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Chapter 16

Photoperiodism in Amphibians and Reptiles

Zachary M. Weil1and David P. Crews

2

1

Departments of Psychology and Neuroscience, Ohio State University, Columbus,OH 43210 and

2Section of Integrative Biology, University of Texas, Austin, TX

78712 USA

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Introduction

As animals moved from the aquatic to terrestrial habitats, novel adaptations developed to

cope with the more pronounced seasonal variations in environmental conditions. This is

especially true for tetrapod vertebrates inhabiting temperate and boreal zones that evolved

mechanisms to time reproduction to coincide with the time of year during which environmental

conditions are mild and conducive to offspring (and parental) survival. Specifically, most small

terrestrial animals breed during the spring and summer when temperatures are mild and food is

relatively abundant. Most temperate-zone amphibians and reptiles display marked annual cycles

in breeding. As noted throughout this volume, organisms across taxa have evolved mechanisms

to attend to photoperiod to monitor seasonal time, presumably because it is a consistent and

relatively noise-free environmental signal.

Photoperiodic signals can be decoded by organisms in essentially two broad ways. The

first relatively straightforward system is an hourglass or interval timer. These systems function

by measuring the total period of light (or dark) and the build-up (or break down) of

photoproducts regulates the downstream neuroendocrine systems. This type of photoperiodic

system exists in some insect species (Lees, 1966; Truman, 1971). The other general class of

photoperiodic systems are those based on the circadian clock (Bnning, 1936). Circadian

theories of photoperiodism propose that organisms have endogenous daily rhythms of

responsiveness and non-responsiveness to the effects of light. Said another way light must fall

during the circadian phase of photoresponsiveness in order to interact with the photoperiod

detection circuitry. This model has been called external coincidence because the circadian

rhythm of photosensitivity has to coincide with external stimuli (light). An alternative model

suggests that light entrains two separate oscillators (e.g., a dawn and dusk oscillator) and the

phase relationship between the two rhythms or internal coincidence determines the responses to

the observed day length (Pittendrigh, 1972; Pittendrigh & Minis, 1964).

In contrast to mammals and most birds, however, ambient temperature is an important

variable for poikilothermic organisms such as amphibians and reptiles in addition to photoperiod

in regulating seasonal physiology. The principle aim of this chapter is to provide a broad

overview of photoperiodic regulation of reproductive and non-reproductive responses in

amphibians and reptiles. This chapter will focus mostly on laboratory investigations of

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photoperiodic and seasonal phenomena where environmental factors can be precisely controlled

and the respective contributions of day length and environmental temperature can be elucidated.

Amphibians.

The class of Amphibia consists of three orders, the anura (frogs and toads), urodele

(salamanders and newts), and caecilians (legless snake- or worm-like amphibian), together

comprising thousands of species (Duellman & Trueb, 1986). Little is known about the caecilians

and so we will focus on the anuran and urodele amphibians. Evolutionarily, amphibians

represent an intermediate between fish and fully terrestrial vertebrates and as such can provide

important insights into the evolution of photoperiodism in higher vertebrates. Additionally,

amphibian species utilize a substantial variety of reproductive strategies, including the dramatic

indirect development; indeed, the term amphibian is derived from the Greek "double" and

life as many species are born in larval states and undergo a metamorphosis into an adult

phenotype.

As a general rule, day length appears to be less of an important player in the regulation of

reproduction in amphibians than it is in other vertebrate taxa. Few data are available in controlled

laboratory studies on the role of photoperiod in biological timing in amphibians. Further, many

of the studies have been conducted used unnatural extreme photoperiods (e.g., constant light, or

LD 1:23) that do not recapitulate the day lengths experienced in nature. However, in some

temperate zone species day length can play an important modulatory role in the regulation of

reproductive responses to other stimuli (Rastogi, Iela, Saxena, & Chieffi, 1976).

Amphibians use a variety of environmental conditions (temperature, rainfall, and

photoperiod) to regulate the timing of reproduction and metamorphosis (Lofts, 1984).

Reproduction in tropical amphibians tends to be continuous or restricted to the rainy season with

most species minimally responsive to day length. It is important to note, however, that this

characteristic of "continuous reproduction" refers to the population and not to the individual.

That is, reproduction at some level is always apparent in the population but this is due to the lack

of reproductive synchrony among the individuals in reproductive readiness. Temperate

amphibians, on the other hand, tend to fall into one of two categories in terms of the timing of

breeding which we will designate here as Determinate and Indeterminate breeding strategies. An

example of the first category is the European common frog (Rana temporaria)that breeds after

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emerging from hibernation in the spring and then undergoes gonadal regression and ceases

spermatogenesis and steroidogenesis for several months, followed by spontaneous regression and

induction of gonadal activity in preparation for the next breeding season. During the quiescent

period the gonads are refractory to stimulation with environmental or endocrine variables (Van

Oordt, 1956). However, most species of amphibians are capable of breeding at any time of the

year if conditions permit. Such Indeterminate breeding species may exhibit seasonal patterns of

reproduction in the field, but are physiologically capable of breeding indefinitely as long as the

environmental conditions remain favorable. This dichotomy thus suggests the lack of a

circannual rhythm of sensitivity to environmental cues.

