78 lm in summer (February). Secretory activity in the transported to the surface in small vesicles and not epididymis also occurs during the summer. The ductus concentrated or stored in granules. Sever (2010) added that deferens is very small, and the authors state that it is difficult with this mode of secretion, histological studies would to see in dissections. The authors conclude that sperm indicate that the epididymis is non-secretory. In the Black storage occurs primarily in the epididymis and anterior Swampsnake (S. pygaea), Sever (2010) reported that the ductus deferens. The unique feature of Sphenodon is the epididymal secretory granules contained glycogen. He added length of the epididymis, which extends from the testis to that the exact function was not determined, but perhaps the anterior third of the kidney. The presence of sperm they produce an environment for sperm storage and/or storage in the epididymis, as opposed to the ductus deferens, maturation, as known in mammals (Sever, 2010). as in snakes, suggests that epididymal sperm storage is a In lizards, seasonal changes in the epididymis are substan- primitive trait in the Lepidosauria and that storage in the tial. Saint Girons and Duguy (1970) examined the seasonality ductus deferens, is the derived condition. of the epididymis in two populations of Lacerta muralis in France. They reported that the seasonal differences in the Efferent testicular ducts of squamates.—In squamates, the morphology of the epididymis were dramatic. In the epididymis and ductus deferens are the two major efferent summer, following the spring mating season, the diameter ducts connecting the testis with the cloaca and copulatory of the epididymis was 40–60 lm and the epithelial height organs (Fox, 1977). These ducts also serve as storage sites for was 15–17 lm. The diameter increased in November at the sperm. The morphology of the efferent ducts has recently same time the seminiferous tubules enlarged. By March, the been reviewed in both snakes (Trauth and Sever, 2011) and seminiferous tubules reached their maximum diameter, the lizards (Rheubert et al., 2015), and the similarities at the epididymis reached 160–200 lm, and the epithelial height gross, histological, and ultrastructural level suggest that these increased to 50–65 lm. Saint Girons and Duguy (1970) also structures are homologous. In both snakes and lizards, the reported that sperm were present in the epididymis only seasonal development and secretory activity is dependent on during spermiogenesis. androgens (Courty and Dufaure, 1979; Gist, 2011). However, Hraoui-Bloquet (1985) described the cycle of the epididy- the seasonal patterns of sperm storage in these ducts differ mis of Lacerta laevis. He reported that the epididymis was substantially between lizards and snakes. The differences in deeply involuted from the end of July to the end of the site of sperm storage and seasonal changes in the efferent September. The epithelial cells did not appear to be secretory. ducts were noted by Volsøe (1944). In snakes, the epididymis Sperm were present in the epididymis in mid-September. appears to be secretory for most, if not all, of the year, and Hypertrophy of the epididymis occurred during October, and sperm are present in the epididymis generally only during by the end of October the epithelial cells were 55 lm high and immediately following spermiogenesis. In snakes, long- and the supranuclear region was filled with coarse grains of term sperm storage occurs in the ductus deferens. In lizards, secretion vacuoles. The coarse granules are characteristic of the diameter, epithelial height, and secretory activity of the Lacertidae (Van der Stricht, 1893). These grains were mixed epididymis vary greatly during the year and this structure with the sperm. Hypertrophy of the epididymis extended to serves as the major site of sperm storage (Volsøe, 1944). the end of June, following the mating period. The role of the The degree of the seasonal changes in the epididymis are secretory material produced in the lizard epididymis is different in snakes and lizards. In Vipera berus, Volsøe (1944) unknown; however, secretions from the epididymis in other found that the diameter varied from a low in June of 160 lm amniotes function in the maturation of the spermatozoa to a high of 250 lm in March and April, the breeding season (Rheubert et al., 2015). of this species. The epithelial height paralleled the changes Not all lizards have dramatic seasonal changes in the with a low of 35 lm to a high of 60 lm. Volsøe (1944) epididymis. Van Loben Sels and Vitt (1984) reported that examined the ductus deferens for secretory granules but was changes in the epithelial height of Urosaurus sp. were not as able to find only faint traces of granules in a few cells in a few large as changes in the diameter. The diameter of the individuals. Volsøe (1944) suggested that the secretory epididymis ranged from a high of 110 lm (June) to a low activity of the ductus epididymis is lost in snakes. of 18 lm (December). The seasonal changes in the height of Fox (1952) examined the epididymis in Thamnophis elegans the epithelial cells of the epididymis were not as dramatic. and reported that seasonal changes in the diameter and They reported that epithelial cell height ranged from a high Aldridge et al.