691

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Turkish Journal of Zoology Turk J Zool(2016) 40 691-703copy TUumlBİTAKdoi103906zoo-1508-53

Reproductive pattern and sex hormones of Calotes emma Gray 1845 andCalotes versicolor Daudin 1802 (Squamata Agamidae)

Worawitoo MEESOOK1 Taksin ARTCHAWAKOM2 Anchalee AOWPHOL1 Panas TUMKIRATIWONG11Department of Zoology Faculty of Science Kasetsart University Bangkok Thailand

2Sakaerat Environmental Research Station Thailand Institute of Scientific and Technological Research Ministry of Science and Technology Nakhon Ratchasima Thailand

Correspondence fscipntkuacth

1 IntroductionLizards (including snakes) are the most speciose living clade of reptiles with almost 7200 species Even excluding snakes lizards are still the most speciose extant reptiles with approximately 4450 species Lizards which belong to the family Agamidae are classified into 52 genera with approximately 400 species (Vitt and Caldwell 2009) 31 species of which are found in Thailand (Laohachinda 2009) The genus Calotes Cuvier 1817 a genus of Agamidae currently contains 23 species (Hartmann et al 2013) There are 7 species of Calotes known to occur on the Southeast Asian mainland C chincollium Vindum 2003 C htunwini Zug and Vindum 2006 C irawadi Zug et al 2006 C jerdoni Guumlnther 1870 C emma Gray 1845 C mystaceus Dumeacuteril and Bibron 1837 and C versicolor Daudin 1802 The latter 3 species are well known to Indochina including Thailand (Zug et al 2006 Hartmann et al 2013)

Male reproductive displays are directly correlated to testicular hypertrophy in the majority of seasonally breeding vertebrates and are accompanied by an elevated

level of circulating sex steroids This is termed an associated reproductive pattern (Crews 1984 1999 Crews et al 1984 Licht 1984 Norris 2013) In contrast in a small number of vertebrate species such as some turtles snakes and bats males mate at a time when their gonads are quiescent (QU) and the levels of circulating sex steroids are low (Garstka et al 1982) This pattern of reproduction is referred to as a dissociated reproductive pattern (Volsoslashe 1944 Crews 1976 1984 1999 Lofts 1977 Licht 1984 Norris 2013) In snakes especially in the family Colubridae various terms have been used to describe the male reproductive patterns observed in different species Volsoslashe (1944) introduced the term prenuptial spermatogenesis to describe sperm production that occurs immediately prior to mating and postnuptial spermatogenesis to describe sperm production that occurs after mating Aldridge et al (2009) introduced the term preovulatory spermatogenesis to describe sperm production immediately prior to ovulation and postovulatory spermatogenesis as sperm production that occurs after ovulation They assumed that androgenesis was associated with the development of secondary sex characters and mating behavior

Abstract We monitored testicular and ovarian morphologies seminiferous tubules the sexual segments of the kidneys (SSK) follicular histologies and male testosterone and female estradiol to define the reproductive pattern of Calotes emma and Calotes versicolor Samples were collected monthly at Sakaerat Environmental Research Station in Thailand for 1 year Testicular hypertrophies occurred at a time characteristic for each species with their time course corresponding well to both active spermatogenesis and the hypertrophied SSK Gravid females were also found at a time characteristic for each species In active reproductive females oviductal eggs were concomitantly encountered with ovarian vitellogenic follicles The previtellogenic and vitellogenic follicles corresponded well to granulosa layer alterations The distinct large pyriform cells were present in the granulosa layer of previtellogenic follicles but disappeared from the vitellogenic follicles Male testosterone levels rose during testicular and SSK hypertrophies and female estradiol levels increased during active reproductive stages of late vitellogenic follicles and gestation We suggest that the reproductive patterns of C emma and C versicolor fall into the same reproductive pattern of annual continual reproduction but that the time courses of such events are different in the 2 Calotes and even in individuals of the same Calotes population

Key words Estradiol sexual segment kidney spermatogenesis testosterone vitellogenesis

Received 28082015 AcceptedPublished Online 29032016 Final Version 24102016

Research Article

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Vitt and Caldwell (2009) added that the currently known diversity of seasonal patterns of tropical squamate reproduction suggests that no single explanation is sufficient Snakes are included in the same order (Squamata) but demonstrate a more diverse reproductive pattern in contrast to lizards Tumkiratiwong et al (2012) studied the reproductive patterns of captive male and female Naja kaouthia monocled cobra Lesson 1831 suggesting that its reproductive pattern exhibited either postnuptial spermatogenesis or a dissociated reproductive pattern

There is no detailed information available on testicular and ovarian cycles plasma sex hormonal profiles during gonadal cycles or male reproduction-associated sexual segment of the kidney (SSK) especially in Calotes emma This paper therefore monitored annual alterations of reproductive organs of males and females of 2 Calotes species ie C emma and C versicolor and investigated male and female sex hormones male testosterone and female estradiol to define the reproductive patterns of 2 such Calotes species We expected that annual male and female reproductive alterations of the testes male SSK and the ovaries would require underlying morphological

and histological investigations and also that annual sex hormonal levels would reveal the reproductive patterns of 2 such Calotes species

2 Materials and methods21 AnimalsWe caught the adult lizards monthly over 1 year by hand or with a noose in the 3 forest types a dry evergreen forest a deciduous dipterocarp forest and an ecotone forest at Sakaerat Biosphere Reserves of the Sakaerat Environmental Research Station (14deg26prime33Primendash14deg32prime50PrimeN 101deg50prime43Primendash101deg57prime21PrimeE 720ndash770 m above sea level) located at Nakhon Ratchasima Province northeastern Thailand We classified adults of C emma and C versicolor based on external morphological differences (Figure 1) The adult stages of the 2 species were diagnosed based on the internal morphology of active male and female gonads (Figure 1) and expressed in terms of the snoutndashvent length (SVL) as (1) male C emma gt578 cm and C versicolor gt540 cm (2) female C emma gt657 cm and C versicolor gt599 cm We attempted to collect 10 adult males and females but in some months the samples were smaller than expected and sometimes we could not collect any samples