Anurans

Most instances of photoperiodism have been reported in temperate-zone anurans that

exhibit an Indeterminate breeding strategy and species with a Determinate strategy in which

gonadal recrudescence and regression are regulated by a circannual clock that renders the

gonadal responses sensitive and refractory, respectively, to day length and temperature signals.

Determinate breeders utilize day length and other environmental cues to time breeding

behavior. Perhaps the best studied in this regard are green frog (Rana esculenta). The breeding

season beginsas temperatures increase in late March and early April. The cessation of breeding

is followed by a decrease in circulating androgens and a concomitant increase in all aspects of

spermatogenesis. Over the following winter androgen concentrations rise in preparation for

breeding in the spring and the spermatogenic tissues degenerate (Rastogi, Iela, Saxena, &

Chieffi, 1976). Importantly, in this species, androgens appear to be important for the regulation

of breeding behavior, but not necessary for the early stages of spermatogenesis (Rastogi, Chieffi,

& Iela, 1972). However, androgens appear to inhibit mitotic division of spermatogonia and also

be necessary for spermatidogenesis (meiotic division of secondary spermatocytes into

spermatids). In the laboratory exposure to intermediate day lengths (12:12 LD) cycles and mild

temperatures (20C) can maintain summer-active gonadal activity indefinitely (at least up to 60

days) (Rastogi et al., 1978). During winter LD 12:12 cycles maintain gonadal activity at 28 C,

however, if temperatures are lowered to 4 C for as few as 7 days spermatogenesis is markedly

inhibited. Short day lengths (LD 3:21) inhibit gonadal activity in late summer (Rastogi et al.,

1978). Conversely, winter gonadal activity can persist in warm temperatures even in short day

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lengths. Taken together, it appears that gonadal regression in winter is mediated by low

temperatures and these frogs exploit a warm fall-early winter with a second breeding event as the

short day lengths cannot solely mediate spermatogenic arrest (Rastogi et al., 1978). Conversely,

spring recrudescence of the gonads requires both warm temperatures and permissive day lengths

(>12L) suggesting that day length prevents inappropriate early breeding in response to early

spring increases in temperature.

It is important to emphasize that the reproductive axis of green frogs canbe transformed

from a Determinate to an Indeterminate breeding strategy. Such flexibility is inherent in

biological systems and usually revealed under specific laboratory conditions. Here the direction

of the flexibility can be instructive. For example, a female rat normally exhibits a 4-5 day estrous

cycle and spontaneously ovulates at a predictable time, but when placed under constant light she

will go into constant estrus and ovulate in response to copulation. However, it is not possible to

transform an induced ovulating species into one that ovulates spontaneously. In the case of green

frogs breeding will become continuous breeding in mild conditions if individuals are removed

from the inhibitory effects of short day lengths For instance, exposure to LD 21:3 in mid-

summer induced spermatogenic arrest after 60 days, but LD 12:12 maintained gonadal activity

throughout. It is likely that these extremely long photoperiods have various nonspecific effects

on the neuroendocrine and circadian systems of these animals. In their natural habitats these

animals never experience day lengths greater than LD 16:8 or LD 8:16 and so it is difficult to

determine what would happen with more natural photoperiods. Whereas such studies are

common in the amphibian literature, they provide evidence that the neuroendocrine-reproductive

axis can process and respond to changes in day length albeit in a permissive rather than direct

causal fashion (Rastogi et al., 1978; Rastogi, Iela, Saxena, & Chieffi, 1976).

Iberian water frogs (Rana perezi) exhibit a reproductive pattern that combines an

Indeterminate breeding strategy with a temporal dissociation between spermatogenesis and

steroidogenesis (Delgado, Gutierrez, & Alonso-Bedate, 1989). Reproduction in this species

appears to be regulated by both exogenous environmental conditions and an endogenous clock.

Frogs captured in the winter and exposed to either warm temperatures (25 C) or long

photoperiods (LD 18:6) increase gonadal size and the number of primary spermatocytes..

However, in the early spring, testicular recrudescence is sensitive only to high temperatures;

frogs exposed to long days in low temperatures are unresponsive (Delgado, Alonso-Gomez, &

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Alonso-Bedate, 1992). Spermatogenesis is reduced by warm temperatures in the winter and

enhanced by short days in the late spring, but is refractory to low temperatures following

breeding. Unlike green frogs, the reproductive cycle of Iberian water frogs appears to be the

product of an interaction between environmental light and temperature. In the winter, low

temperatures and short photoperiods stimulate androgen production and inhibit spermatogenesis.