—Spermatogenesis and mating in squamates 247 of 15 lm in June to a low of 12 lm in December and the packed with sperm in March when the testis was inactive. epididymis contained sperm only during spermiogenesis. This pattern of sperm storage in S. pygaea was not universal. The seasonal changes in the ductus deferens are different Sever (2010) presented a photomicrograph (his fig. 1) of the in snakes and lizards. In snakes, the ductus deferens under- epididymis of this species in October, the time following goes seasonal changes in epithelial height; however, much of peak spermiogenesis and before the spring mating period. the variation appears to be passive, due to the presence of The anterior tubules of the epididymis have scattered sperm, sperm. For example, Volsøe (1944) examined the ductus whereas the more posterior tubules close to the ductus deferens in Viper berus and found that the diameter, at the deferens are packed with sperm. The presence of sperm in the level of the kidney, varied from 130 lm to 575 lm. He epididymis in the spring snakes may represent an overabun- reported that the highest values were found late in April and dance of sperm in the efferent ducts in general rather that May, when the duct was distended with sperm. The height of true sperm storage in the epididymis as occurs in lizards. the epithelium varied from 13 lmto37lm and was lowest In some snakes, sperm are absent from the efferent ducts when the width was its largest, suggesting that the variation during the year. For example, in Austrelaps superbus, some is probably due only to a passive distension of the duct. He adult males lack sperm in the summer following the mating reported that no signs of secretory activity were present; season (Shine, 1977). Almeida-Santos et al. (2004) reported however, Siegel et al. (2009) showed carbohydrate-filled that sperm are present in the ductus deferens throughout the secretory vacuoles in the apical region of epithelial cells year in Crotalus durissus (268S). They added, however, that lining the ductus deferens through use of transmission there was seasonal difference in the concentration of sperm electron microscopy in Agkistrodon piscivorus, another viper- in the ductus. They reported that the sperm count following id. spermiation (41x105 per mm3) was greater than the sperm In Thamnophis sirtalis, Fox (1952) reported that the count in the winter (38x105 per mm3). In this population, epithelial cells of the ductus deferens were flattened (at times mating is restricted to the autumn. Almeida-Santos et al. wider than tall) when the duct was filled with sperm (fall to (2004) suggested that the reduction in the sperm count may spring) and the cells became elongate (much taller than be due to mating activity. wide) and crowded together in the summer when the sperm The lack of long-term sperm storage in snakes was also content was diminished. He added that the fluctuations reported by Saint Girons and Pfeffer (1971) in Boiga cyanea, looked more like a passive stretching or compression of the Enhydris enhydris, Homalopsis buccata, and Natrix piscator from cells than actual changes in cellular volume. Cambodia. Saint Girons and Pfeffer (1971) reported that In lizards, Hraoui-Bloquet (1985) reported that in Lacerta these snakes had prenuptial spermatogenesis, and following laevis, the development of the ductus deferens paralleled the mating and the cessation of spermiogenesis, sperm were development of the epididymis. The ductus deferens was absent from ductus deferens. involuted in July and began to develop in October. The length of sperm storage in the efferent ducts does not Spermatozoa, mixed with the secretory granules from the appear to be related to phylogeny. Saint Girons (1976) found epididymis, appeared in October and remained in the ductus that sperm are present in the ductus throughout the year in deferens until May or June. Spermatogenesis in Lacerta laevis Vipera aspis, V. latastei, and V. seoanei and absent, following began in October, was suppressed during hibernation, then the spring mating season, in the ductus in the summer in V. resumed in the spring (Hraoui-Bloquet, 1985). ammodytes, V. berus, and V. ursinii. The difference in sperm Sperm storage in male squamates.—Sperm storage in the storage pattern may be due to the seasonal pattern of efferent ducts differs in snakes and lizards. In snakes, the spermatogenesis and mating; the former group has postnup- ductus deferens is the major site for long-term sperm storage tial and latter group has prenuptial spermatogenesis. (W. Fox, 1952; H. Fox, 1977), whereas the epididymis Shine (1977) suggested a similar relationship between the generally has sperm present primarily during spermiogenesis. pattern of spermatogenesis and sperm storage and the timing The seasonal presence and abundance of sperm in the of the mating season in several species of Australian elapids. efferent ducts is variable. In many species, sperm are present In four viviparous species (Austrelaps superbus, Hemiaspis in the ductus deferens during the entire year. For example, signata, Notechis scutatus, and Parasuta gouldii), spermiogen- Marshall and Woolf (1957) reported that the epididymis of esis occurred in the summer, and sperm were present in the Vipera berus had sperm only when sperm were being ductus deferens all year. These species mate in the autumn produced, whereas the ductus deferens had sperm the entire and spring; however, in another live-bearing species (Pseu- year. In Typhlops vermicularis, Fox (1965) examined the dechis porphyriacus), spermiogenesis and mating occurred in efferent ducts in late winter and early spring (February the spring. The ductus deferens retained sperm only during through May). He reported that sperm filled the epididymis spring. of the February and March specimens, when spermiogenesis Intwooviparouselapids(Pseudonaja nuchalis and P. was occurring; however, by April when spermiogenesis was textilis), Shine (1977) reported that spermiogenesis occurred complete, sperm were absent from the upper three-fourths of in spring (and possibly also in summer), and these species the epididymis. Fox (1965) found a few sperm in the distal retained spermatozoa in the ductus deferens all year. Both epididymis at the level of the most caudal testicular lobe; species of Pseudonaja mated in spring, and possibly through however, he noted that the entire ductus deferens remained summer (Shine, 1977). highly convoluted and was characteristically filled with Gorman et al. (1981) reported a different cycle in sperm. Acrochordus granulatus (108N). They reported that the repro- Some snakes appear to store sperm for extended periods in ductive cycle of both sexes was seasonal and highly the epididymis. Sever et al. (2002) reported that in Seminatrix synchronized, and they added that following the mating pygaea, the epididymis and the entire ductus deferens were season the ductus deferens lacked sperm. 248 Copeia 108, No. 2, 2020

