Journal of Herpetology, Vol. 48, No. 2, 162–171, 2014Copyright 2014 Society for the Study of Amphibians and Reptiles

Reproductive Biology of Sceloporus consobrinus (Phrynosomatidae): Male Germ CellDevelopment and Reproductive Cycle Comparisons within Spiny Lizards

JUSTIN L. RHEUBERT,1,2 KATHERINE TOUZINSKY,3 DAVID M. SEVER,4 ROBERT D. ALDRIDGE,1 ANTHONY J. WILMES,1 DUSTIN S. SIEGEL,5

AND KEVIN M. GRIBBINS3

1Department of Biology, Saint Louis University, 3507 Laclede Avenue, St. Louis, Missouri 63103 USA3Department of Biology, Wittenberg University, PO Box 720, Springfield, Ohio 45504 USA

4Department of Biological Sciences, Southeastern Louisiana University, SLU 10736 Hammond, Louisiana 70402 USA5Department of Biology, Southeast Missouri State University, Cape Girardeau, Missouri 63701 USA

ABSTRACT.—Reproductive cycles of lizards have long been studied in both field and laboratory scenarios. However, comparisons of

spermatogenic cycles and germ cell development strategies in different populations across a large geographic range have yet to be explored. The

purpose of this study is to A) describe the spermatogenic cycle and germ cell development strategy of a population of Sceloporus consobrinus insoutheast Louisiana, B) compare this cycle to a more northern population of this species, and C) compare the reproductive cycles of species

within Sceloporus (N = 21). In S. consobrinus from Louisiana, recrudescence begins in the fall (October and November), and the peak of

spermatogenesis is reached the following spring/summer (May, June, July). This spermatogenic cycle is similar to that of a more northernpopulation of S. consobrinus from Missouri. Within the genus Sceloporus, there are two seasonal patterns of spermatogenesis: initiation of

spermatogenesis in the summer/fall and initiation of spermatogenesis in the spring. In both summer/fall and spring spermatogenic patterns,

spermiogenesis occurs in the spring and may continue into the summer. The seasonal timing of recrudescence is an extremely plastic trait that

has evolved multiple times throughout the Sceloporus clade. However, there appears to be an association of summer/fall and springrecrudescence with latitude. Tropical populations have a higher frequency of spring recrudescence and temperate populations have a higher

frequency of summer/fall recrudescence.

Gribbins and Gist (2003) described a novel germ cell

development strategy exhibited during spermatogeneis in

reptiles (squamates, testudines, crocodilians). Although testicu-

lar architecture in reptiles resembles that of other amniotes

(Leblond and Clermont, 1952; Russell et al., 1990; Kumar, 1995),

germ cells mature and migrate toward centralized lumina

within seminiferous tubules as large cohorts similar to that

found in anamniotes (but lacking the cyst/lobule testicular

organization of anamniotes; Lofts, 1964). This ‘‘atypical’’ germ

cell development strategy for an amniotic testis was termed

‘‘temporal,’’ because spermatogenic events are separated tem-

porally (for review, see Gribbins, 2011). In the ‘‘typical’’ type of

germ cell development exhibited by mammals and birds, the

seminiferous tubules can be divided along their peripheral axis

into sections, which have specific germ cell generations

observed together in consistent sequential cellular associations.

The number of stages present is also species specific. This spatial

organization allows germ cells to be released into the lumen in

waves along the length of each seminiferous tubule (Russell et

al., 1990). Consequently, this has been termed the spatial germ

cell development strategy (for review, see Gribbins, 2011).

Recent work on a variety of reptiles (Squamata, Gribbins et

al., 2008; Rheubert et al., 2009a; Testudines, Gribbins et al., 2003;

Crocodilia, Gribbins et al., 2006; Wang et al., 2008) suggests that

the temporal germ cell development strategy is common among

reptiles. Rheubert et al. (2009b) have proposed two hypotheses

for the evolution of the germ cell development strategy in

reptiles: 1) retention of the anamniote germ cell development

strategy in basal amniotes and modern reptiles and convergence

of the spatial development in birds and mammals; or 2) spatial

development evolved in early amniotes with reversals in

crocodilians, lepidosaurians, and chelonians. The former hy-

pothesis leads to a more parsimonious solution when optimized

to the evolutionary history of vertebrates (Rheubert et al.,

2009b).

The temporal germ cell development strategy leads to

discrete episodes of spermatogenesis in reptiles. Previous

research has shown that lizards exhibit prenuptial spermato-

genesis (sperm are produced immediately prior to the mating

season). However, it has been noted that there are variations in

lizard spermatogenic cycles (Tinkle, 1969; Adolph and Porter,

1993; Villagran-Santa Cruz et al., 2009). In some species,

recrudescence (the onset of mitotic divisions of germ cells)

begins in the summer/fall, whereas in other species, recrudes-

cence begins in the spring (Licht, 1972). However, the starting

time of recrudescence appears to be fixed within a species

independent of elevation and latitude (Table 1). This was

investigated most thoroughly by Angelini and Picariello (1975)

who developed the terms thermorigostatic and thermopsicror-

igostatic to provide more descriptive terminology explaining

the variation observed in reproductive strategies. Also, they

showed a relationship between spermatogenic cycle and

effective temperature, a measure of temperateness developed

by Bailey (1960).

Tinkle (1969) and Tinkle et al. (1970) concluded that natural

histories evolve in direct correlation to reproductive effort

(using clutch size as a measurement of reproductive effort).

Tinkle (1969) alluded to the idea that early maturing species that

have multiple broods and have small clutch sizes may have an

extended breeding period and that those species typically occur

in tropical climates. Although temperature apparently plays a

major role in the spermatogenic cycle, few studies have

compared reproductive cycles from various geographic regions

in lizard models. Three distinct reproductive strategies have

been described within the genus Sceloporus: 1) spring spermato-

genesis; 2) summer/fall spermatogenesis; and 3) continuous

spermatogenesis. In all of these cycles, spermiogenesis occurs

immediately prior to mating; thus, all three cycles are prenuptial

by definition.