Rising photoperiods stimulate further release of androgens in low temperatures in the late winter

and through breeding indicating that rising photoperiods in the spring are necessary for mating

behavior. Following breeding, warm temperatures and long photoperiods inhibit androgen

production and stimulate spermatogenesis. Once started spermatogenesis proceeds to

completion independent of photoperiod signaling (Delgado, Alonso-Gomez, & Alonso-Bedate,

1992). Taken together, these data indicate that day length interacts with temperature and an

endogenous oscillator to time breeding and spermatogenesis.

Urodeles

Much as with anuran amphibians, early experiments with urodeles consistently failed to

detect photoperiodism in the regulation of reproduction (e.g., (Ifft, 1942)). Since then there have

been few studies that have examined the question of photoperiodism in urodeles. Red-backed

salamanders (Plethodon cinereus)breed twice a year, once in early spring and again in late

September and early October. In between the two breeding periods the gonads grow and

produce sperm. In spring, when spermatogenesis is just beginning warm temperatures greatly

increase gonadal growth and this effect is slightly potentiated by exposure to long days.

Similarly, warm temperatures stimulate spermatogenesis with maximal effect in long days

(Werner, 1969). Early in the winter the gonads are refractory to both warm temperatures and

long photoperiods but later in the winter quiescent period, long days and warm temperatures

stimulate gonadal growth and slightly increase spermatogenesis. Neither warm temperatures nor

photoperiod alone stimulate either gonadal growth or spermatogenesis (Werner, 1969). Similar

results have been reported for marbled newts (Triturus marmoratus)that also exhibit gonadal

growth and enhanced spermatogenesis following exposure to long days at warm temperatures

(Fraile, Paniagua, & Rodriguez, 1988).

Regeneration

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A striking characteristic of urodeles is their ability to regenerate limbs. In vermillion

spotted newts (T. viridiscens)day length alters the rate of forelimb regeneration after bilateral

amputation. Exposure to constant light increases, while constant darkness inhibits, limb

regeneration, with 1 LD 5:9 cycles falling intermediate (Maier & Singer, 1977) . The effects of

light appears to interact with circulating prolactin concentrations as treating newts in constant

light with prolactin has no effect on forelimb regeneration. However, exogenous prolactin does

markedly speed regeneration in newts housed in constant darkness (Maier & Singer, 1981).

Reception of Photoperiod Information

The anatomical site of photoperiod transduction in amphibians remains somewhat

unspecified. The lateral eyes, pineal gland, and associated frontal organ are all competent

photoreceptors (Oksche, 1984; Oksche & Hartwig, 1979). Removal of either the eyes or the

pineal gland eliminated the stimulatory effects of long day lengths in green frogs (Rastogi, Iela,

Saxena, & Chieffi, 1976) and enhanced ovarian function in Iberian water frogs (Alonso-Gomez,

Tejera, Alonso-Bedate, & Delgado, 1990). In skipping frogs (R. cyanophlictis)blinding or

parietal shielding (blocking pineal photoreceptors directly) each increased ovarian size alone and

in combination (Udaykumar & Joshi, 1996). On the other hand marbled newts lacking eyes and

intact animals respond identically to photoperiod stimulation (Fraile, Paniagua, & Rodriguez,

1988).

The role of the pineal melatonin system in the interpretation of day length information in

amphibians has yet to be fully explicated even though melatonin was originally described as the

hormone responsible for blanching of leopard frog (Rana pipiens)skin in the darkness (McCord

& Allen, 1917). The pineal gland does not seem to be the principle source of circulating

melatonin in amphibians. Most amphibians exhibit strong daily rhythms of melatonin in the

blood with peaks during the scotophase (d'Istria, Monteleone, Serino, & Chieffi, 1994; Delgado

& Vivien-Roels, 1989; Gern & Norris, 1979; Rawding & Hutchison, 1992) and both temperature

and day length contribute to the pattern of melatonin production (Delgado & Vivien-Roels,

1989). However, in anurans the concentration of melatonin in the pineal itself is markedly lower

than in the retina. Further blood dynamics of circulating melatonin most closely mirror the

concentrations of melatonin in the retina (Delgado & Vivien-Roels, 1989). In green frogs daily

rhythms in the expression of the rate limiting enzyme N-acetyl transferase do not relate to

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circadian rhythms of melatonin content in the pineal (d'Istria, Monteleone, Serino, & Chieffi,

1994). However, pinealectomy abolished nightime peaks in melatonin rhythms in melatonin, but

not diurnal concentrations in tiger salamanders (Ambystoma tigrinum)(Gern & Norris, 1979).

Melatonin administration to amphibians is generally associated with gonadal inhibition.

The gonadosomatic index (GSI) decreases with daily injections of melatonin in tree frogs (Hyla

cinera)(De Vlaming, Sage, & Charlton, 1974) and marsh frogs (Rana ridibunda)(Delgado,

Gutierrez, & Alonso-Bedate, 1983). Exogenous melatonin counteracts the stimulatory effects of

blinding on the GSI in skipper frogs (Rana cyanophlyctis)(Joshi & Udaykumar, 2000).