Figure 1 External morphologies of the representatives of 2 Calotes species Top C versicolor A no patch of granular scales in front of forelimb insertion bottom left C emma B crescent-shaped patch of small granular scales in front of forelimb insertion and C large postorbital spine present Bottom middle dissections of urogenital morphology of male Calotes T testis Vd vas deferens K kidney bottom right female Calotes OvaF ovarian follicles OviE oviductal eggs Lines were drawn from a total preparation (in ventral view)

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The months that lacked samples in each species are shown in parentheses as follows males of C emma (May July and August) and C versicolor (December) females of C emma (February to March May and December) and C versicolor (March to May July October and December) We anesthetized the samples with diethyl ether and collected blood samples by puncturing the cardiac chamber however it was not possible to acquire samples from those lizards with very small cardiac chambers Blood samples were kept at 4 degC in heparinized vials and centrifuged within 2ndash3 h following blood collection The blood was centrifuged at 1600 times g for 10 min Plasma was then aspirated off and frozen at ndash79 degC for later analysis of plasma levels in male testosterone and in female estradiol22 External and internal reproductive morphologiesWe measured SVL which ranges from the snout tip to the anterior margin of the vent We sacrificed male and female lizards to investigate the general reproductive morphologies (Figure 1 bottom middle male bottom right female) and measured follicular size and testicular mass Each specimen was weighed to the nearest 001 g its SVL was measured to the nearest 005 mm23 Testicular male SSK and ovarian histologiesTestes male SSK and ovaries were excised and fixed in a 10 vv buffered neutral formalin solution processed by the paraffin technique (Avwioro 2011) The tissue was cut in a cross-section to 6 microm in thickness using a LEICA RM2145 (Nussloch Germany) Sections were stained with hematoxylin and eosin The reproductive stage of adult females was determined on the basis of the presence or absence of types of pyriform cells in the granulosa layer Follicles with the pyriform cells which appeared in the granulosa layer were considered to be previtellogenic follicles and follicles not containing the pyriform cells were considered to be vitellogenic follicles (Tumkiratiwong et al 2012) Females having vitellogenic follicles andor oviductal eggs were considered to be at the active reproductive stage while females having only previtellogenic follicles were considered to be at the QU reproductive stage Females with regressed follicles were considered to be at the postparturient stage Males with the appearance of sperm bundles andor free sperm in the seminiferous tubules (ST) were assessed as being at the active reproductive stage while males without those attributes were assessed as being at the inactive reproductive stage (Tumkiratiwong et al 2012) Males with SSK that had strongly eosinophilic-stained granules were regarded as being at the active reproductive stage (Sever et al 2002)24 Categorization of female individuals based on reproductive statusSimilar-sized follicles were organized into distinct groups The total number and diameter of follicles belonging to

the group with the largest-sized follicles were recorded from both ovaries The follicular size was measured with a Vernier caliper (0ndash150 times 002 mm) Follicles greater than or equal to 25 mm in diameter represented vitellogenic status (Shanbhag and Prasad 1993)

Since ovarian follicular development ovulation and gestation occur asynchronously in a population individuals with differing reproductive statuses are encountered Therefore the data from the reproductive phase were classified based on the reproductive status of individuals rather than on a monthly classification as follows (1) the QU stage previtellogenic follicles sized lt25 mm in diameter (2) the early vitellogenic (EV) stage initiation of vitellogenesis with follicles sized 25ndash50 mm in diameter and without oviductal eggs (3) the late vitellogenic (LV) stage late period of vitellogenesis with follicles sized gt50 mm in diameter (4) the early gestation (EG) stage oviductal eggs with previtellogenic follicles sized lt25 mm in diameter (5) the midgestation (MG) stage oviductal eggs with vitellogenic follicles sized 25ndash50 mm in diameter and (6) late gestation (LG) stage oviductal eggs with vitellogenic follicles sized gt50 mm in diameter (modified from Radder et al 2001)25 Measurement of testosteroneThe plasma level of testosterone was measured by a 125I radioimmunoassay (RIA) We added a 500-microL sample of plasma to 50 mL of dichloromethane in a screw-top glass extraction tube We then capped the mixture and mixed it for 60 min by gentle inversion with an end-over-end rotator and then centrifuged the sample for 5 min at 1600 times g to separate the layers The upper phase was aspirated without disturbing the interface 20 mL of the lower phase was then transferred to a clean 12 times 75 mm glass tube and evaporated to dryness under a gentle stream of nitrogen at 37 degC Finally we reconstituted the extract with 200 microL of testosterone buffer The testosterone extraction procedure was performed using a Coat A Count Testosterone RIA Kit (Diagnostic Products Los Angeles CA USA) The intra- and interassay variations expressed as coefficients of variation (CVs) were 84 and 79 respectively The approximate sensitivity of this assay was 40 pgmL The cross-reactivity with androstenedione was 05 The spiking recovery values averaged 983 plusmn 06 Dilutions of 50 25 and 125 of the undiluted concentration of 7300 pgmL were 3490 pgmL 1700 pgmL and 780 pgmL respectively26 Measurement of estradiolThe plasma level of estradiol was measured by a 125I RIA We added 250 microL of plasma to 20 mL of diethyl ether in a screw-top glass extraction tube and then capped and mixed it by gentle inversion with an end-over-end rotator for 30 min It was centrifuged for 5 min at 1600 times g to separate the layers The lower (aqueous) phase was

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frozen using dry ice the organic phase was then decanted into another vial and evaporated to dryness under a gentle stream of nitrogen at 37 degC Finally the extract was reconstituted with 250 microL of estradiol buffer The estradiol extraction procedure was performed using a Coat A Count Estradiol (TKE2) RIA kit (Diagnostic Products) The intra- and interassay variations calculated as CVs were 53 and 64 respectively The approximate sensitivity of this assay was 10 pgmL The specificity of cross-reactivity with estradiol was 032 The spiking recovery values averaged 968 plusmn 33 Dilutions of 50 25 125 and 625 of the undiluted concentration of 2309 pgmL were 1144 pgmL 600 pgmL 279 pgmL and 148 pgmL respectively27 Statistical analysisTesticular masses testosterone levels ovarian weights estradiol levels and diameters of the largest follicle were expressed as the mean plusmn standard error of the mean (SEM) The KolmogorovndashSmirnov test and Levenersquos test were used to determine if the data were normally distributed and the homogeneity of variance respectively Nonparametric tests were used as all data mentioned above were nonnormal and heteroscedastic Therefore the KruskalndashWallis H test was used to test for differences in the ovarian weights the estradiol levels and the diameters of the largest follicle among the reproductive stages of nongestation periods The MannndashWhitney U test was then used to compare differences

in the ovarian weights the estradiol levels and the diameters of the largest follicle between each reproductive stage of the various follicular growths of C emma only because of the insufficient sample size There was no statistical analysis of differences in male testosterone levels or testicular masses based on months due to the small amount of data available Spearmanrsquos correlation coefficients were used to determine relationships between the testicular mass and testosterone and the diameter of the largest follicles and estradiol levels The level of significance was set to P lt 00528 Ethical aspectsThis study was approved by the Ethics Committee of the Department of National Parks Wildlife and Plant Conservation Ministry of Natural Resources and Environment Thailand (License No 090930218344)