2Corresponding Author. E-mail: justin.rheubert00@gmail.comDOI: 10.1670/12-156

Sceloporus consobrinus (Leache and Reeder, 2002) from

Missouri exhibits fall spermatogenesis with recrudescence

beginning in September and testicular regression beginning in

June and July (Matter, 1987). McKinney and Marion (1985)

found that there was variation in testicular cycles within the

geographic range of what was formerly Sceloporus undulatus but

has since been divided into S. consobrinus (Missouri) and S.

undulatus (Alabama) (sensu Leache and Reeder, 2002; Leache,

2009). McKinney and Marion (1985) illustrated that testicular

recrudescence occurred at similar times, but regression began

earlier in the Alabama population (May to July) than in the

more northern Missouri population (June and July).

Some species of spiny lizards, such as Sceloporus occidentalis

(Goldberg, 1974), start recrudescence in the fall months prior to

winter hibernation and spermatogenesis is arrested until

spermiogenesis begins in the spring. This reproductive cycle is

what Saint Girons (1982) referred to as mixed spermatogenesis.

Others, such as Sceloporus horridus, exhibit a spring recrudes-

cence with spermiogenesis beginning immediately after prolif-

eration and meiosis (Valdez-Gonzalez and Ramırez-Bautista,

2002). Continuous spermatogenesis has only been observed in

the low-latitude, high-elevation, viviparous, Sceloporus bicantha-

lis (Hernandez-Gallegos et al., 2002; Gribbins et al., 2011) and

the low-latitude, low-elevation, oviparous, Sceloporus variabilis

(Ramırez-Bautista et al., 2006a).

In the current study, we examined the reproductive cycles of

male lizards from the same species across a latitudinal gradient.

The purpose of this study is threefold: 1) to investigate the

spermatogenic cycle of a southern population of S. consobrinuswhile examining the germ cell development strategy in

comparison with other vertebrate models; 2) to test the

hypothesis that spermatogenic cycles of S. consobrinus vary

across a latitudinal gradient; and 3) to examine spermatogenic

cycles of lizards within the Sceloporus genus within a phyloge-

netic context.

MATERIALS AND METHODS

Adult male S. s. consobrinus were collected monthly insoutheastern Louisiana from Sandy Hollow Nature Reserve(30849.100N, 90824.450W) near Amite, Tangipahoa Parish fromAugust to November 2008 and March to August 2009. Threecollection attempts per month were made for S. consobrinusbetween December 2008 and February 2009, but no individualswere observed. Specimens were euthanized with a 0.2-ccintracoelomic injection of sodium pentobarbital within 24 h ofcapture and reproductive tracts (testis, testicular ducts, kidneys,cloacae) were removed and preserved in Trump’s fixative (EMS,Hatfield, PA) as approved by the Institute of Animal Care andUse Committee and Southeastern Louisiana University whereeuthanizations and dissections were performed.

Testicular tissues were allowed to fix for at least 24 h thendehydrated with an increasing concentration of ethanolsolutions. Tissues were then infiltrated with a 1 : 2 solutionof Spurr’s plastic (EMS, Hatfield, PA): 100% ethanol solution, a1 : 1 solution of Spurr’s plastic: 100% ethanol solution, andthen with pure Spurr’s plastic for 12 h. Fresh plastic was thenprepared, and tissues were embedded in small beam capsulesand allowed to cure for 48 h in a Fisher IsotemperatureVacuum Oven (Fisher Scientific, Pittsburgh, PA). Blocks weresectioned at 3lm using an LKB-Ultramicrotome III (LKBProdukter AB, Bromma, Sweden). Tissues were then stainedwith a toluidine blue/basic fuschin composite stain for generalcytology (Hayat, 1993).

Slides were examined using a Zeiss compound lightmicroscope (Carl Zeiss Microimaging, Inc., Thornwood, NY)to determine germ cell morphologies and spermatogenic stages.Photographs were taken with an attached SPOT digital cameraand 5.0 software (Diagnostic System Laboratories, Webster, TX).Composite micrographs were constructed and analyzed usingAdobe Photoshop CS5 (Adobe Systems, San Jose, CA).

Reproductive and geographic data on Sceloporus species wereobtained from the literature and included: spermatogenic mode

TABLE 1. Reproductive data for Sceloporus. Data obtained from other sources is marked with an asterisk (*). Parity: V = viviparous. O = oviparous;Clutches: S = single, M = multiple clutches per year, na = information not available; Latitude = 8N; Elevation in meters.

Species Parity mode* Clutches* Recrudescence Latitude Elevation Reference

S. bicanthalis V na Continuous 19 3,200 Hernandez-Gallegos et al. (2002)S. gadoviae O S Spring 17 600 Lemos-Espinal et al. (1999)S. graciosus O na Fall 40 1,300* Woodbury and Woodbury (1945)S. grammicus V na Spring 19 2,294 Jimenez-Cruz et al. (2005)S. horridus O M Spring 18 1,500 Valdez-Gonzalez and Ramırez-Bautista (2002)S. jarrovi V na Spring 31 6,500 Goldberg (1971)S. magister O M* Spring 35 150* Vitt and Ohmart (1974)S. malachiticus V S Spring 9 800–3,200 Marion and Sexton (1971)S. melanorhinus O S Spring 19 10–584 Ramırez-Bautista et al. (2006b)S. mucronatus V S Spring 19 3,200–3,400 Mendez de la Cruz et al. (1988)S. occidentalis O S Fall 34 1,584 Goldberg (1974)

Fall 37 180* Wilhoft and Quay (1961)S. orcutti O S na 33 244* Mayhew (1963)S. poinsetti V S Fall 31 na Ballinger (1973)S. pyrocephalus O M Spring 18 550 Ramırez-Bautista et al. (2002)S. scalaris O S Fall 31 2,600 Newlin (1976)S. spinosus O M* Fall 19 500 Ramırez-Bautista et al. (2012)