Exogenous melatonin induces gonadal regression in black spined toads (B. melanosticus)when

administered either in the morning or the evening, but not at both phases of the day (Chanda &

Biswas, 1982). Similarly, in Indian green frogs (R. hexadactlya)melatonin inhibited

spermatogenesis under some dosing schedules, but not others (Kasinathan & Gregalatchoumi,

1988). Melatonin also inhibits in vitroovulation of Argentine common toads (Bufo arenarum)

(de Atenor, de Romero, Brauckmann, Pisan, & Legname, 1994). However, other in vivoand in

vitro studies have reported no effects or stimulatory actions of exogenous melatonin including in

seasonally breeding frogs such as Iberian water frog sand European common frogs (Alonso-

Bedate, Carballada, & Delgado, 1990; Alonso-Bedate, Delgado, & Carballada, 1988).

Reptiles

Reptiles are the most diverse of the vertebrate taxa, including the snakes, lizards, turtles,

Tuatara, and crocodilians. Ancestral reptiles gave rise to both birds and mammals 250-300

million years ago (Godwin & Crews, 2002; Padian & Chiappe, 1998). The study of reptilian

neuroendocrine systems is guided by the implicit assumption that modern reptiles exhibit

phenotypic properties similar to the putative systems of ancestral amniotes. Extant traits

including poilkiothermy, temperature-dependent sex determination, oviparity, and

underdeveloped cerebral cortices likely also occurred in common ancestral amniotes (Godwin &

Crews, 2002). Additionally, many reptilian species restrict breeding to a specific time of the

year, but substantial diversity exist in the mechanisms that underlie these rhythms (Crews, 1999).

Studying reptilian brains is therefore advantageous for researchers interested in photoperiodism

and seasonality in general, and also important to understand the evolutionary processes that

shaped seasonality across vertebrate taxa.

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Photoperiodic control of reproduction in reptiles was initially reported in the mid 1930s.

Long day lengths produced out-of-season gonadal development in both green anoles (Anolis

carolinensis)(Clausen & Poris, 1937) and red-eared slider turtles (Trachemys scripta elegans)

(Burger, 1937). The overall goal of this section therefore is to review what has been learned in

the eighty years since those initial discoveries and to also address remaining unanswered

questions facing the field.

The control of seasonal breeding in reptiles is accomplished by a complex interplay

between photoperiod and a variety of non-photic cues including temperature, food, and water

availability. Additionally, some species of reptiles appear to exhibit a circannual clock that

interacts with other proximate cues. In general the reproductive patterns of reptiles are

characterized by those species that exhibit an Associated Reproductive strategy in which gonadal

growth, steroidogenesis and gametogenesis precedes and are temporally associated with breeding

or a Dissociated Reproductive strategy in which gonadal growth, steroidogenesis, and

gametogenesisfollowbreeding; in the latter instance the gametes produced are then used in the

next breeding season (Crews, 1984; Whittier & Crews, 1987). We will consider the control of

seasonal breeding in both of these general types of breeders.

Photoperiodism and Breeding Cycles in Associated Breeders

There have been reports of photoperiodic modulation of reproductive and non-

reproductive traits in all reptile groups (Burger, 1937; Haldar & Pandey, 1989; McPherson,

1981; Mendonca & Licht, 1986). Both the annual breeding cycle and the control of these

rhythms by photoperiodic and non-photoperiodic cues in reptiles have been best studied in the

green anole lizard and as such we will consider this species in detail. The green anole is a small

iguanid lizard that inhabits the temperate zone throughout the southeastern United States (Conant

& Collins, 1998). Although the timing of reproduction will vary with latitude, in general male

green anolesare reproductively quiescent from late September until late in January. At that

point the males emerge from hibernation and begin establishing and defending territories.

Females become active approximately one month later (see below) and breeding commences

shortly thereafter. The breeding season lasts until approximately the middle of August at which

point testicular collapse occurs.

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Early studies based on the experimental methodology that had established a role for the circadian

clock in plant and insect photoperiodism (e.g. night interruption [presentation of relatively brief

light pulses during various parts of the daily cycle; can distinguish between hourglass and

circadian models because the timing but not the absolute amount of light is the important

parameter], resonance [light:dark cycles with periods of multiples of 12 such as 6:18, 6:30, 6:42;

these cycles can detect the presence of a circadian rhythm in photoresponsiveness because

without changing the amount of light increasing dark phases can render these rhythms inductive)

and T-cycle experiments [similar to resonance experiments but with light:dark cycles within the

range of entrainment], (Nanda & Hamner, 1958; Pittendrigh & Minis, 1964; see below) initially

failed to detect circadian involvement in the photoperiodic response of the green anole lizard

(i.e., the total amount of light, rather than the timing in which it was administered, appeared to be

the key determinant of reproductive responses to day length). An hour of light placed at various

phases of the circadian clock did not alter gonadal responses to short (LD 6:18) photoperiods in

either the regressive or recrudescent phases. T-cycles of various lengths (24-60 hours) failed to

induce gonadal development with 8 hour photophases. Additionally, photocycles with 6 hours of

light and increasing dark periods were all non-stimulatory while photoperiods with 16 hours of

light and increasing dark cycles were all inductive. These data were interpreted as support for

the hypothesis that the anoles utilized an hourglass-type system (Underwood, 1978; Underwood

& Hall, 1982). However, further studies on the anole circadian system indicated that locomotor

rhythms became disorganized or exhibit splitting under very short photoperiods or the resonance

and T-cycle experiments in the earlier experiments (Underwood, 1983a). This disorganization

apparently rendered detection of a circadian regulation of photoperiod transduction impossible

(Underwood & Hyde, 1990).