3 Results31 Annual alterations in male and female reproductive morphologies of Calotes Annual changes in testicular morphological events between C emma and C versicolor are depicted in Figure 2 The representative C emma testes were recrudesced in December continued to hypertrophy from January to April and in June and completely regressed from September to November In the representative C versicolor the testes became hypertrophied from January to September and regressed from October to November

Figure 2 Schematics of annual changes in testicular size Top C emma bottom C versicolor Notes T testis Vd vas deferens K kidney JanndashDec denotes from January to December All scale bars equal 5 mm

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Annual changes in ovarian morphological events (Figure 3) and annual changes in the number of follicular types and egg types (Table 1) of the representative 2 Calotes species are shown In the representative C emma we found that in January April from June to July and from August to November follicles were in the EV EG LV and QU stages respectively (Figure 3 top Table 1) In the representative C versicolor we found that in January and February the ovaries contained only QU follicles in June and August QU and EV follicles and oviductal eggs in September QU follicles and oviductal eggs in November only QU follicles (Figure 3 bottom Table 1)32 Annual histological alterations in ST SSK and ovaries of Calotes In the male representative C emma the ST and SSK were hypertrophied from January to April and in June then regressed from September to November becoming active again in December (Figure 4 top left and top right respectively) Spermatozoal masses were contained inside the hypertrophied ST Spermatogonia initially appeared in November additionally a few types of germ cells occurred but there were still no active spermatozoa in December (Figure 4 top left) In the representative C versicolor both ST and SSK were active from January to September but were inactive from November to December (Figure 5 top left and top right)

We found that in the female representative C emma active ovaries contained follicles of vitellogenic stages in January and June but inactive ovaries contained previtellogenic and atretic follicles in August (Figure 4 bottom left and bottom right) In the representative C versicolor ovaries contained previtellogenic and atretic follicles in January and both previtellogenic and EV follicles in June (Figure 5 bottom left and bottom right) Additionally we found corpus luteum in the ovaries of 1 representative in September (Figure 5 bottom left and bottom right) We also observed cellular alterations in the granulosa layer of both C emma and C versicolor It was demonstrated that inside the granulosa layer of the previtellogenic follicles both types of small and pyriform cells appeared However both such cell types disappeared in the granulosa layer of the vitellogenic follicles (Figures 4 and 5 bottom right)33 Annual plasma testosterone levels and testicular masses in Calotes Annual variations in plasma testosterone levels and testicular masses between the 2 Calotes species are depicted (Figures 6a and 6b) In the representative C emma plasma testosterone levels and testicular masses initially increased in December peaked in March and thereafter gradually reduced from April to November (Figure 6a) In the representative C versicolor plasma testosterone

Figure 3 Schematics of seasonal changes in ovarian size Top C emma bottom C versicolor Notes OvaF ovarian follicles OviE oviductal eggs Ovi oviduct All scale bars equals 5 mm JanndashNov denotes from January to November

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levels and testicular masses initially increased in January peaked in April and thereafter gradually reduced from May to November (Figure 6b) The changes in testicular masses tended to correspond well to the changes in plasma testosterone levels between the 2 species in the genus Calotes (r = 0789 P = 0001 and r = 0732 P = 0001 for C emma and C versicolor respectively)34 Annual plasma estradiol and ovarian cycles in Calotes Variations in ovarian masses and the diameter of the largest follicle were observed between the 2 Calotes species

during their reproductive stages (Table 2) The ovarian weights and the diameters of the largest follicles differed significantly from the QU stage to the LV stage regarding the growth of ovarian follicles (P = 0001) in C emma It was also observed that the growth of ovarian follicles concomitantly occurred with the growth of oviductal eggs during the gestation period of the 2 species

Variations in plasma E2 levels in relation to the diameters of the largest follicles between the 2 Calotes species during reproductive stages are graphically depicted (Figures 7a and 7b) Regarding the groups of follicular sizes

Table 1 Annual changes in numbers of follicular and egg types according to Calotes species

Follicular size and eggs Jan Feb Apr May Jun Jul Aug Sep Oct Nov

C emmalt25 mm 21 - 18 - 18 22 41 33 24 2325ndash50 mm 10 - - - 4 1 - - - -gt50 mm - - - - 8 5 - - - -Oviductal eggs - - 4 - - - - - - -C versicolorlt25 mm 16 28 - - 28 - 19 16 - 1025ndash50 mm - - - - 5 - - - - -gt50 mm - - - - - - - - - -Oviductal eggs - - - - 5 - 6 5 - -

Notes JanndashNov denotes January to November respectively

Figure 4 Photomicrographs of annual changes in C emma Top left testes top right male SSK bottom left ovaries bottom right granulosa layers (GL) Notes SZ spermatozoa ST seminiferous tubules SSK sexual segments of kidney AF atretic follicle PF previtellogenic follicle VF vitellogenic follicle P pyriform cells S small cells

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of C emma plasma E2 levels were the difference between the QU and the EV stages (U = 900 P = 0090) the QU and the LV stages (U = 200 P = 0009) and the EV and the LV stages (U = 200 P = 0083) Additionally the diameters of the largest follicles were the difference between the QU and the EV stages (U = 000 P = 0004) the QU and the LV stages (U = 000 P = 0004) and the EV and the LV stages (U = 000 P = 0021) The correlation coefficient between plasma E2 and the diameter of the largest follicle was 082 (P = 0001) and 032 (P = 0365) of C emma and C versicolor respectively