The studies by Saint Girons (1976), Shine (1977), and species rather than the result of enhanced sperm storage Gorman et al. (1981) support the hypothesis that not only ability. are the times of production of spermatozoa adapted to the In lizards with seasonal spermatogenesis, the length of timing of the mating patterns but so is the retention of time that sperm are stored is variable. In the majority of spermatozoa in the ductus deferens. Schuett et al. (2002) species, the evacuation of sperm from the epididymis occurs reported an unusual pattern of sperm storage in the ductus immediately or within a few months (2–3) following the deferens of Crotalus scutulatus. This species has a summer and cessation of spermiogenesis (Anguis fragilis, Herlant, 1933; spring mating season, and Schuett et al. (2002) reported that Uromastix acanthinura, Kehl, 1944; Anolis carolinensis, Fox, sperm were present in the ductus in all months, but not in all 1958; Leiopelma fuscum, Wilhoft and Reiter, 1965; Xantusia individuals. Snakes with a spring mating season are normally riversiana, Goldberg and Bezy, 1974; Urosaurus ornatus,Van associated with long-term sperm storage (Aldridge and Loben Sels and Vitt, 1984; Podarcis muralis, Kwiat and Gist, Duvall, 2002). Perhaps the lack of sperm in some individuals 1987; Sceloporus mucronatus, Estrada-Flores et al., 1990; was the result of successful matings. The mechanism of how Pseudocordylus melanotus, Flemming, 1993; Heloderma suspec- sperm are removed from the efferent ducts (expulsion or tum, Goldberg and Lowe, 1997; Hemicordylus capensis, van resorption) and why (perhaps lack of endocrine support) is Wyk and Mouton, 1998; and many others). unknown. One difficulty in determining long-term sperm storage in In lizards, the epididymis appears to be the major site for lizards is that sperm are not always removed from the sperm storage (Fox, 1952); however, some studies report that epididymis, even though the epithelium regresses and is no sperm are also present in the ductus deferens (Goldberg and longer secretory. In the tropical lizard Tropidurus itambere, Miller, 1985; Sever and Hopkins, 2005). Goldberg and Miller Ferreira et al. (2009) reported that sperm, in varying (1985) studied the reproductive cycle of Anniella pulchra in a amounts, were present throughout the year. However, later Mediterranean climate region of southern California. They in the paper they state sperm are present in the epididymis reported that testicular cycle was extended with recrudes- primarily during spermatogenesis, and following the cessa- cence beginning in the summer (June and July) and tion of spermatogenesis, sperm are lost from the epididymis spermiogenesis occurring from October through January. within a month (Ferreira et al., 2009). A similar situation was Despite the long period of spermiogenesis, Goldberg and reported by Du Toit et al. (2003) in Chamaesaura anguina. Miller (1985) stated that sperm were not present in the They found that, following a summer spermatogenesis and ductus deferens until February, and by July, following the summer/autumn mating period, some males (50%) still spring mating season, the ductus was empty. possessed sperm in the winter and by spring, one-third of

Sever and Hopkins (2005) examined the reproductive cycle the males still had sperm. Thus, the presence of residual of Scincella lateralis from South Carolina. They reported that sperm may be interpreted as sperm storage for an additional mating season (i.e., Pseudocordylus melanotus, Flemming, spermatogenesis is seasonal, with spermiogenesis occurring 1993; N. metallicus, Swain and Jones, 1994; Smaug giganteus, in the spring. They also examined sperm storage in the van Wyk, 1995; Hemiergis decresiensisandNiveoscincus excurrent ducts and found that sperm storage in the coventryi, Murphy et al., 2006). epididymis and anterior ductus deferens is seasonal; howev- er, sperm are present in the posterior ductus deferens Sperm storage in female squamates.—One factor that influ- throughout the year. An examination of the photomicro- ences female sperm storage in squamates is the number of graphs of the ductus deferens presented in their figure 1 clutches/litters per breeding season. Temperate climate (Sever and Hopkins, 2005) showed that sperm are present in snakes have, almost universally, one clutch (or litter) per August and October specimens. However, the diameters of season. Based on North American studies, the single the tubules in August and October are approximately half exception to this rule is Thamnophis sauritus from Alabama that of the tubules in June, suggesting that these sperm are (308N). In their study, Langford et al. (2011) reported that residual sperm and not sperm stored for future matings. Sever two females produced two litters in the same season. In and Hopkins (2005) confirmed our conclusion by stating that lizards, however, the majority of oviparous, spring-breeding mating occurs in the spring following spermiation and lizards have several clutches per season. development of the SSK. The retention of sperm in the Oviductal sperm storage in snakes was recently reviewed ductus deferens is consistent with the partial development of by Siegel et al. (2011) and