When later studies used longer photoperiods and night break manipulations, it became

apparent that the circadian system controlled the reception of photoperiod information as long as

the perceived length of the photoperiod was 10-11 hours or greater in night break, resonance, or

T-cycle experiments. Again, as in previous experiments, shorter photoperiods were not

permissive to a circadian involvement in day length measurement (Underwood & Hyde, 1990).

Light pulses late in the subjective night either from night break, resonance, or T-cycle

experiments were associated with induction of reproductive recrudescence and fat deposition

(Ferrell & Meier, 1981; Underwood & Hyde, 1990).

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Temperature and photoperiod acting at different times of the cycle

Temperature is the critical regulator of testicular recrudescence, with temperatures a few

degrees below the preferred body temperature impairing spermatogenesis, whereas a few degrees

over the body temperature induces rapid testicular degeneration (Licht & Basu, 1967).

Stimulation of reproductively inactive adult malegreen anoleswith a LD 14:10 photoperiod

enhances testicular recrudescence when animals are housed at 32 C, but not when maintained at

20 C; shorter photoperiods do not accelerate testicular recrudescence even at warmer

temperatures (Licht, 1967a); see also (Fox & Dessauer, 1958). Interestingly, a 12 hour cycle

alternating between 32 and 20 C was stimulatory when the warmer temperatures coincided

with the light cycle, but not when the elevated temperatures occurred at night (Licht, 1966; Licht,

1967a).

Toward the end of the breeding season as fall approaches photoperiod becomes the

critical factor regulating testicular activity, with shortening day lengths at the end of the breeding

season responsible for testicular collapse. Studies indicate that a photoperiod below LD13.5:10.5

inhibits testicular activity, whereas photoperiod above LD 13.5:10.5 maintains testicular activity

(Licht, 1970) . Several lines of evidence suggest that induction of testicular recrudescence

during the winter when animals normally are in hibernation is independent of day length. First,

during the early part of the fall when day lengths are still long (but decreasing) green anoles

exhibit a relative refractoriness to the stimulatory effects of photoperiod (Crews & Licht, 1974;

Licht, 1967b) Additionally, the low winter temperatures are permissive to photoperiod

responsiveness (Licht, 1966). Finally, once gonadal regeneration begins it is independent of

further photoperiod stimulation (Licht, 1967b).

In addition to photoperiodic and temperature effects on gonadal activity, there is a

circannual rhythm in the responsivity of green anoles to both environmental factors. Early in the

fall long day lengths maintain gonadal recrudescence, however, this effect is greater when the

photoperiod treatment started in October then if it is introduced several weeks earlier, suggesting

a relative photorefractoriness. Additionally, high temperatures accelerate testicular

recrudescence independent of day length after mid-October. Together these data indicate that

soon after the initial regression of the reproductive tract there is a relative refractory period to

both temperature and day length (Crews & Licht, 1974; Licht, 1967b);. Finally, photoperiod

and temperature regimens that do not accelerate gonadal recrudescence do eventually result in

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gonadal development the following spring. Full spermatogenesis, however, never occurs in

green anoles without exposure to warm temperatures for at least part of the day (Licht, 1967a, ,

1967b). Gonadal quiescence appears to be mediated by reduced hypothalamic gonadotropin

secretion rather than at the level of the pituitary or gonads themselves (Crews and Licht, 1974).

a similar finding has been reported in mammals (Kriegsfeld, Drazen, & Nelson, 1999;

Shanbhag, Radder, & Saidapur, 2000).

Aside from the pronounced dependence on environmental temperature this type of

rhythm is similar to those seen in warm-blooded vertebrates. For instance, hamsters can breed

indefinitely under stimulatory day lengths; gonads regress in response to short days and will

either spontaneously recrudesce the following spring or can be stimulated to do so sooner with

exposure to long days (Goldman, 2001; Zucker & Morin, 1977). Additionally, reptiles display

the familiar vertebrate pattern of an endogenous oscillator that interacts with environmental

information to regulate breeding cycles. However, the relative contribution of day length

signaling and circannual rhythms are difficult to tease apart based on the extant literature. Fence

lizards (Sceloropus undulatus)and parthenogenetic whiptail lizards (Cnemidophorous

uniparens)appear to regulate reproductive cycles independently of photoperiod and via

endogenous fluctuations in the sensitivity to temperature (Cuellar & Cuellar, 1977; Marion,

1970; Moore, Whittier, & Crews, 1984); whereas the response of checkered water snakes (Natrix

piscator) to photoperiod is dependent on ambient humidity (Haldar & Pandey, 1989). The green

anole studies presented here were conducted on lizards that had been captured in the weeks after

the conclusion of the breeding season (e.g., Fox & Dessauer, 1958; Licht, 1966). Therefore it is

difficult to tell whether endogenous oscillators were entrained by photoperiod exposure prior to

the previous breeding season or a true circannual clock exists.