4 DiscussionAnnual variations in the timing of male and female reproductive stages were encountered among populations of C emma and C versicolor even in individuals of the same population Annual changes in testicular sizes were categorized into 2 phases in both species (1) an active hypertrophied testicular phase and (2) an inactive

regressed testicular phase However the timing of those 2 events appeared asynchronously in the 2 Calotes species even in individuals of the same population (data not shown here) The testes were hypertrophied with active spermatozoal production from December to June and January to September in the representative C emma and C versicolor respectively A study by Gouder and Nadkarni (1979) showed that males of C versicolor widely distributed in India were spermatogenetically active from April to September Active vitellogenic follicles and oviductal eggs were concomitantly encountered in C emma and C versicolor Therefore these 2 Calotes species exhibited polyautochrony and multiclutches Radder et al (2001) reported that Indian garden lizards C versicolor showed polyautochrony and multiclutches The representative gravid lizards were found in April in C emma and in June and from August to September in C versicolor Gravid C versicolor whose habitat is in India was encountered from May to October (Shanbhag and Prasad 1993) We

Figure 5 Photomicrographs of annual changes in C versicolor Top left testes top right male SSK bottom left ovaries bottom right granulosa layers (GL) Notes SZ spermatozoa ST seminiferous tubules SSK sexual segments of kidney AF atretic follicle PF previtellogenic follicle VF vitellogenic follicle P pyriform cells S small cells CL corpus luteum

Figure 6 Annual profiles (mean plusmn SEM) of testosterone levels and testicular masses (a) C emma (b) C versicolor Notes JanndashDec denotes January to December The numbers (in parentheses) represent the number of analyzed samples in each month

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are likely to suggest after studying the annual alterations in the male and female reproductive morphologies of the 2 Calotes species that the active reproductive events of both males and females of the Calotes species lasted nearly 1 year with only a few months of reproductive arrest which is especially seen in C versicolor

According to our investigations on annual histological alterations in ST and male SSK timing of the 2 Calotes species both ST and SSK were in active spermatogenic and

hypertrophied stages respectively which corresponded well with the timing of the testicular hypertrophied stage mentioned above In other words the timing of arrested spermatogenesis and regressed SSK was in accordance with that of the regressed testes Likewise we confirmed that the 2 Calotes species have an active reproductive stage that is much longer than the inactive reproductive stage

SSK is present in a variety of male snakes and lizards but is absent in both turtles (Regaud and Policard 1903)

Table 2 Changes in ovarian weight and diameter of the largest follicle in Calotes during the reproduction cycle

Reproductive stages N Ovarian weights Diameter of the largest follicle

C emma (20)QU 11 002 plusmn 000a 178 plusmn 006a

EV 4 011 plusmn 009b 350 plusmn 033b

LV 4 171 plusmn 049c 802 plusmn 085c

EG 1 002 240MG - - -LG - - -C versicolor (9)QU 4 002 plusmn 000 152 plusmn 199EV 1 02 428LV - - -EG 3 002 plusmn 000 222 plusmn 069MG 2 011 plusmn 009 436 plusmn 036LG - - -

Data are presented as mean plusmn SEM The differences in superscript alphabets indicate the significant differences among the reproductive stages of nongestation at P lt 001 (N sample sizes)Notes QU quiescent EV early vitellogenic LV late vitellogenic EG early gestation MG mid-gestation and LG late gestation

Figure 7 Changes in the plasma levels of estradiol and the diameter of the largest follicle (a) C emma (b) C versicolor Notes QU quiescent EV early vitellogenic LV late vitellogenic EG early gestation MG mid-gestation LG late gestation Data are presented as mean plusmn SEM The differences in superscript alphabets (estradiol levels) and in the numbers of asterisks (diameters of the largest follicles) indicate the significant differences between the various follicular sizes at P lt 001 The number (in parentheses) represents the analyzed samples in each month

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and crocodilians (Fox 1952) Bishop (1959) found that the testes of the male garter snake Thamnophis sirtalis were spermatogenically active during the same time as the hypertrophied SSK during the active reproductive period the diameter of the SSK tubule was 5 times greater than that of the SSK tubule during the inactive reproductive period However SSK development in female lizards has been reported in the genus Cnemidophorus (Del Conte 1972 Del Conte and Tamayo 1973) and Scincella laterale (Sever and Hopkins 2005) They suggested that the females had a low level of natural androgens which caused the SSK development (Del Conte and Tamayo 1973 Sever and Hopkins 2005) In the present study we did not monitor the annual seasonal alterations in female SSK The hypertrophy of the SSK is synchronous with androgen secretion and spermatogenic activity (Sever and Hopkins 2005) Norris (2013) also stated that the SSK of sexually active squamates undergoes hypertrophy and is under the influence of androgens In this study we did not investigate any alterations in annual SSK with annual androgen secretion but we did demonstrate that the hypertrophy and the regression of SSK changed seasonally and synchronously with the active spermatogenic event and the spermatogenic arrest respectively In the Iberian rock lizard Lacerta monticola SSK secretions form a copulatory plug that adheres to the femalersquos cloaca following copulation to occlude oviductal openings however such a plug does not prevent subsequent mating nor does it reduce the femalersquos attractiveness (Moreira and Birkhead 2003)

With our investigations on annual alterations in female ovarian morphologies between 2 Calotes we found that individuals in the same Calotes species showed different timing of reproductive events throughout a 1-year period (the data are not shown here) Additionally there was quite clear evidence that QU EV and LV follicles and oviductal eggs overlapped among individuals within the same populations of both Calotes species Female reproductive status is definitely distinguishable between the 2 Calotes species Gravid lizards were encountered in 1 individual of C emma in April and in 3 individuals of C versicolor in June August and September Shanbhag et al (2000) reported that female C versicolor showed inactive reproduction from December to April and gravidity was encountered from May to October