in lizards by Siegel et al. (2015). the SSK reported by Sever and Hopkins (2005) and suggests The basic structure of the oviduct is similar in snakes and that androgen levels are somewhat elevated following the lizards; however, location of sperm storage is variable. Eckstut spring/summer mating season. et al. (2009) reviewed sperm storage location in female In lizards with continuous spermatogenic cycles, sperm are squamates and concluded that infundibular sperm storage present in the epididymis throughout the year (e.g., Agama was the ancestral mode, with some female lizards and snakes agama; Marshall and Hook, 1960). In Tropidurus torquatus,a acquiring sperm storage in the more caudal portions of the species in which spermatogenesis is described as cyclic, oviduct. sperm are present in the epididymis throughout the year Schuett (1992) described sperm storage in female snakes (Vieira et al., 2001). A closer examination of the data, (particularly temperate zone pitvipers) as short-term sperm however, reveals that sperm are present in the testes of the storage, generally occurring over weeks to two months, and majority of males for ten months (April–January) of the year. long-term sperm storage, generally occurring longer than six In the late summer and fall (February–March), the testes are months. The length of time sperm is stored in females is not relatively smaller, and spermatids are the prevalent cell type. necessarily related to pre-/postnuptial spermatogenesis. For Thus, the continuous presence of sperm in the epididymis example, in Crotalus viridis, spermatogenesis is often de- may be due to the extensive spermatogenic season in this scribed as postnuptial because it occurs in the summer Aldridge et al.—Spermatogenesis and mating in squamates 249 following spring ovulation. However, mating is restricted to (Fox, 1977). The function of the SSK secretions are unknown the summer/autumn and thus females must store sperm in in many taxa, but Friesen et al. (2013) recently demonstrated the oviducts until spring fertilization (Rahn, 1942). that mating plugs are unequivocally produced through Some female snakes seem to be able to store sperm for very secretions of the SSK in Thamnophis sirtalis. Interestingly, long periods of time. Strugariu (2007) reported that a these plugs did not prevent subsequent male suitors from Coronella austriaca, collected in early spring (mid-May) of mating but instead prevented sperm leakage out of the 2005, gave birth to two males and four females in September female cloaca post coitus (Friesen et al., 2013). of the following year. If this individual mated in the spring of There are two major differences between the SSK in snakes 2005, the sperm were viable for one year. and lizards; one is the degree of seasonal change in SSK In lizards, a number of studies have examined the ability of development, and the second is the relationship between a single mating to fertilize multiple clutches within a spermatogenesis and development of the SSK. Seasonal breeding season. Cuellar (1966) isolated female Uta stans- changes in the number and staining intensity of the buriana following the spring mating season. These females secretory granules varies considerably depending on whether had three separate clutches of eggs laid during a three-month a snake is examined in mating or the non-mating season period. In these females, fertility decreased dramatically in (Fox, 1977); however, the SSK is always prominent through- subsequent clutches (clutch 1 was 95%, clutch 2 was 53%, out the year. In lizards, however, the SSK varies dramatically and clutch 3 was 0% fertile). Cuellar (1966) concluded that during the mating and non-mating season, and, as Fox sperm were functional for at least 81 days. He did not (1977) points out, in many lizards the SSK is hardly determine if reduced fertility was due to sperm quality or distinguishable from the uriniferous tubules during the quantity. non-mating season. The second difference is the temporal In Urosaurus ornatus, Villaverde and Zucker (1998) reported relationship between spermatogenesis and the SSK. As that females stored viable sperm for at least three months. pointed out Sanyal and Prasad (1966), SSK activity in lizards They added that later clutches had much reduced fertility in is always synchronized with testicular activity. control and experimental groups. They added that later clutches had much reduced fertility in control and experi- Endocrinology of reproduction.—In a recent review of the cues mental groups, indicating that this strategy was much below that squamates use to time reproduction, Van Dyke (2015) the optimum (i.e., 65% [with males] and 48% [without summarized the importance of the hypothalamic-pituitary- males] of females with viable clutches down to 9% and 13%). gonadal axis (HPGA) in regulating reproductive cycles. He Similar decreases in fertilization and or viability were included mechanisms for how environmental and physio- Acanthodactylus schreiberi logical stimuli may serve as proximate cues for activation of reported for (Zotos et al., 2012), Ctenophorus fordi (Uller et al., 2013), Hemidactylus frenatus the HPGA. He added that animals should initiate reproduc- (Murphy-Walker and Haley, 1996), and others. tion based on cues that communicate: a) environmental In other species of lizards, long-term sperm storage may be conduciveness for successful reproduction, and survival of required in both males and females. Srinivas et al. (1995) offspring and (usually) parents, b) physiological capability of reported that male Psammophilus dorsalis undergo spermato- parents to reproduce, and c) likelihood of successful mating. genesis in the spring and summer and by August, spermato- Having reliable cues may be important to males because genesis and sperm storage in the epididymis ends. In this the