The reproductive cycle of the female green anole is somewhat different in its regulation

by temperature and photoperiod cues. The cycle can be divided into three distinct phases

(Crews, 1975, , 1980; Crews & Garrick, 1980): (1) Previtellogenesis from November to

February is marked by inactive ovaries with small translucent unyolked follicles and regressed

oviducts, (2) Vitellogenesis starts in March and continues until the end of the breeding season.

This stage is characterized by follicular development and ovulation every 10 to 14 days, (3) The

third stage regression is associated with follicular atresia and ovarian collapse (Crews, 1975;

Crews & Licht, 1974; Licht, 1973).

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Long day lengths can stimulate ovarian recrudescence late in the fall or during the

winter, but immediately after breeding there is a period of photo- and thermal-refractoriness

(Crews & Licht, 1974; Licht, 1973);. This is very similar to the pattern of reproductive cyclicity

in side-blotched lizards(Uta stansburiana) (Tinkle & Irwin, 1965) and to mammalian patterns.

The period of refractoriness may be related to the presence of large highly vascularized atretic

follicles during the post-breeding period. The atretic follicles may directly inhibit the ovarian

sensitivity to gonadotropins-induced by exogenous cues (Crews & Licht, 1974). In addition to

photoperiod, temperature, humidity, and social stimuli are significant (Crews, Rosenblatt, &

Lehrman, 1974). For example, long days and warm temperatures during the photophase induce

ovarian recrudescence during the winter and this effect is potentiated by exposure to intact males

(Crews, 1975; Crews, Rosenblatt, & Lehrman, 1974). High relative humidity is essential

important for this photothermal regimen to stimulate gonadal activity in anole lizards and the

checkered water snake (Haldar & Pandey, 1989).

Detection and Transduction of Photoperiod Signals

The circuitry through which photoperiod information is communicated to reptile tissues

is not well defined. The pineal gland seems critical to the expression of photoperiodic

responsiveness and this is likely secondary to the role of the pineal as both photoreceptor and

endogenous oscillator (see below). The lizard pineal gland differs from mammalian pineal glands

in several important ways. First, the principal pineal cells (analogous to the pinealocytes of birds

and mammals) are both secretory in nature (as in mammals), but also are rudimentary

photoreceptors. Additionally, there are populations of nerve cells that project axons to tectal and

tegmental structures and receives synaptic input from various brain regions (Quay, 1979).

Pineal glands in lizards appear to be the critical site for the detection of photoperiod

information. Retinal photoreceptors are not necessary for the regulation of circadian rhythms or

day length determinations in green anole lizards. Extraretinal photoreceptors maintained gonadal

responses to day length and the phase response curve for circadian responses to environmental

light was actually enhanced by blinding (Underwood, 1985b; Underwood & Calaban, 1987b).

Removal of the parietal eye also does not alter the reception of photoperiodic information

(Underwood, 1985c). Conversely, circadian rhythms of locomotor activity can be entrained in

pinealectomized and/or blinded lizards by 24-hour cycles of either light or temperature

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(Underwood, 1986a; Underwood & Groos, 1982) indicating that extraretinal and extrapineal

photoreceptors exist in lizards. However, shielding the heads of blinded-pinealectomized Texas

spiny lizards (Sceloporous olivaceous) causes them to free run (Underwood & Menaker, 1976).

The anatomical sites of these receptors have not been determined, but it is known that the

parietal eye cannot be the location as pinealectomy includes removal of the parietal eye.

The lizard pineal is a rhythmic oscillator that is directly responsive to light and maintains

daily rhythms of melatonin production in culture under constant lighting conditions (Menaker &

Wisner, 1983; Underwood, 1983b), although the pineal gland of desert iguanas (Diposaurus

dorsalis)becomes arrhythmic under constant conditions (Janik & Menaker, 1990). Additionally,

removal of the pineal gland leads to splitting of locomotor rhythms of anoles suggesting that the

pineal is a master oscillator that when removed allows other subordinate oscillators to be

expressed (Underwood, 1983a, , 1983b). Melatonin may be the key output signal for the

circadian system particularly because exogenous melatonin can shift the circadian rhythm of

locomotor activity (Hyde & Underwood, 1995; Underwood, 1986a) possibly by binding to

receptors in the SCN (Bertolucci, Sovrano, Magnone, & Foa, 2000).