We found that in the previtellogenic follicles (QU and EV) of females of both Calotes species the granulosa layer contained 2 types of cells pyriform and small cells Uribe et al (1996) stated that in squamates the follicular epithelium or granulosa initially consists of small cuboidal cells but differentiates during the previtellogenic phase and becomes multilayered and polymorphic by the presence of unique flask-shaped pyriform cells intermediate cells and

small cells These pyriform cells differentiate from small somatic follicular cells early in follicular development via the intermediate-cell stage to become nurse cells in direct contact with the developing oocytes (Maurizii et al 2004) Differentiation of the small cells into pyriform cells appears to be linked to the progressive appearance of glycoproteins with terminal α-N-acetylgalactosamine residues on the cell surface which may be involved in fusion between the oocyte and the follicle cell membranes as well as maintenance of the differentiated pyriform cells The pyriform cells are connected to the oocyte via intercellular bridges containing a cytoskeleton of α-tubulin and cytokeratin microtubules (Maurizii et al 2004) Tumkiratiwong et al (2012) also demonstrated that the previtellogenic follicles of the captive monocled cobra Naja kaouthia had many pyriform cells in the granulosa layer but fewer in the vitellogenic follicles In this study the pyriform cells disappeared when the follicles entered the vitellogenic stage Andreuccetti (1992) studied the differentiation of pyriform cells and their contribution to oocyte growth in 3 lizards namely Tarentola mauritanica Cordylus wittifer and Platysaurus intermedius and a colubrid snake Coluber viridiflavus and revealed that pyriform cells differentiate from small follicle cells via intermediate cells after establishing an intercellular bridge with the oocyte Once pyriform cells are differentiated they display ultrastructural features indicative of synthetic activity including abundant ribosomes Golgi membranes vacuoles mitochondria and lipid droplets These cellular components extend to the apex of the cell at the level of the intercellular bridge suggesting that constituents of pyriform cells may be transferred to the oocyte Pyriform cells and the oocytes may fulfill similar vitellogenic functions The establishment of an intercellular bridge may represent a crucial event in the development of an integrated system in which pyriform cells and oocytes cooperate Norris (2013) reported that the squamate granulosa contains the pyriform cells which are in direct contact with the developing oocyte and are apparently involved with early steps in oocyte development soon after the onset of vitellogenesis As ovulation approaches the granulosa cells as well as some thecal cells accumulate cholesterol-positive lipids and proliferate and luteinize to form corpora lutea following ovulation Follicular atresia is a common occurrence in reptilian ovaries as in other vertebrates (Norris 2013) as we found the corpora lutea in the follicle in accordance to oviductal egg appearances of a representative C versicolor collected in September Additionally as shown in Table 1 several atretic follicles lt25 mm in diameter were commonly encountered in C emma and C versicolor

Based on both morphological and histological investigations we found that testes ST and SSK were

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concomitantly active and were associated with high levels of plasma testosterone We also demonstrated that there were high correlations between levels of plasma testosterone (T) and testicular mass where annual changes occurred in the same direction as the testicular size and time of spermatogenetic events among the males of the Calotes species Radder et al (2001) reported that in the male tropical or oriental garden lizard C versicolor plasma T is highest during the breeding season which correlated with testis mass and reproductive behavior Changes in T levels are associated with high spermatogenetic activity Radder et al (2001) also stated that the changes in plasma T levels during different phases of the male reproductive cycle in C versicolor follow a reproductive pattern of a prenuptial type of spermatogenesis that is similar to that of some other species of lizards the spiny-tailed lizard Uromastix hardwicki (Arslan et al 1978a) the viviparous lizard Lacerta vivipara (Courty and Dufaure 1982) the western shingleback lizard Tiliqua (Trachydosaurus) rugosa (Bourne et al 1986) the male lizard Podarcis s sicula (Ando et al 1990) Podarcis s sicula Raf (Ando et al 1992) the white-throated savanna monitor Varanus albigularis (Phillips and Millar 1998) and the male brown anoles Anolis sagrei (Tokarz et al 1998)

We found that estradiol (E2) levels increased in vitellogenic females its high levels were associated with the presence of the largest vitellogenic follicles in the 2 Calotes species Radder et al (2001) reported that in female C versicolor with overlapping reproductive events such as vitellogenesis and gestation E2 was at low levels when the ovaries were regressed and at high levels at vitellogenic follicular recruitment reaching peak level at the time of preovulatory follicles The same patterns of E2 secretion were found again when the second set of follicles underwent vitellogenesis (Radder et al 2001) Surprisingly Amey and Whittier (2000) reported that in female bearded dragons Pagona barbata plasma E2 was low or nondetectable across all reproductive states In C versicolor E2 levels were low in nonreproductive females with small previtellogenic follicles and those in the EG phase (Radder et al 2001) We do not discuss the level of the plasma progesterone (P) during the gestation period as its level was not detectable in this study The gravid lizards in EG exhibited low plasma E2 but high P levels and the highest P levels coincided with eggshell production P levels declined after eggshell formation as reported in other gravid individuals in several species of lizards that do not possess vitellogenic follicles of the subsequent clutch including C versicolor (Radder et al 2001) Uromastix hardwicki (Arslan et al 1978b) Agama atra (Van Wyk 1984) Eumeces obsoletus Scelporus undulatus and Crotaphytus collaris (Masson and Guillette 1987) and Psammodromus algirus (Diaz et al 1994) However a

decline in P levels in MG with vitellogenic follicles did not seem to facilitate recruitment or growth of the subsequent set of vitellogenic follicles in gravid Sceloporus jarrovi (Guillette et al 1981)