M* Fall 19 537–2,254 Valdez-Gonzalez and Ramırez-Bautista (2002)S. torquatus V na Spring 19 2,600 Guillette and Cruz (1993)S. undulatus O M Fall 33 300* McKinney and Marion (1985)

Fall 36 120* Atland (1941)S. utiformes O na Spring 19 600 Ramırez-Bautista and Gutierrez-Mayen (2003)S. variabilis O na Fall 19 1,000 Benabib (1994)

Fall 18–19 3–50 Ramırez-Bautista et al. (2006a)S. woodi O M na 29 na Jackson and Telford (1974)

SPERMATOGENESIS IN SCELOPORUS 163

(fall recrudescence, spring recrudescence, continuous), paritymode, latitude, and elevation. Only reproductive studies thatincluded an entire annual cycle or could state definitively theonset of recrudescence and regression were used. Reproductivecycles were divided into fall, spring, and continuous cycles. Fallreproductive cycles show evidence of recrudescence during thefall months and exhibit a short 1–2-month hiatus followingtesticular regression, spring reproductive cycles show evidenceof recrudescence during the spring months with typically a 4–6-month hiatus following testicular regression, and continuousreproductive cycles show no evidence of testicular regressionthroughout the year. Data gathered from the literature werecoded based on latitude, parity mode, and spermatogenic cycle.For phylogenetic analyses, the region inhabited was dividedinto temperate and tropical regions based on 23.58 N latitude asa dividing line. Data were optimized onto two pre-existingphylogenetic hypotheses proposed by Wiens and Reeder (1997)and Leache (2010) using a strict parsimony optimization inMesquite version 2.75 (Maddison and Maddison, 2011). Thesetwo phylogenetic hypotheses were left unrooted because ofconflicting results posed by out-group determination.

RESULTS

Testicular Structure and Germ Cell Morphology.—The testes aresuspended by the mesorchium within the pleuroperitonealcoelom posterio-dorsal to the liver. Deep to the visceralpleuroperitoneum, the testes are covered by the tunicaalbuginea (Fig. 1, Ta). Seminiferous tubules (Fig 1, St) liejuxtapositioned to one another and are lined with a seminifer-ous epithelium, containing developing germ cells and Sertolicells, surrounding a centralized lumen.

Premitotic cells, spermatogonia, lie within the basal compart-ment of the seminiferous epithelium. Spermatogonia A (Fig. 2,SpA) contain dispersed chromatin and are ovoid in shape.Spermatogonia A divide mitotically to give rise to spermatogo-nia B (Fig. 2, SpB), which also contain dispersed chromatin butusually show a more round morphology. Both spermatogonialtypes are present within the seminiferous epithelium of allmonths sampled during this study. Spermatogonia B dividemitotically and decrease in size as they transform into pre-leptotene spermatocytes (Prophase I of meiosis; Fig. 2, PL).

These spermatocytes have well-defined nuclear membranesand prominent nuclei. Also, they are the smallest of the dividinggerm cells. During the first prophase the chromatin becomesmore filamentous and darker staining in leptotene spermato-cytes (Fig. 2, LP). The nucleus begins to increase in size, and

cytoplasmic volume decreases as meiotic cells progress duringdevelopment. Chromatin fibers thicken further in zygotene cells(Fig. 2, ZY) and as the chromosomes condense, larger areas ofnucleoplasm form in pachytene spermatocytes (Fig. 2, PA). Thechromatin fibers finish condensation and reside just below thenuclear membrane in diplotene cells (Fig. 2, DI), and the nuclearmembrane begins to degenerate. The fully condensed chromo-somes line up at the metaphasal plate in metaphase I of meiosis(Fig. 2, M1). After completion of the first nuclear and cytokinesisevents, the secondary spermatocytes (Fig. 2, SS) are muchsmaller in cell size than the preceding primary spermatocytes.The second meiotic division (Fig. 2, M2) follows the samegeneral patterns as meiosis I with the chromosomal amount andcell size halved when compared to primary spermatocytes.

After completion of meiosis II the germ cells begin thestepwise differentiation into mature spermatozoa that can bebroken into seven steps based on the terminology of Russell etal. (1990) for mammalian species. Step 1 spermatids (Fig. 2, S1)mark the beginning of spermiogenesis with a well-definednucleus and the presence of an acrosomal vesicle. Step 2spermatids (Fig. 2, S2) have acrosomal vesicles that makecontact with the nuclei causing a slight indentation in the apexof each nucleus. During the Step 3 spermatid stage (Fig. 2, S3),the acrosomal granule becomes visible within the acrosomalvesicle near the nuclear membrane. The acrosomal vesicle isfully developed in Step 4 spermatids (Fig. 2, S4), and the germcell begins elongation. Elongation continues as the acrosomalvesicle flattens the apex of the nucleus in Step 5 spermatids (Fig.2, S5). During this time, the chromatin condenses, giving thenucleus a darker staining appearance. In Step 6 spermatids (Fig.2, S6), the nucleus is almost homogenous and the flagellum isclearly visible. The nucleus is fully elongated and homogenousin Step 7 spermatids (Fig. 2, S7), giving the spermatid its filiformshape just prior to shedding into the lumen as a maturespermatozoon (Fig. 2, MS).

FIG. 1. Cross-section of the testis in Sceloporus consobrinus showingthe seminiferous tubules (St) and surrounding tunica albuginea (Ta).Scale = 100 lm.

FIG. 2. Germ cell types found within the seminiferous epithelium ofSceloporus consobrinus. Spermatogonia A (SpA), Spermatogonia B (SpB),Pre-leptotene (PL), Leptotene (LP), Zygotene (ZY), Pachytene (PA),Diplotene (DI), Meiosis I (M1), Secondary spermatocyte (SS), Meiosis II(M2), Step 1 spermatid (S1), Step 2 spermatid (S2), Step 3 spermatid (S3),Step 4 spermatid (S4), Step 5 spermatid (S5), Step 6 spermatid (S6), Step7 spermatid (S7), mature spermatozoon (MS). Scale = 10 lm.