maintenance of active spermatogenesis and/or secondary species, females produce clutches from June to December. sexual characters may be costly. As Sacchi et al. (2016) Consequently, females must store sperm in the oviduct for pointed out, the maintenance of high T-levels may impose clutches produced from September to December. The authors metabolic and immunological costs. Metabolic costs include did not report on the hatching success of the later clutches. the maintenance of metabolic rate, sexual characters, and Srinivas et al. (1995) also examined sperm storage in females activity, and this may cause a degradation of body condition and found that large amounts of sperm were stored in the and/or survival rate (see Sacchi et al., 2016 for references). anterior vaginal region of the oviduct from August–Decem- Second, elevated androgen levels may compromise immune- ber. Fewer sperm were present in the oviduct in January– competence (via the hypothalamic-pituitary-adrenal axis) March and by April, before the female reproductive season, and thus increase vulnerability to parasites (see Sacchi et al., all of the sperm were absent from the oviduct. Oviductal 2016 for references). Sacchi et al. (2016) concluded that there sperm storage in lizards for two or more reproductive seasons is a trade-off between testosterone plasma level and immune has not been definitively shown in any taxon. function, in accordance with the immune-competence handicap hypothesis (Folstad and Karter, 1992). Sexual segment of the kidney.—The SSK has recently been In snakes with postnuptial spermatogenesis, mating may reviewed for snakes (Aldridge et al., 2011) and lizards occur many months (generally the following spring) follow- (Rheubert et al., 2015). The basic anatomy, secretions, and ing the completion of (summer/autumn) spermatogenesis. hormonal control are identical in both groups which led Increases in plasma androgen (primarily testosterone) levels Sever et al. (2002) to propose that the SSK is a synapomorphy are associated with each process (Aldridge, 1979; Weil and for squamates. The SSK is primarily a male trait but has also Aldridge, 1981; Aldridge et al., 1990). Weil (1985) proposed been found in females in two species of lizards, Cnemidopho- that the sources of androgens for these processes were rus lemniscatus (Del Conte and Tamayo, 1973) and Scincella different. He suggested that androgens from the seminiferous laterale (Sever and Hopkins, 2005). The SSK is androgen tubules (i.e., Sertoli cells) were responsible for androgens dependent, with ontogenic development (Krohmer et al., necessary for spermatogenesis and androgens from the 2004) and seasonal hypertrophy (Bishop, 1959; Fox, 1977) interstitial cells of Leydig were responsible for development dependent on elevated plasma androgen levels. The SSK of the SSK. We suggest that each represents a separate HPGA secretions are transferred to the female during copulation with each regulated by separate proximate cues. 250 Copeia 108, No. 2, 2020

It seemed reasonable to suggest that because all lizards of injury, increased energy used in development and were prenuptial, there was a single source of androgens in maintenance of secondary sex characteristics, increased lizards used for stimulating both spermatogenesis and energy used associated with an increase in the general development of mating structures (the SSK) and mating metabolic rate (androgen effect), and resulting decreased behavior. Recent studies, however, by Boretto et al. (2014) growth (see Aldridge and Duvall, 2002). and Melo et al. (2019) suggest that lizards are similar to In a more recent study, Graham et al. (2008) reported that snakes in having two sources and patterns of secretion of the mating season of the cottonmouth (Agkistrodon piscivo- androgens, Sertoli cells and interstitial cells of Leydig, which rus) occurs in the summer/autumn and spring in some regulate the same structures as in snakes. populations, while in other populations, mating occurs Boretto et al. (2014) examined the viviparous liolaemid primarily (or only) in the summer/autumn. In their popula- lizard Phymaturus punae (~298S) from Argentina. They tion, the mating season was restricted to the summer/ reported that ultrastructural features observed in Sertoli and autumn. They added that sperm storage in the male Leydig cells indicate that both types of cells have the extended into the spring in most, if not all, males, thus potential to synthesize steroid hormones. They suggested making a spring mating possible. Graham et al. (2008) that the Sertoli cells controlled the spermatogenic cycle and predicted that ‘‘future studies of A. piscivorus, and other Leydig cells, the mating period (Boretto et al., 2014). They pitvipers, will likely show similar geographic variation in the added that the temporal asynchrony in steroid activity in P. timing and frequency of the mating season.’’ We agree with punae suggests that this mechanism must be important to this statement and contend that two components of the initiate spermatogenesis in spring, controlled by the steroid reproductive cycle, hypertrophy of the SSK and long-term activity of Sertoli cells, as Leydig cells are inactive. sperm storage, which are maintained by androgens (Take- Melo et al. (2019) studied the oviparous tropidurid lizard waki and Hatta, 1941; Bishop, 1959), enable males to mate Eurolophosaurus nanuzae (~198S) in southern Brazil. Their when they encounter receptive females. data suggest that spermatogenesis and SSK development may Graham et al. (2008) reported that the SSK in cotton- be controlled differently. They reported that testicular mouths was hypertrophied throughout the active season, activity was continuous throughout the year, while the SSK with SSK peak diameter coinciding with maximum sper- activity varied seasonally, with maximum hypertrophy matogenesis. They also reported the testosterone levels occurring during the mating season (Melo et al., 2019). peaked during the summer coinciding with peak spermato- Whether the differences in spermatogenesis and develop- genesis and SSK diameter. Following the summer mating ment of the SSK are due to different androgen thresholds or season, testosterone levels dropped to basal levels and