In the present study there were variations in the timing of breeding between the 2 Calotes species and even within populations of the same species We could not relate the copulation timing of 2 such Calotes species to gonadal activity or sex hormonal surges as the timing of natural mating could not be observed during the times we collected data Lizard species that inhabit temperate zones have mostly exhibited seasonal reproduction (Fitch 1970 Licht 1984 Pianka and Vitt 2003) The 10 lizard species that have been studied widely to date exhibit an associated reproductive pattern (Lovern 2011) that is green anoles Anolis carolinensis (Crews 1980 Lovern et al 2004) brown anoles Anolis sagrei (Lee et al 1989 Tokarz 1998) eastern fence lizards Sceloporus undulates (Cox et al 2005) mountain spiny lizards Sceloporus jarrovi (Woodley and Moore 1999) tree lizards Urosaurus ornatus (French and Moore 2008) wall lizards Podarcis sicula (Putti et al 2009) common lizards Lacerta vivipara (Vercken and Clobert 2008) little striped whiptail lizards Cnemidophorus inornatus (Crews 2005) garden lizards Calotes versicolor (Shanbhag 2003 Lovern 2011) and leopard geckos Eublepharis macularius (Rhen et al 2005) The temperate Florida populations of the brown anole Anolis sagrei show a strong seasonality in reproduction (Lee et al 1989) while the tropical Caribbean (Licht and Gorman 1970 Sexton and Brown 1977) and Hawaiian populations of this species (Goldberg et al 2002) show a less-pronounced seasonality in which reproductively active individuals can be found throughout the year Although individuals within a population of many tropical lizard species can breed at any time no individuals within the population breed year-round (Lovern 2011) Additionally Vitt and Caldwell (2009) stated that the belief used to be that tropical squamates had continuous reproduction in aseasonal tropical environments or reproduced during the wet season in a wetndashdry seasonal tropical environment Many tropical lizard species namely the anoles Anolis acutus (Ruibal et al 1972) Anolis limifrons (Sexton et al 1971) and Anolis opalinus (Jenssen and Nunez 1994) as well as the gecko Cyrtodactylus malyanus the flying lizard Draco melanopogon (Inger and Greenberg 1966) and the parthenogenetic oviparous whiptail lizard Cnemidophorus nativo (Menezes et al 2004) showed slightly more frequent breeding during the wet season than during the dry season (Jenssen and Nunez 1994)

Reproductive patterns can be described in a variety of ways but not all species fit neatly into such categorizations However 2 general types of reproductive patterns are

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recognized in terms of prenuptial and postnuptial reproductive patterns (Lance 1998) Prenuptial reproductive pattern terms such as gonadal recrudescence sex steroid production and gametogenesis occur in advance of mating whereas postnuptial reproductive patterns occur following mating In other words in a high-elevation population of Sceloporus grammicus in Parque Nacional de Zoquiapan in central Mexico an active reproductive event occurring in the early fall is described as dissociated from testicular recrudescence in males but is associated with the initiation of ovarian recrudescence in females (Guillette and Casas-Andreu 1980 1981 Zuniga-Vega et al 2008) This is in contrast to S grammicus from Teotihuacan Mexico in which testicular recrudescence and breeding occur in the summer and fall at the onset of female ovarian recrudescence (Jimenez-Cruz et al 2005) In S mucronatus from Valle de la Cantimplora Mexico peak testicular recrudescence and mating occur during the summer prior to ovarian recrudescence which does not occur until several months later (Ortega-Leon et al 2009) This is distinct from many fall-breeding

populations elsewhere (Mendez-De La Cruz et al 1994 Villagran-Santa Cruz et al 1994) The examples above demonstrate that gonadal activity and mating behavior are clearly variable but hormone analyses have not been performed in these species and so endocrine relationships cannot be assessed at this point

In conclusion we suggest that the males and females of the 2 Calotes species have much more prolonged active reproductive phases than inactive reproductive phases The reproductive patterns of C emma and C versicolor were classified into the same reproductive pattern of continual reproduction

AcknowledgmentsWe thank the Department of Zoology of Kasetsart University for financial support We also thank the staff of Sakaerat Environmental Research Station Nakhon Ratchasima Province for devoting time for research collaboration We also thank Mrs Sureerat Sangkrut for drawing all illustrations In addition we wish to thank the anonymous referees for many helpful suggestions

References

Aldridge RD Goldberg SR Wisniewski SS Bufalino AP Dillman CB (2009) The reproductive cycle and estrus in the colubrid snakes of temperate North America Contemp Herpetol 4 1-31

Amey AP Whittier JM (2000) Seasonal patterns of plasma steroid hormones in males and females of the bearded dragon lizard Pogona barbata Gen Comp Endocrinol 117 335-342

Andreuccetti P (1992) An ultrastructural study of differentiation of pyriform cells and their contribution to oocyte growth in representative Squamata J Morphol 212 1-11

Ando S Panno ML Ciarcia G Imbrogno E Buffone M Beraldi E Sisci D Angelini F Botte V (1990) Plasma sex hormone concentrations during the reproductive cycle in the male lizard Podarcis s sicula J Reprod Fertil 90 353-360

Ando S Ciarcia G Panno ML Imbrogno E Tarantino G Buffone M Beraldi E Angelini F Botte V (1992) Sex steroids levels in the plasma and testis during the reproductive cycle of lizard Podarcis s sicula Raf Gen Comp Endocrinol 85 1-7

Arslan MJ Lobo J Zaidi AA Jalali S Qazi MH (1978a) Annual androgen rhythm in the spiny-tailed lizard Uromastix hardwicki Gen Comp Endocrinol 36 16-22

Arslan MJ Zaidi P Lobo J Zaidi AA Qazi MH (1978b) Steroid levels in preovulatory and gravid lizards (Uromastix hardwicki) Gen Comp Endocrinol 34 300-303

Avwioro G (2011) Histochemical uses of haematoxylin-a review J Pham Clin Sci 1 24-34

Bishop JE (1959) A histological and histochemical study of the kidney tubule of the common garter snake Thamnophis sirtalis with special reference to the sexual segment in the male J Morphol 104 307-358

Bourne AR Taylor JL Watson TG (1986) Annual cycles of plasma and testicular androgens in the lizard Tiliqua (Trachydosaurus) rugosa Gen Comp Endocrinol 61 278-286

Courty Y Dufaure JP (1982) Circannual testosterone dihydrotestosterone and androstenedione in the plasma and testis of Lacerta vivipara a seasonally breeding viviparous lizard Steroids 39 517-529

Cox RM Skelly SL Leo A John-Alder HB (2005) Testosterone regulates sexually dimorphic coloration in the eastern fence lizard Sceloporus undulatus Copeia 2005 597-608

Crews D (1976) Hormonal control of male courtship behavior and female attractively in the garter snake (Thamnophis sirtalis parietalis) Horm Behav 7 451-460

Crews D (1980) Interrelationships among ecological behavioral and neuroendocrine processes in the reproductive cycle of Anolis carolinensis and other reptiles Adv Stud Behav 11 1-74

Crews D (1984) Gamete production sex hormone secretion and mating behavior uncoupled Horm Behav 14 22-28

Crews D (1999) Reptilian reproduction overview In Knobil E Neil JD editors Encyclopedia of Reproduction New York NY USA Academic Press pp 239-243