164 J. L. RHEUBERT ET AL.

Sceloporus consobrinus Germ Cell Cycle.—The testes of S.consobrinus are spermatogenically active during March. Sper-matogonial cells (Fig. 3A, Sp) are observed in close proximity tothe basal lamina (Fig. 3A, black arrowhead). Meiotic cells (Fig.3A, Me) and spermiogenic cells (Fig. 3A, Ss) are seen within theseminiferous epithelium. During April, spermatogonial cells(Fig. 3B, Sp) line the basement membrane and meiotic cells (Fig.3B, Me), and spermiogenic cells (Fig. 3B, Ss) are found withinthe germinal epithelium. In May, spermatogonial cells (Fig. 3C,Sp) again are found juxtapositioned to the basement membrane(Fig. 3C, black arrowhead). During this time, meiotic cells (Fig.3C, Me) make up the majority of the seminiferous epitheliumalthough a few spermiogenic cells (Fig. 3C, Ss) are observed.Mature sperm (Fig. 3C, Ms) are found in the May seminiferouslumina from April spermatids, which have completed spermio-genesis and have been spermiated. June is characterized by anincrease in spermiogenic cells (Fig. 3D, Ss) and a considerabledecrease in the number of meiotic cells (Fig. 3D, Me). Again,spermatogonial cells (Fig. 3D, Sp) line the basal lamina (Fig. 3D,black arrowhead). June is marked by increases in elongatingspermatids and by large amounts of mature spermatozoa (Fig.3D, Ms) within the lumen. The numbers of meiotic cells (Fig. 4A,Me) and spermiogenic cells (Fig. 4A, Ss) have decreased by July.Although mature spermatozoa (Fig. 4A, Ms) are still found

within the lumen, the amount seems to have decreasedsubstantially (though no statistical analyses were performed).During August (Fig. 4B) and September (Fig. 4C), spermatogo-nial cells (Fig. 4B,C, Sp) and a few remnant deteriorating germcells are present within the germinal epithelium, and the lumenis completely absent or has decreased in size. In September, thenumber of spermatogonial cells filling the epithelium hasincreased indicating testicular recrudescence. By October, themajority of germ cells have entered the meiotic phase (Fig. 4D,Me); however, no mature spermatozoa are observed within thelumen. In November testes, individual variation of spermato-genic activity is observed between different specimens. Indi-viduals caught on the same day exhibit differing stages withinthe seminiferous epithelium with one individual containing theearly stages of meiotic cells (Fig. 5A, Me), one individualshowing the later stages of meiotic cells (Fig. 5B, Me), and oneindividual that has even begun spermiogenesis (Fig. 5C, Ss).

Seasonal Activity and Germ Cell Development Strategy.—Sper-matogenic recrudescence in S. consobrinus is marked by thebeginning of spermatogonial proliferation in September (Fig.6A, S. consobrinus). By November, spermiogenesis and spermi-ation were observed in one individual. Spermatogenesis beginsin fall and continues through midsummer of the following year.A short testicular quiescent period was observed in August.

FIG. 3. Cross-section of seminiferous tubules of Sceloporus consobrinus during A) March, B) April, C) May, and D) June. Germ cell types includespermatogonia (Sp), meiotic cells (Me), spermiogenic cells (Ss), and mature spermatozoa (Ms). Basal lamina (black arrowhead). Scale = 50 lm.

SPERMATOGENESIS IN SCELOPORUS 165

Although all germ cell types can be found within theseminiferous epithelium during the spermiogenically activemonths, a large prolific event is observed in the fall with theprimary spermatocytes transforming to spermiogenic cells inthe spring. Spermiogenesis is completed by June, and a largespermiation event is observed during June and July. This patternof testicular cycle represents a temporal release of germ cellswithin an amniotically structured testis.

Sceloporus Reproductive Cycles.—Variation can be observedwithin the reproductive cycles of the Sceloporus genus (Fig. 6A,Btop panel) with fall (Fig. 6A) and spring (Fig. 6B) cyclesdominating (continuous is only observed in one species, S.bicanthalis; Hernandez-Gallegos et al., 2002, and potentially apopulation of S. variabilis; Ramırez-Bautista et al., 2006a).However, factoring out individual variation (which was shownpreviously in this paper) and annual variation (James, 1991;Castilla et al., 1992) and accounting for arbitrary delineations formonths (i.e., difference between 31 August and 1 September),and giving a 6 1-month variance (Fig. 6A; B middle panel), nomonthly differences can be observed (Fig. 6A;B) within aspecies. However, three major trends appear within Sceloporusspermatogenic cycles: 1) fall spermatogenesis (Fig. 6A); 2)spring spermatogenesis (Fig. 6B); and 3) continuous spermato-

genesis (not shown). The two populations of S. consobrinus(Missouri and Louisiana) exhibit fall spermatogenesis furthersuggesting spermatogenic cycles are fixed within a species.

Optimization onto the phylogenetic hypothesis proposed byWiens and Reeder (1997) resolved the ancestral character statesfor Sceloporus as spring spermatogenic cycle, inhabiting tropicalareas, and oviparous. The same holds true for the Leache (2010)hypothesis except that spermatogenic cycle is unresolved. Thephylogenetic hypothesis proposed by Wiens and Reeder (1997)resulted in optimizations for spermatogenic mode, regioninhabited, and parity mode as more parsimonious with 7, 6,and 3 steps, respectively, compared to 7, 7, and 4 steps forLeache (2010). From these results, it is clear that spermatogeniccycle provides little information in terms of evolutionary historyof Sceloporus, because a fall spermatogenic cycle has evolvednumerous times independently within Sceloporus on both theWiens and Reeder (1997) and Leache (2010) topologies.