different androgen sources requires additional research. remained low throughout the spring (Graham et al., 2008). Based on these studies, we suggest that the HPGA may be We suggest that, using the SSK as a bioassay of androgen similar in all squamates, but with one significant difference. stimulation, the hypertrophy of the SSK in the spring in the In male lizards, both HPGA appear to be mostly inactive cottonmouth indicates that basal plasma androgens levels between reproductive events (i.e., the SSK tubules resemble may be sufficient to maintain development of the SSK and the uriniferous tubules) and sperm is lost in the efferent sperm storage throughout the year. ducts. In snakes, the SSK may vary in size and secretory The source of androgens that stimulate SSK development activity; however, it is always hypertrophied (i.e., under and support sperm storage is not well understood. Krohmer androgen stimulation), and, in most species, sperm are stored (2004) reviewed the role of androgens in snakes. He noted continuously in the ductus deferens. that adult male gartersnakes (Thamnophis sirtalis parietalis) The environmental cues that regulate the HPGA appear to continue to exhibit courtship and mating behavior for be easily modified evolutionarily, in both males and females. several years following castration (if exposed to an extended Van Dyke (2015) suggested that females with associated period of low temperature dormancy). He added that cycles (prenuptial) may respond differently to cues than castrated males also continue to have measurable concentra- females with dissociated (postnuptial) cycles. We suggest that tions of plasma testosterone. Krohmer (1985) suggested that in most lizards, the cues that males and females use are androgens of either a neural or adrenal source may be an generally different because, in general, males begin sper- important source. He observed that, in water snakes (Nerodia matogenesis weeks to months prior to the onset of spp.), the adrenal glands of castrated males increased in mass vitellogenesis (i.e., activation of the HPGA). (~57%) during the active season. He proposed that following castration, the reduction in circulating testosterone increases Mating season in species with postnuptial spermatogenesis.—In gonadotropin-releasing, follicle-stimulating, and luteinizing this paper, we used mating data for the entire species to hormones, and that these stimulate the adrenals to increase determine spermatogenic patterns. We realize that there may the production of androgens (Krohmer, 1985). Clearly, more be populations within a species with different mating studies on the source of androgens during the reproductive seasons. In species with postnuptial spermatogenesis, there cycle of snakes are needed. are three observed mating strategies: mating in the summer/ autumn only, mating in the spring only, and mating in the Phylogenetic comparative analyses.—In the absence of state- summer/autumn and spring. Aldridge and Duvall (2002) dependent reconstructions (see Maddison et al., 2007), we suggested that males should limit the time they are recovered traditionally understood character histories for physiologically prepared to mate because of the potential parity mode, including an ancestral state of oviparity and costs associated with this behavior. Some potential costs multiple transitions to viviparity in lizards and snakes (see include: energy spent in searching for receptive females, King and Lee, 2015). However, we still recovered moderate reduced feeding during the mating season, increased expo- support for an early origin of viviparity in colubroid snakes sure to predators, increased male–male combat and potential with a subsequent reversal to oviparity, as well as reversals Aldridge et al.—Spermatogenesis and mating in squamates 251 from oviparity to viviparity in anguid (Elgaria) and phryno- oviparous temperate lineages to viviparous temperate line- somatid (Sceloporus) lizards. These are corroborated by recent ages, offering at least partial confirmation to the hypothesis estimates using state-dependent models (Pyron and Bur- that colder climates promote the adaptation toward vivipar- brink, 2014; Wright et al., 2015; Harrington and Reeder, ity. We hypothesize that a similar mechanism affects sperm 2017), reinforcing the potentially complex history of production and mating season and is, perhaps, related to squamate reproductive modes. As noted by Griffith et al. parity-mode transitions. (2015), to fully understand the complex evolution of parity Within snakes, there is no significant relationship between mode, a coding scheme that takes into account detailed parity mode and the evolution of postnuptial spermatogen- morphology/physiology will need to be undertaken in the esis, considering continuous and prenuptial spermatogenesis future. together as a single state. In contrast, within snakes, there is a The ancestral history of climatic preference is similar to strong correlation between climatic origin and mating other recent reconstructions (e.g., Pyron, 2014), but consid- strategy (Fig. 3). Note that this test required the input data erably more uncertain given the number of transitions to contain all combinations of states, yet no snake in our involved and the complex biogeographic history of the dataset exhibited the state 0, 1. Thus, we randomly changed group. Overall, we find moderate support for Temperate Zone one