Crews D Camazine B Diamond M Mason R Tokarz R Garstka WR (1984) Hormonal independence of courtship behavior in the male garter snake Horm Behav 14 29-41

Crews D (2005) Evolution of neuroendocrine mechanisms that regulate sexual behavior Trends Endocrinol Metab 16 354-361

Del Conte E (1972) Granular secretion in the kidney Rss of female lizards Cnemidophorus l lemniscatus (Sauria Teiidae) J Morphol 137 181-191

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Vitt and Caldwell (2009) added that the currently known diversity of seasonal patterns of tropical squamate reproduction suggests that no single explanation is sufficient Snakes are included in the same order (Squamata) but demonstrate a more diverse reproductive pattern in contrast to lizards Tumkiratiwong et al (2012) studied the reproductive patterns of captive male and female Naja kaouthia monocled cobra Lesson 1831 suggesting that its reproductive pattern exhibited either postnuptial spermatogenesis or a dissociated reproductive pattern

There is no detailed information available on testicular and ovarian cycles plasma sex hormonal profiles during gonadal cycles or male reproduction-associated sexual segment of the kidney (SSK) especially in Calotes emma This paper therefore monitored annual alterations of reproductive organs of males and females of 2 Calotes species ie C emma and C versicolor and investigated male and female sex hormones male testosterone and female estradiol to define the reproductive patterns of 2 such Calotes species We expected that annual male and female reproductive alterations of the testes male SSK and the ovaries would require underlying morphological

and histological investigations and also that annual sex hormonal levels would reveal the reproductive patterns of 2 such Calotes species

2 Materials and methods21 AnimalsWe caught the adult lizards monthly over 1 year by hand or with a noose in the 3 forest types a dry evergreen forest a deciduous dipterocarp forest and an ecotone forest at Sakaerat Biosphere Reserves of the Sakaerat Environmental Research Station (14deg26prime33Primendash14deg32prime50PrimeN 101deg50prime43Primendash101deg57prime21PrimeE 720ndash770 m above sea level) located at Nakhon Ratchasima Province northeastern Thailand We classified adults of C emma and C versicolor based on external morphological differences (Figure 1) The adult stages of the 2 species were diagnosed based on the internal morphology of active male and female gonads (Figure 1) and expressed in terms of the snoutndashvent length (SVL) as (1) male C emma gt578 cm and C versicolor gt540 cm (2) female C emma gt657 cm and C versicolor gt599 cm We attempted to collect 10 adult males and females but in some months the samples were smaller than expected and sometimes we could not collect any samples

Figure 1 External morphologies of the representatives of 2 Calotes species Top C versicolor A no patch of granular scales in front of forelimb insertion bottom left C emma B crescent-shaped patch of small granular scales in front of forelimb insertion and C large postorbital spine present Bottom middle dissections of urogenital morphology of male Calotes T testis Vd vas deferens K kidney bottom right female Calotes OvaF ovarian follicles OviE oviductal eggs Lines were drawn from a total preparation (in ventral view)

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The months that lacked samples in each species are shown in parentheses as follows males of C emma (May July and August) and C versicolor (December) females of C emma (February to March May and December) and C versicolor (March to May July October and December) We anesthetized the samples with diethyl ether and collected blood samples by puncturing the cardiac chamber however it was not possible to acquire samples from those lizards with very small cardiac chambers Blood samples were kept at 4 degC in heparinized vials and centrifuged within 2ndash3 h following blood collection The blood was centrifuged at 1600 times g for 10 min Plasma was then aspirated off and frozen at ndash79 degC for later analysis of plasma levels in male testosterone and in female estradiol22 External and internal reproductive morphologiesWe measured SVL which ranges from the snout tip to the anterior margin of the vent We sacrificed male and female lizards to investigate the general reproductive morphologies (Figure 1 bottom middle male bottom right female) and measured follicular size and testicular mass Each specimen was weighed to the nearest 001 g its SVL was measured to the nearest 005 mm23 Testicular male SSK and ovarian histologiesTestes male SSK and ovaries were excised and fixed in a 10 vv buffered neutral formalin solution processed by the paraffin technique (Avwioro 2011) The tissue was cut in a cross-section to 6 microm in thickness using a LEICA RM2145 (Nussloch Germany) Sections were stained with hematoxylin and eosin The reproductive stage of adult females was determined on the basis of the presence or absence of types of pyriform cells in the granulosa layer Follicles with the pyriform cells which appeared in the granulosa layer were considered to be previtellogenic follicles and follicles not containing the pyriform cells were considered to be vitellogenic follicles (Tumkiratiwong et al 2012) Females having vitellogenic follicles andor oviductal eggs were considered to be at the active reproductive stage while females having only previtellogenic follicles were considered to be at the QU reproductive stage Females with regressed follicles were considered to be at the postparturient stage Males with the appearance of sperm bundles andor free sperm in the seminiferous tubules (ST) were assessed as being at the active reproductive stage while males without those attributes were assessed as being at the inactive reproductive stage (Tumkiratiwong et al 2012) Males with SSK that had strongly eosinophilic-stained granules were regarded as being at the active reproductive stage (Sever et al 2002)24 Categorization of female individuals based on reproductive statusSimilar-sized follicles were organized into distinct groups The total number and diameter of follicles belonging to

the group with the largest-sized follicles were recorded from both ovaries The follicular size was measured with a Vernier caliper (0ndash150 times 002 mm) Follicles greater than or equal to 25 mm in diameter represented vitellogenic status (Shanbhag and Prasad 1993)