Several species of Sceloporus have migrated (immigrated orextended) into temperate regions from tropical regions. Fromtree to tree comparison (compare Fig. 7A or D to Fig. 7B or E,respectively), it appears as though the fall spermatogenic cycleis linked to species that have moved into temperate regions. Theonly exceptions are Sceloporus jarrovi (spring and temperate), S.

FIG. 4. Cross-section of seminiferous tubules of Sceloporus consobrinus during the months of A) July, B) August, C) September, and D) October.Germ cell types include spermatogonia (Sp), meiotic cells (Me), spermiogenic cells (Ss), and mature spermatozoa (Ms). Basal lamina (blackarrowhead). Scale = 50 lm.

166 J. L. RHEUBERT ET AL.

magister (spring and temperate), Sceloporus spinosus (fall andtropical), and S. variabilis (fall and tropical). However, nostatistical analyses were performed, and data represented on‘‘pruned’’ phylogenetic hypotheses should be interpreted withcaution.

Further examination using characterizing optimization ontophylogenies suggests that parity mode (oviparous to viviparouson the Leache, 2010 topology; many transitions unresolved onthe Wiens and Reeder, 1997 topology) evolved multiple timeswithin Sceloporus (Fig. 7) but does not appear to be correlatedwith spermatogenic cycle to any degree from tree to treecomparison (compare Fig. 7A or D to Fig. 7C or F, respectively)on either the Wiens and Reeder (1997) or Leache (2010)topologies. Again, no statistical analyses were performed, anddata represented on pruned phylogenetic hypotheses should beinterpreted with caution.

DISCUSSION

The testicular organization of S. consobrinus is consistent withreports on amniotic vertebrates (i.e., seminiferous tubules linedwith a germinal epithelium in which germ cells develop).Spermatogenesis begins in September with proliferation of thespermatogonia. Spermiogenesis is first observed in Novemberalthough the majority of spermiogenesis and spermiationlargely occurs in June and July (spermiation is observed priorto this and sperm are found in the testicular ducts as early asMarch; JLR, unpubl. data). This model of germ cell develop-ment, in which germ cells develop in tandem with one anotherand are released in large episodic events, is consistent with whathas been observed in all reptiles studied to date and someprimates (Luetjens et al., 2005; Gribbins et al., 2008; Wang et al.,2008; Rheubert et al., 2009b). This method differs from thetypical mammalian model in which germ cells are arranged indevelopmental order along the peripheral axis of a seminiferoustubule (Russell et al., 1990).

Two predominate spermatogenic cycles can be observedwithin genus Sceloporus: a fall recrudescence or spring recru-descence (see Fig 6). Whether a spring or fall spermatogeniccycle is observed, all lizards exhibit prenuptial spermatogenesis(Aldridge et al., unpubl. data). Saint Girons (1982) used the term‘‘mixed’’ spermatogenesis to describe the fall spermatogeniccycle. However, a review of the literature and terminologysuggests this is a confusing and nondefinitive term. Angeliniand Picariello (1975) used the term thermorigostatic (spermato-genesis with stasis during the warmest and coldest months ofthe year), as a more accurate description. However, neither thisnor other terms proposed by Angelini and Picariello (1975) hasbeen adopted; therefore, we use the terms spring and fall todescribe recrudescence.

Sceloporus consobrinus from various latitudinal regions exhibitsimilar spermatogenic cycles. Although these cycles areindistinguishable from one another because of variation (seeFig. 6), data collected in southeastern Louisiana more closelyresembles the Missouri population examined by McKinney andMarion (1985) and Matter (1987) with regression starting later inthe year (July and August). This finding is interesting as Leache(2009) found evidence (nuclear DNA) that S. consobrinus isparaphyletic, and this population shares a more recent commonancestor with S. undulatus. However, mitochondrial DNAsupports S. consobrinus being monophyletic. Furthermore, thisdiffers from the findings of Villagran-Santa Cruz et al. (2009),who investigated populations of S. mucronatus at different

FIG. 5. Cross-section of seminiferous tubules showing individualvariation found during November in Sceloporus consobrinus. A)Individual only exhibiting spermatogonia (Sp) and early meiotic cells(Me). B) Individual exhibiting spermatogonia (Sp) and late meiotic cells(Me). C) Individual exhibiting spermatogonia (Sp), meiotic cells (Me),and spermiogenic cells (Ss). Basal lamina (black arrowhead). Scale = 50lm.

SPERMATOGENESIS IN SCELOPORUS 167

elevations and found that the timing of peak spermatogenesisdiffered (although recrudescence and quiescence did not). Thissuggests that correlations between spermatogenic variables andlatitude (or potentially climate or elevation) at small (i.e.,populational) scales vary and have implications for evolution-ary constraints. This evolutionary restriction may have dramaticeffects in terms of climate change as Sinervo et al. (2010) havepredicted.

Although previous data suggest that spermatogenic cycles areinfluenced by certain abiotic factors, such influence may nothold true in nature. Previous research has shown thatphotoperiod (Licht, 1972; Marion, 1982), rainfall (Saint Girons,1982), and temperature (Licht, 1966) can effect spermatogeniccycles in laboratory settings. However, populations inhabitingdifferent areas with different climatic influences exhibit similarspermatogenic cycles. Furthermore, our present findings sug-gest that species inhabiting similar geographic regions (even insympatry) exhibit differences in spermatogenic cycles. Our datasuggest that spermatogenic cycles are plastic and have evolvedmultiple times (Fig. 7) with no single driving force.