species with the state 1, 1 to 0, 1. The highest rates not origins early in Squamata, with multiple, more recent involving the artifactual 0, 1 state represent transitions from transitions to tropical environments (Supplementary Fig. 1; temperate prenuptial/continuous to temperate postnuptial. see Data Accessibility). However, changes in both directions This strongly supports both the hypothesis that movement are relatively frequent, both within and among clades. We into tropical areas is associated with a shift to continuous find moderate to strong support for an ancestrally temperate spermatogenesis across squamates, and that temperate state for snakes as a whole, and Colubroidea in particular environments select for postnuptial spermatogenesis in (Supplementary Fig. 1; see Data Accessibility). snakes (Fig. 3). Our results for mating strategy confirm previous specula- In lizards, as in snakes, there is no significant correlation tion (see above) about the evolutionary history of spermato- between parity mode and mating strategy, suggesting that genesis in relation to mating (i.e., prenuptial, postnuptial, transitions from the ancestral state of prenuptial spermato- and continuous spermatogenesis), but these results offer a genesis to the derived state of continuous sperm production new evolutionary sequence in snakes. As noted, all lizards in males are not linked with shifts to viviparity in females. As exhibit either prenuptial (most species) or continuous in snakes, there is a very strong evolutionary correlation in spermatogenesis (some tropical lineages), and prenuptial lizards between climatic origin and mating strategy (Fig. 3). spermatogenesis is strongly supported as the ancestral state The highest rates of transition are from temperate continu- for Squamata (Supplementary Fig. 2; see Data Accessibility). ous to tropical continuous, and then from tropical contin- Numerous transitions to continuous spermatogenesis occur uous to tropical prenuptial. This again confirms strong through time (both ancient and recent) in a variety of lizard support for the hypothesis that tropical conditions promote families, seemingly related to occurrence in tropical environ- the adoption of continuous spermatogenesis in both lizards ments (see below). A single transition to postnuptial and snakes (Fig. 3). spermatogenesis is recovered early in snakes, possibly as For the threshold model, we again evaluated lizards and early as the most recent common ancestor of Serpentes (Supplementary Fig. 2; see Data Accessibility). The earliest snakes separately, with mating strategy coded as a binary nodes with strong support for postnuptial spermatogenesis as variable as described above. For lizards, there is a significant the ancestral state are the pitvipers (Crotalidae) and Colu- positive correlation between mean annual temperature broidea excluding Viperidae, Crotalidae, and Homalopsidae experienced by a species and the probability that the species (Supplementary Fig. 2; see Data Accessibility). exhibits continuous spermatogenesis (r ¼ 0.51;Fig.4). Within Colubroidea, excluding Viperidae, Crotalidae, and Though there is some variance across the phylogeny, a Homalopsidae, there are multiple reversions to prenuptial majority of species with mean annual temperature greater spermatogenesis in both temperate and tropical species (Fig. than 208C exhibit continuous spermatogenesis, reinforcing 3; Supplementary Figs. 1, 2; see Data Accessibility). There are the hypothesis that adaptations to tropical climates promote also transitions to continuous spermatogenesis in several the adoption of a continuous mating strategy (Fig. 4). tropical lineages, most notably Central and South American Within snakes, an essentially identical pattern is found in Dipsadidae(Fig.3;SupplementaryFigs.1,2;seeData comparison to lizards. There is a strong negative correlation Accessibility). These results support the idea that postnuptial between mean annual temperature experienced by a species spermatogenesis was an early-evolving trait for snakes that and the probability that the species exhibits continuous significantly influenced the trajectory of their mating spermatogenesis (r ¼ –0.58) and a transition to postnuptial strategies, in a different manner than for lizards. However, spermatogenesis (Fig. 4). As in lizards, nearly all snakes with both groups still appear to exhibit a preferential shift to mean annual temperatures greater than 208C exhibit pre- continuous spermatogenesis in lineages that are exclusively nuptial or continuous spermatogenesis (Fig. 4). Nearly all tropical, suggesting that warmer climates release the con- species with temperatures below 208C exhibit the snake- straint of seasonal spermatogenesis and the need for long- specific postnuptial condition (Fig. 4). An unusual exception term sperm storage in the ductus deferens (Fig. 3). We test to this pattern is the Old World viperid genus Vipera, for this hypothesis further in the remaining sections. which all six species have prenuptial spermatogenesis. These As suggested by numerous previous studies (e.g., Tinkle species are highly temperate, including V. berus (discussed and Gibbons, 1977; Pyron and Burbrink, 2014), there is a above) with the lowest recorded mean annual temperature in significant correlation between parity mode and climatic our dataset of 2.18C. Thus, there may be an additional origin across squamates. The highest rate of change is from evolutionary mechanism promoting prenuptial spermato- 252 Copeia 108, No. 2, 2020