As in mammals, the pineal is necessary for the proper transduction of photoperiod signals

in reptiles. However, melatonin replacement cannot recapitulate the physiological effects of day

length as it can in mammals. In green anole lizards males will undergo testicular recrudescence

regardless of day length at all times of the year including during the photorefractory period

(Underwood, 1985a). A similar pattern is observed in Indian garden lizards (Calotes versicolor).

Injections of melatonin during the photo- or scotophase (or both) will not reverse the effect of

pinealectomy (Haldar & Thapliyal, 1977; Thapliyal & Haldar, 1979) Administration of

melatonin to intact animals also did not alter gonadal responses (Underwood, 1985a). Chronic

release melatonin capsules, however, will block the progonadal effects of pinealectomy in short

inhibitory day lengths. This is consistent with data from ruin lizards that exhibit bimodal activity

patterns with peaks in the early morning and late afternoon; in the autumn and spring, however,

activity is unimodal (Foa et al., 1994; Foa, Tosini, & Avery, 1992). Pinealectomy or constant

release melatonin in constant conditions induces the unimodal spring/fall phenotype (Bertolucci,

Sovrano, Magnone, & Foa, 2000). Additionally, phase response curves to melatonin are not

apparent in seasons other than summer (Bertolucci & Foa, 1998). Considered together, these

data suggest that in addition to being a key regulator of photoperiodic responses the pineal gland

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in lizards also is involved in the expression of circadian rhythms, although this function varies

during different times of the year.

It is apparent that the pineal gland is the key receptor for photoperiod information, but

that melatonin is not the sole neuroendocrine signal of day length information. Although

melatonin replacement does not rescue the apparent photoperiod insensitivity in pinealectomized

green anoles under all conditions, a potential role for pineal melatonin in the regulation of intact

reptile photoperiodism cannot be ruled out. At a minimum, the pineal or circulating melatonin

content can provide information about the phase of the pineal oscillator. Indeed, the pineal

melatonin rhythm (PMR) is a powerful transducer of environmental information (particularly

light and temperature) into a physiological one. The phase, amplitude, and duration of the

pineal melatonin rhythm in iguanid lizards can be affected by photoperiod and thermoperiod

duration, light intensity, and amplitude of temperature rhythms (Underwood, 1985d; Underwood

& Calaban, 1987a; Vivien-Roels, Pevet, & Claustrat, 1988). Under appropriate photoperiod and

temperature cycles (e.g., 12:12 LD and cooler temperatures during the lights on period) strong

daily peaks of melatonin can occur during lights on (Underwood & Calaban, 1987a).

Light at night does not acutely suppress melatonin production in green anole lizards

(Underwood, 1986b) or box turtles (Terrapene carolina triunguis) (Vivien-Roels, Pevet, &

Claustrat, 1988). When daily melatonin rhythms are assessed under various T-cycle, resonance,

or nightbreak conditions, neither the amplitude nor the duration of the melatonin signal predict

whether the treatment would stimulate gonadal regrowth (Hyde & Underwood, 1993). Instead,

the only aspect of the melatonin rhythm that covaries significantly with reproductive responses is

the phase at which the melatonin peak occurred. Photoperiod (and temperature) regimens that

produce melatonin peaks during the early or late phase of the lights off-period stimulated

gonadal growth, whereas those regimens that produced a peak towards the middle of the dark

period are inhibitory (Hyde & Underwood, 1993). Finally, the same types of manipulations

(e.g., temperature amplitude, light intensity) that alter melatonin rhythms also alter the

expression of circadian clock genes (Magnone, Jacobmeier, Bertolucci, Foa, & Albrecht, 2005;

Malatesta et al., 2007; Vallone et al., 2007). Together, these data suggest that the phase

relationship between melatonin and some endogenous oscillator (e.g., an internal coincidence

system) determines reproductive responses to day length and temperature. However, an external

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coincidence system cannot be ruled out and neither can a direct role of pineal synaptic

connections on other CNS targets.

Dissociated Breeders

In most seasonal vertebrates increases in gonadal steroids and spermatogenesis occur

shortly before the onset of the breeding season and are necessary for the full expression of

reproductive behaviors. However, there are examples of vertebrates with dissociated

reproductive patterns in which the gonads are quiescent during winter hibernation and the

breeding season and become activated only after the conclusion of the mating season (Crews,

1984; Whittier & Crews, 1987). Red-sided garter snakes (Thamnophis sirtalis)display such a

dissociated pattern as the gonads do not become active until after the brief summer breeding

season; sex behavior is not dependent on circulating androgens. Instead, the increase in sex

steroid hormones the previous summer appear to program the brain and regulate mating the

following spring (Crews, 1991; Crews, Robker, & Mendonca, 1993). These animals are not

photoperiodicper seas the major cue for the expression of spring mating behavior is prolonged

exposure to low temperatures (Lutterschmidt, LeMaster, & Mason, 2006; Whittier, Mason,

Crews, & Licht, 1987).