All Sceloporus examined in this study that exhibit fallrecrudescence are associated with higher latitudes, but this isnot the case for spring cycles (all spring cycles are not alwaysassociated with lower latitudes). It may be that all cyclesoriginated in the spring and then, for some species, evolved tofall cycles as more temperate regions were inhabited (e.g.,Sceloporus orcutti, S. jarrovi, and Sceloporus magister). Theevolution of extended spermatogenic cycles in more temperate

areas seems evident in S. bicanthalis (which exhibits continuousspermatogenesis), because this species inhabits a temperateregion within a tropical latitude (elevation 1,850–4,250 m).However, deeper investigations are needed to understand whyall high-elevation species have not evolved a longer spermato-genic cycle. Tinkle (1969) suggested that the tropics providelong periods favorable for reproduction. However, if this werethe case for spermatogenesis, why is the spermatogenic cycleextended in the temperate regions where conditions favorablefor reproduction are not extended? Smith and Hall (1974) statedthat cold-induced species have protracted reproductive cycles,which is clearly not the case with male spermatogenic cycles. Itmay be possible that spermatogenic cycles evolved to beginproliferation earlier such that mature spermatozoa are presentfor the first mating opportunities in the spring or that sexualselection pressures are more prominent in temperate species assuggested by Gribbins et al. (2011).

The notion that climate is the sole deciding factor in thetiming of spermatogenesis seems obsolete in light of new data.Historical researchers such as Volsøe (1944), Licht (1966), andAngelini and Picariello (1975) have published works onspermatogenic cycles alluding to climate being the majordriving force, and Licht (1972) went as far to say, ‘‘Of all theenvironmental factors, temperature appears to be the singlemost important and widespread.’’ However, these researcherslacked sample size and likely looked at extreme cases and foundcorrelations that were not tested rigorously. With the accumu-lation of data over the past 30 years, more in-depth investiga-

FIG. 6. Reproductive cycles in the Sceloporus genus. A) Fall spermatogenesis showing spermatogenic cycles (top), 6 1-month variation (middle)with gray bars showing a 6 1-month deviation, and spermatogenic cycle overlap (bottom) with red bars demonstrating areas of overlap between thereproductive cycles of the various species. B) Spring spermatogenesis showing spermatogenic cycles (top), 61-month variation (middle) with graybars showing a 6 1-month deviation, and spermatogenic cycle overlap (bottom) with red bars demonstrating areas of overlap between thereproductive cycles of the various species.

168 J. L. RHEUBERT ET AL.

FIG. 7. Character optimization of spermatogenic cycle, region inhabited, and parity mode across the molecular and morphological hypothesisproposed by Wiens and Reeder (1997) and the purely molecular hypothesis proposed by Leache (2010).

SPERMATOGENESIS IN SCELOPORUS 169

tions can be used to falsify the assumption of climate being themajor or only driving force for spermatogenesis. An in-depthanalysis into a single genus or species (e.g., S. consobrinus) thatoccupies a variety of climates (although similar in respect tosquamates as a whole) has shown that there are exceptions tothis eneralization.

It appears that, although an extreme change in a single abioticfactor can influence spermatogenesis, a suite of characterswithin the organism’s niche play a large role in the timing ofspermatogenesis, similar to what Shine and Berry (1978)suggested for the evolution of viviparity. Even though ‘‘outliers’’are observed showing exceptions to the generalizations, moredata on various reproductive strategies (including femalestrategies for which data are lacking) may lead to results thatcould potentially give researchers insights into how life-historystrategies are arranged and what mechanisms underlie varia-tion in reproductive strategies.

Acknowledgments.—The authors thank C. Murray, J. Lee, andJ. Babin for their assistance with collection of specimens. JLRexpresses gratitude to O. Hernandez-Gallegos for his input anddiscussions during the preparation of the manuscript. Collec-tions and tissue preparation also received support fromNational Science Foundation grant DEB-0809831 to DMS andcompetitive research grants from Wittenberg University.

LITERATURE CITED

ADOLPH, S. C., AND W. P. PORTER. 1993. Temperature, activity, and lizardlife histories. American Naturalist 142:273–295.

ANGELINI, F., AND P. PICARIELLO. 1975. The course of spermatogenesis inReptilia. Accademi di Scienze Gisiche E Matematiche 9:1–48.

ATLAND, P. D. 1941. Annual reproductive cycle of the male fence lizard.Journal of the Elisha Mitchell Scientific Society 57:73–83.

BAILEY, H. P. 1960. A method of determining the warmth andtemperateness of climate. Geografiska Annaler 42:1–16.

BALLINGER, R. E. 1973. Comparative demography of two viviparousiguanid lizards (Sceloporus jarrovi and Sceloporus poinsetti). Ecology54:269–283.

BENABIB, M. 1994. Reproduction and lipid utilization of tropicalpopulations of Sceloporus variabilis. Herpetological Monographs 8:160–180.

CASTILLA, A. M., L. J. BARBADILLO, AND D. BAUWENS. 1992. Annual variationin reproductive traits in the lizard Acanthodactylus erythrurus.Canadian Journal of Zoology 70:395–402.

GOLDBERG, S. R. 1971. Reproductive cycle of the ovoviviparous iguanidlizard Sceloporus jarrovi Cope. Herpetologica 27:123–131.

———. 1974. Reproduction in mountain and lowland populations of thelizard Sceloporus occidentalis. Copeia 1974:176–182.

GRIBBINS, K. M. 2011. Reptilian spermatogenesis: a histological andultrastructural perspective. Spermatogenesis 3:250–269.

GRIBBINS, K. M., AND D. H. GIST. 2003. Cytological evaluation ofspermatogenesis within the germinal epithelium of the maleEuropean wall lizard, Podarcis muralis. Journal of Morphology 258:296–306.

GRIBBINS, K. M., D. H. GIST, AND J. D. CONGDON. 2003. Cytologicalevaluation of spermatogenesis and organization of the germinalepithelium in the male slider turtle, Trachemys scripta. Journal ofMorphology 255:337–346.

GRIBBINS, K. M., R. M. ELSEY, AND D. H. GIST. 2006. Cytological evaluationof the germ cell development strategy within the testis of theAmerican alligator, Alligator mississippiensis. Acta Zoologica 87:59–69.