Fig. 3. Plots of climate (left) and mating strategy (right), showing summary from 100 stochastically mapped character histories (pie charts at nodes). Phylogeny from Tonini et al. (2016) with branches colored by a representative history from each of the distributions. Insets show the significant Pagel binary-correlation models between climate and mating strategy for lizards and snakes, illustrating the transition rates between states scaled to the maximum rate across both clades. genesis in highly temperate clades for which breeding temperature has a strong effect on this transition with most seasons are very short and highly periodized. lizard and snake species experiencing mean annual temper- Taken as a whole, our results indicate that prenuptial atures greater than 208C adopting continuous sperm produc- spermatogenesis is the ancestral state of Squamata with tion. Further research is now needed to pinpoint the multiple transitions to continuous spermatogenesis in physiological mechanisms underlying these changes. tropical lizards. Snakes are characterized by a single likely Numerous, and rather randomly appearing, transitions in origin of postnuptial spermatogenesis early in their history, both temperate and tropical snake lineages from postnuptial and from which more tropical lineages have transitioned to spermatogenesis back to the ancestral condition of prenup- continuous sperm production, while other temperate and tial spermatogenesis suggest that additional evolutionary tropical lineages have reverted to the ancestral prenuptial mechanisms may be acting in snakes. These may be state. Evolutionary correlations reveal that mean annual potentially related to more xeric conditions for high- Aldridge et al.—Spermatogenesis and mating in squamates 253

Fig. 4. Phenograms of lizards (top) and snakes (bottom), showing time-scaled phylogeny from Tonini et al. (2016) with tip height scaled to mean annual temperature for each species’ range. The insets show the distribution of estimated correlation between temperature and mating strategy for each clade using the threshold model of Felsenstein et al. (2012). For both groups, the horizontal dashed line at 208C shows the approximate point at which mating strategies shift, with the majority of lizards with continuous and snakes with continuous or prenuptial spermatogenesis associated with higher temperatures. Of note is the clade of viperine vipers (Vipera) associated with lower temperatures (~2–158C) that nonetheless exhibit prenuptial spermatogenesis. 254 Copeia 108, No. 2, 2020

Table 5. Possible scenario for evolution of Serpentes. evolution of this taxon, and this hypothesis was supported 1) The snake clade evolved within the lizard clade (Wiens et al., by our phylogenetic analyses. The lack of definitive post- 2012). nuptial spermatogenic cycles in all, or almost all, lizards 2) The earliest snakes were initially burrowers (Wiens et al., suggests that the conversion from pre- to postnuptial 2012). spermatogenic cycles (i.e., the development of long-term 3) Burrowing forced a change from heliothermy, common in sperm storage in males) may be a rare development in lizards, to thigmothermy, common in primitive snakes (Cooper, squamate evolution, another hypothesis supported by our 1994). phylogenetic analyses. 4) Burrowing reduced the importance of the visual systems and enhanced the vomerolfactory system (Cooper, 1994). This DATA ACCESSIBILITY switch may have contributed to the reliance on pheromones Supplemental material is available at https://www. for sexual recognition. copeiajournal.org/ch-19-230. 5) Snakes may have inherited tropotaxis from ancestral varanid lizards (Auffenberg, 1994; Cooper, 1994), and used the vomeronasal organs to determine the direction of pheromones ACKNOWLEDGMENTS of reproductive females (Mason, 1992; Parker and Mason, We thank Shai Meiri and Anat Feldman (TAU) for access to 2011). previously unpublished temperature data generated from the 6) Burrowing may have contributed to the lack of territoriality and, Roll et al. (2017) range maps. We thank Stan Trauth for consequently, increased the unpredictability of sexual comments on an earlier draft of this manuscript. We also encounters (Devine, 1984). Less predictability of sexual recognize our institutions of employment for continued encounters may have selected for the ability to store sperm support of our research. over long periods of time in the ductus deferens in males. 7) Long-term sperm storage permitted the timing of the mating LITERATURE CITED season to evolve independently from the timing of spermatogenesis and vitellogenesis (Whittier and Tokarz, Abts, M. L. 1988. Reproduction in the saxicolous desert 1992). lizard, Sauromalus obesus: the female reproductive cycle. 8) Prenuptial spermatogenesis may have secondarily evolved in at Copeia 1988:382–393. least five families of temperate snakes (Table 2). Aldridge, R. D. 1979. Seasonal spermatogenesis in sympatric Crotalus viridis and Arizona elegans in New Mexico. Journal of Herpetology 13:187–192. temperature arid- or desert-adapted lineages such as Chilo- Aldridge, R. D. 1993. 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Herpe- 2014), the adaptive strategies of males and females appear to tological Monographs 16:1–25. differ in response to temperature; i.e., our results do not Aldridge, R. D., W. P. Flanagan, and J. T. Swarthout. 1995. support a strong correlation between mating strategy and The reproductive biology of the diamondback water snake parity mode. (Nerodia rhombifer) from Veracruz, Mexico, with compari- sons of tropical and temperate snakes. Herpetologica 51: Conclusions: Possible scenario for evolution of snake reproduc- 182–192. tive cycles.—Snakes and lizards differ in several major Aldridge, R. D., S. R. Goldberg, S. S. Wisniewski, A. P. respects. Many snakes (primarily those with postnuptial Bufalino, and C. B. Dillman. 2009. The reproductive cycle spermatogenesis) have the ability to store sperm throughout and estrus in the colubrid snakes of temperate North the year; lizards do not. Snakes store sperm primarily in the America. 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