However, a role for pineal melatonin has been suggested in the regulation of spring

breeding. Depending upon the time of year the pineal is removed, spring courtship behavior is

either disrupted or enhanced. Pinealectomy prior to hibernation prevents males from courting

females on spring emergence but if conducted on males that are actively courting, there is no

effect on behavior (Crews, Hingorani, & Nelson, 1988; Mendonca, Tousignant, & Crews, 1996a,

1996b; Nelson, Mason, Krohmer, & Crews, 1987). Interestingly, in those few males that fail to

court on emergence, pinealectomy induces robust courtship behavior. Measurements of

circulating concentrations of melatonin reveal significantly higher levels in males that are

actively courting but in males that fail to court, the circadian rhythm is absent. Shielding the

eyes disrupts the retinal melatonin rhythm and abolishes the effect of pinealectomy (Crews,

Hingorani, & Nelson, 1988; Mendonca, Tousignant, & Crews, 1996a, , 1996b; Nelson, Mason,

Krohmer, & Crews, 1987). Exogenous melatonin fails to reinstate mating behavior (Mendonca,

Tousignant, & Crews, 1996b). It is likely that melatonin communicates temperature rather than

photoperiod information as these animals are nonresponsive to day length. Circulating

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melatonin also varies with temperature in the diamondback water snake (Nerodia rhombifera)

(Tilden & Hutchinson, 1993).

Conclusions and Future Directions

The study of amphibian and reptile photoperiodism has revealed evolutionary processes

that underlie seasonality in all vertebrate taxa. Changes in the photoperiodic machinery over

evolutionary time, including a reduced number of pineal efferents and a greater reliance on the

pineal itself for the production of melatonin, could not be appreciated by studying mammalian or

avian systems alone. Of particular interest is the manner by which day length signals are

communicated to all target tissues. In mammals, melatonin productions is under the direct

control of a multisynaptic pathway from the retina and it serves as the principle if not the only,

cue that transduces photoperiodic information from an environmental signal into a physiological

one. However, in all other vertebrate taxa the source, targets and control of production and

release of melatonin are all more equivocal and subject to regulation by non-photic cues. In any

case, to fully appreciate the way in which photoperiodic measurement of seasonal time evolved it

is critical to studies taxa that retain traits similar to those of ancestral vertebrates.

There is certainly much more work to do as the physiological substrates that underlie

photoperiodism in reptiles and particularly amphibians have not been fully described.

Additionally, research on photoperiodism in these taxa has dropped off in recent years and as

such many of the modern neuroendocrine and molecular techniques have not been used in these

species although there are some notable exceptions (e.g., (Inai, Nagai, Ukena, Oishi, & Tsutsui,

2003; Izzo et al., 2006; Magnone, Jacobmeier, Bertolucci, Foa, & Albrecht, 2005; Malatesta et

al., 2007; Neal & Wade, 2007). Finally, the demonstrated diversity in the reproductive strategies

present in these two vertebrate taxa combined with studies of additional species with differing

life history characteristics will deepen our understanding of the causes and constraints of

reproduction in vertebrate in general.

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Figure Captions.

1. Summary of the relationships among photoperiod, temperature and photoperiod in the

regulation of reproduction in green anole lizards. Shaded bars indicate the part of the year when

the lizards are most responsive to photoperiod in the wild..Reproduced by permission of the

Ecological Society of America

2. Average ovarian and oviducal responses ( SEM) to either 10 or 50 g of exogenous follicle

stimulating hormonefollowing removal of an atretic or non-atretic previtellogenic follicle in the

green anole lizard. Dashed lines indicate responses in saline-injected animals. Atretic follicles

may mediate the photo- and thermorefractoriness exhibited by female green anoles after the

mating season (Crews & Licht, 1974). Copyright 1974, The Endocrine Society.

3. An underlying circannual rhythm in environmental sensitivity is indicated by the more rapid

rate of ovarian recrudescence in winter dormant female green anole lizard to a stimulatory

environmental regimen as the period of normal breeding nears. Dates indicate time of year. In all

studies reproductively inactive females were exposed to an unseasonal environmental conditions

(14:10 LD photic cycle with a corresponding 32:23 C daily thermal cycle and constant 60-70%

relative humidity. (Crews and Garrick, 1980). Courtesy Society for the Study of Amphibians

and Reptiles.

4. Seasonal patterns of photoperiod, ambient temperatures, testes size, circulating androgens,

and gonadotropins and spermatogenic activity in green frogs. (Rastogi, Iela, Saxena, & Chieffi,

1976).Reprinted with permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.

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Figures.

Figure 1 (Licht, 1970)

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Figure 2 From (Crews & Licht, 1974).

10 50

0

10

20

30

40

50

60

70

CA-X

Foll.-X

Ovary

Control

mg/gBodyWeight

10 50

5

10

15

20CA-X

Foll.-X

Oviduct

Control

Dose of FSH (g)

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Figure 3 From (Crews & Garrick, 1980)

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Figure 4 From (Rastogi, Iela, Saxena, & Chieffi, 1976)