GRIBBINS, K. M., J. L. RHEUBERT, M. H. COLLIER, D. S. SIEGEL, AND D. M.SEVER. 2008. Histological analysis of spermatogenesis and the germcell development strategy within the testis of the male western

cottonmouth snake, Agkistrodon piscivorus leucostoma. Annals ofAnatomy–Anatomischer Anzeiger 190:461–476.

GRIBBINS, K., M. ANZALONE, M. COLLIER, G. GRANADOS-GONZALEZ, M.VILLAGRAN-SANTA CRUZ, AND O. HERNANDEZ-GALLEGOS. 2011. Temporalgerm cell development strategy during continuous spermatogenesiswithin the montane lizard, Sceloporus bicanthalis (Squamata; Phry-nosomatidae). Theriogenology 76:1090–1099.

GUILLETTE, L. J., JR., AND F. R. MENDEZ DE LA CRUZ. 1993. The reproductivecycle of the viviparous Mexican lizard Sceloporus torquatus. Journal ofHerpetology 27:168–174.

HAYAT, M. A. 1993. Stains and Cytochemical Methods. Plenum Press,New York.

HERNANDEZ-GALLEGOS, O., F. R. MENDEZ-DE LA CRUZ, M. VILLAGRAN-SANTA

CRUZ, AND R. M. ANDREWS. 2002. Continuous spermatogenesis in thelizard Sceloporus bicanthalis (Sauria: Phrynosomatidae) from highelevation habitat of central Mexico. Herpetologica 58:415–421.

JACKSON, J. F., AND S. R. TELFORD JR. 1974. Reproductive ecology of theFlorida scrub lizard, Sceloporus woodi. Copeia 1974:689–694.

JAMES, C. D. 1991. Annual variation in reproductive cycles of scincidlizards (Ctenotus) in Central Australia. Copeia 1991:744–760.

JIMENEZ-CRUZ, E., A. RAMIREZ-BAUTISTA, C. M. JONATHON, M. LIZANA-AVIA,and A. N.-M.D. OCA. 2005. Reproductive cycle of Sceloporusgrammicus (Squamata: Phrynosomatidae) from Teotihuacan, Mexico.Southwestern Naturalist 50:178–187.

KUMAR, M. 1995. Spermatogenesis in the house sparrow, Passerdomesticus: histological observations. Pavo 33:1–4.

LEACHE, A. D. 2009. Species tree discordance traces to phylogeographicclade boundaries in North American fence lizards (Sceloporus).Systematic Biology 58:547–559.

———. 2010. Species trees for spiny lizards (genus Sceloporus):identifying points of concordance and conflict between nuclear andmitochondrial data. Molecular Phylogenetics and Evolution 54:162–171.

LEACHE, A. D. AND T. W. REEDER. 2002. Systematics of the eastern fencelizard (Sceloporus undulatus): a comparison of parsimoney, likelihood,and Bayesian approaches. Systematic Biology 51:44–68.

LEBLOND, C. P., AND Y. CLERMONT. 1952. Spermiogenesis of rat, mouse,hamster, and guinea pig as revealed by the periodic acid-fuschinsulgurous acid technique. American Journal of Anatomy 90:167–215.

LEMOS-ESPINAL, J. A., G. R. SMITH, AND R. E. BALLINGER. 1999. Reproductionin Gadow’s spiny lizard, Sceloporus gadoviae (Phrynosomatidae),from arid tropical Mexico. Southwestern Naturalist 44:57–63.

LICHT, P. 1966. Reproduction in lizards: influence of temperature onphotoperiodism in testicular recrudescence. Science 154:1668–1670.

———. 1972. Environmental physiology of reptilian breeding cycles:role of temperature. General and Comparative Endocrinology 3,Suppl.:477–488.

LOFTS, B. 1964. Seasonal changes in the functional activity of theinterstitial and spermatogenetic tissues of the green frog, Ranaesculenta. General and Comparative Endocrinology 4:550–562.

LUETJENS, G. M., G. F. WEINBAUER, AND J. WISTUBA. 2005. Primatespermatogenesis: new insights into comparative testicular organisa-tion, spermatogenic efficiency and endocrine control. BiologicalReviews 80:475–488.

MADDISON, W. P., AND D. R. MADDISON. 2011. Mesquite: a modular systemfor evolutionary analysis. Version 2.75 [Internet]. Available from:http://mesquiteproject.org.

MARION, K. R. 1982. Reproductive cues for gonadal development intemperate reptiles: temperature and photoperiod effects on thetesticular cycle of the lizard Sceloporus undulatus. Herpetologica 38:26–39.

MARION, K. R., AND O. J. SEXTON. 1971. The reproductive cycle of thelizard Sceloporus malachiticus in Costa Rica. Copeia 1971:517–526.

MATTER, J. M. 1987. The Reproductive Biology of the Male Fence Lizard,Sceloporus undulatus hyacinthinus: Seasonal Conditions and theEffects of Hormone Treatment. Unpubl. Master’s thesis, Saint LouisUniversity, St. Louis, MO.

MAYHEW, W. W. 1963. Reproduction in the granite spiny lizard, Sceloporusorcutti. Copeia 1963:144–152.

MCKINNEY, R. B., AND K. R. MARION. 1985. Plasma androgens and theirassociation with the reproductive cycle of the male fence lizard,

170 J. L. RHEUBERT ET AL.

Sceloporus undulatus. Comparative Biochemistry and Physiology PartA: Physiology 82:515–519.

MENDEZ DE LA CRUZ, F. R., L. J. GUILLETTE JR., M. VILLAGRAN SANTA CRUZ,AND G. CASAS-ANDREU. 1988. Reproductive and fat body cycles of theviviparous lizard, Sceloporus mucronatus (Sauria: Iguanidae). Journalof Herpetology 22:1–12.

NEWLIN, M. E. 1976. Reproduction in the bunch grass lizard, Sceloporusscalaris. Herpetologica 32:171–184.

RAMIREZ-BAUTISTA, A., AND G. GUTIERREZ-MAYEN. 2003. Reproductiveecology of Sceloporus utiformis (Sauria: Phrynosomatidae) from atropical dry forest of Mexico. Journal of Herpetology 37:1–10.

RAMIREZ-BAUTISTA, A., O. RAMOS-FLORES, AND J.W. SITES JR. 2002.Reproductive cycle of the spiny lizard Sceloporus jarrovi (Sauria:Phrynosomatidae) from north-central Mexico. Journal of Herpetol-ogy 36:225–233.

RAMIREZ-BAUTISTA, A., R. GARCIA-COLLAZO, L. J. GUILLETTE, AND G. C.CARPENTER. 2006a. Reproductive, fat, and liver cycles of male andfemale rose-bellied lizards, Sceloporus variabilis, from coastal areas ofsouthern Veracruz, Mexico. Southwestern Naturalist 51:163–171.

RAMIREZ-BAUTISTA, A., V. H. LUJA, C. BALDERAS-VALDIVIA, AND R. ORTIZ-PULIDO. 2006b. Reproductive cycle of male and female spiny lizards,Sceloporus melanorhinus, in a tropical dry forest. SouthwesternNaturalist 51:157–162.

RAMIREZ-BAUTISTA, A., B. P. STEPHENSON, X, HERNANDEZ-IBARRA, U.HERNANDEZ-SALINAS, R. CRUZ-ELIZALDE, A. LOZANO, AND G. R. SMITH.2012. Reproductive strategy of male and female eastern spiny lizardsSceloporus spinosus (Squamata: Phrynosomatidae) from a region ofthe Chihuahuan desert, Mexico. Acta Herpetologica 7:239–252.

RHEUBERT, J. L., H. H. MCHUGH, M. H. COLLIER, D. M. SEVER, AND K. M.GRIBBINS. 2009a. Temporal germ cell development strategy duringspermatogenesis within the testis of the ground skink, Scincellalateralis (Sauria: Scincidae). Theriogenology 72:54–61.

RHEUBERT, J. L., E. H. POLDEMANN, M. E. ECKSTUT, M. H. COLLIER, D. M.SEVER, AND K. M. GRIBBINS. 2009b. Temporal germ cell developmentstrategy during mixed spermatogenesis within the male Mediterra-nean gecko, Hemidactylus turcicus (Reptilia: Gekkonidae). Copeia2009:791–798.

RUSSELL, L. D., S. A. P. HIKIM, R. A. ETTLIN, AND E. D. LEGG. 1990.Histological and Histopathological Evaluation of the Testis. CacheRiver Press, Clearwater, FL.

SAINT GIRONS, H. 1982. Reproductive cycles of male snakes and theirrelationships with climate and female reproductive cycles. Herpeto-logica 38:5–16.

SHINE, R., AND J. F. BERRY. 1978. Climatic correlates of live-bearing insquamate reptiles. Oecologia 33:261–268.

SINERVO, B., F. MENDEZ-DE-LA-CRUZ, D. B. MILES, B. HEULIN, E. BASTIAANS,M. VILLAGRAN-SANTA CRUZ, R. LARA-RESENDIZ, N. MARTINEZ-MENDEZ,M. L. CALDERON-ESPINOSA, R. N. MEZA-LAZARO ET AL. 2010. Erosion oflizard diversity by climate change and altered thermal niches.Science 328:894–899.

SMITH, H. M., AND W. P. HALL. 1974. Contributions to the concepts ofreproductive cycles and the systematics of the scalaris group of thelizard genus Sceloporus. Western North American Naturalist 34:97–104.

TINKLE, D. W. 1969. The concept of reproductive effort and its relation tothe evolution of life histories of lizards. American Naturalist 103:501–516.

TINKLE, D. W., H. M. WILBUR, AND S. G. TILLEY. 1970. Evolutionarystrategies in lizard reproduction. Evolution 24:55–74.

VALDEZ-GONZALEZ, M. A., AND A. RAMIREZ-BAUTISTA. 2002. Reproductivecharacteristics of the spiny lizards, Sceloporus horridus and Sceloporusspinosus (Squamata: Phrynosomatidae) from Mexico. Journal ofHerpetology 36:36–43.

VILLAGRAN-SANTA CRUZ, M., O. HERNANDEZ-GALLEGOS, AND F. R. MENDEZ-DE

LA CRUZ. 2009. Reproductive cycle of the lizard Sceloporus mucronatuswith comments on intraspecific geographic variation. Western NorthAmerican Naturalist 69:437–446.

VITT, L. J., AND R. D. OHMART. 1974. Reproduction and ecology of aColorado River population of Sceloporus magister (Sauria: Iguanidae).Herpetologica 30:410–417.

VOLSØE, H. 1944. Structure and seasonal variation of the malereproductive organs of Vipera berus (L.). Spolia Zoologica MuseiHauniensis V Skrigter, Univeritetets Zoologiske Museum, Copenha-gen, Denmark.

WANG, L., X. WU, D. XU, R. WANG, AND C. WANG. 2008. Development oftestis and spermatogenesis in Alligator sinensis. Journal of AppliedAnimal Research 34:23–28.

WIENS, J. J., AND T. W. REEDER. 1997. Phylogeny of the spiny lizards(Sceloporus) based on molecular and morphological evidence.Herpetological Monographs 11:1–101.

WILHOFT, D. C., AND W. B. QUAY. 1961. Testicular histology and seasonalchanges in the lizard, Sceloporus occidentalis. Journal of Morphology108:95–106.

WOODBURY, M., AND A. M. WOODBURY. 1945. Life-history studies of thesagebrush lizard Sceloporus g. graciosus with special reference tocycles in reproduction. Herpetologica 2:175–196.

Accepted: 25 March 2013.

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