Since ovarian follicular development ovulation and gestation occur asynchronously in a population individuals with differing reproductive statuses are encountered Therefore the data from the reproductive phase were classified based on the reproductive status of individuals rather than on a monthly classification as follows (1) the QU stage previtellogenic follicles sized lt25 mm in diameter (2) the early vitellogenic (EV) stage initiation of vitellogenesis with follicles sized 25ndash50 mm in diameter and without oviductal eggs (3) the late vitellogenic (LV) stage late period of vitellogenesis with follicles sized gt50 mm in diameter (4) the early gestation (EG) stage oviductal eggs with previtellogenic follicles sized lt25 mm in diameter (5) the midgestation (MG) stage oviductal eggs with vitellogenic follicles sized 25ndash50 mm in diameter and (6) late gestation (LG) stage oviductal eggs with vitellogenic follicles sized gt50 mm in diameter (modified from Radder et al 2001)25 Measurement of testosteroneThe plasma level of testosterone was measured by a 125I radioimmunoassay (RIA) We added a 500-microL sample of plasma to 50 mL of dichloromethane in a screw-top glass extraction tube We then capped the mixture and mixed it for 60 min by gentle inversion with an end-over-end rotator and then centrifuged the sample for 5 min at 1600 times g to separate the layers The upper phase was aspirated without disturbing the interface 20 mL of the lower phase was then transferred to a clean 12 times 75 mm glass tube and evaporated to dryness under a gentle stream of nitrogen at 37 degC Finally we reconstituted the extract with 200 microL of testosterone buffer The testosterone extraction procedure was performed using a Coat A Count Testosterone RIA Kit (Diagnostic Products Los Angeles CA USA) The intra- and interassay variations expressed as coefficients of variation (CVs) were 84 and 79 respectively The approximate sensitivity of this assay was 40 pgmL The cross-reactivity with androstenedione was 05 The spiking recovery values averaged 983 plusmn 06 Dilutions of 50 25 and 125 of the undiluted concentration of 7300 pgmL were 3490 pgmL 1700 pgmL and 780 pgmL respectively26 Measurement of estradiolThe plasma level of estradiol was measured by a 125I RIA We added 250 microL of plasma to 20 mL of diethyl ether in a screw-top glass extraction tube and then capped and mixed it by gentle inversion with an end-over-end rotator for 30 min It was centrifuged for 5 min at 1600 times g to separate the layers The lower (aqueous) phase was

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frozen using dry ice the organic phase was then decanted into another vial and evaporated to dryness under a gentle stream of nitrogen at 37 degC Finally the extract was reconstituted with 250 microL of estradiol buffer The estradiol extraction procedure was performed using a Coat A Count Estradiol (TKE2) RIA kit (Diagnostic Products) The intra- and interassay variations calculated as CVs were 53 and 64 respectively The approximate sensitivity of this assay was 10 pgmL The specificity of cross-reactivity with estradiol was 032 The spiking recovery values averaged 968 plusmn 33 Dilutions of 50 25 125 and 625 of the undiluted concentration of 2309 pgmL were 1144 pgmL 600 pgmL 279 pgmL and 148 pgmL respectively27 Statistical analysisTesticular masses testosterone levels ovarian weights estradiol levels and diameters of the largest follicle were expressed as the mean plusmn standard error of the mean (SEM) The KolmogorovndashSmirnov test and Levenersquos test were used to determine if the data were normally distributed and the homogeneity of variance respectively Nonparametric tests were used as all data mentioned above were nonnormal and heteroscedastic Therefore the KruskalndashWallis H test was used to test for differences in the ovarian weights the estradiol levels and the diameters of the largest follicle among the reproductive stages of nongestation periods The MannndashWhitney U test was then used to compare differences

in the ovarian weights the estradiol levels and the diameters of the largest follicle between each reproductive stage of the various follicular growths of C emma only because of the insufficient sample size There was no statistical analysis of differences in male testosterone levels or testicular masses based on months due to the small amount of data available Spearmanrsquos correlation coefficients were used to determine relationships between the testicular mass and testosterone and the diameter of the largest follicles and estradiol levels The level of significance was set to P lt 00528 Ethical aspectsThis study was approved by the Ethics Committee of the Department of National Parks Wildlife and Plant Conservation Ministry of Natural Resources and Environment Thailand (License No 090930218344)

3 Results31 Annual alterations in male and female reproductive morphologies of Calotes Annual changes in testicular morphological events between C emma and C versicolor are depicted in Figure 2 The representative C emma testes were recrudesced in December continued to hypertrophy from January to April and in June and completely regressed from September to November In the representative C versicolor the testes became hypertrophied from January to September and regressed from October to November

Figure 2 Schematics of annual changes in testicular size Top C emma bottom C versicolor Notes T testis Vd vas deferens K kidney JanndashDec denotes from January to December All scale bars equal 5 mm

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Annual changes in ovarian morphological events (Figure 3) and annual changes in the number of follicular types and egg types (Table 1) of the representative 2 Calotes species are shown In the representative C emma we found that in January April from June to July and from August to November follicles were in the EV EG LV and QU stages respectively (Figure 3 top Table 1) In the representative C versicolor we found that in January and February the ovaries contained only QU follicles in June and August QU and EV follicles and oviductal eggs in September QU follicles and oviductal eggs in November only QU follicles (Figure 3 bottom Table 1)32 Annual histological alterations in ST SSK and ovaries of Calotes In the male representative C emma the ST and SSK were hypertrophied from January to April and in June then regressed from September to November becoming active again in December (Figure 4 top left and top right respectively) Spermatozoal masses were contained inside the hypertrophied ST Spermatogonia initially appeared in November additionally a few types of germ cells occurred but there were still no active spermatozoa in December (Figure 4 top left) In the representative C versicolor both ST and SSK were active from January to September but were inactive from November to December (Figure 5 top left and top right)

We found that in the female representative C emma active ovaries contained follicles of vitellogenic stages in January and June but inactive ovaries contained previtellogenic and atretic follicles in August (Figure 4 bottom left and bottom right) In the representative C versicolor ovaries contained previtellogenic and atretic follicles in January and both previtellogenic and EV follicles in June (Figure 5 bottom left and bottom right) Additionally we found corpus luteum in the ovaries of 1 representative in September (Figure 5 bottom left and bottom right) We also observed cellular alterations in the granulosa layer of both C emma and C versicolor It was demonstrated that inside the granulosa layer of the previtellogenic follicles both types of small and pyriform cells appeared However both such cell types disappeared in the granulosa layer of the vitellogenic follicles (Figures 4 and 5 bottom right)33 Annual plasma testosterone levels and testicular masses in Calotes Annual variations in plasma testosterone levels and testicular masses between the 2 Calotes species are depicted (Figures 6a and 6b) In the representative C emma plasma testosterone levels and testicular masses initially increased in December peaked in March and thereafter gradually reduced from April to November (Figure 6a) In the representative C versicolor plasma testosterone

Figure 3 Schematics of seasonal changes in ovarian size Top C emma bottom C versicolor Notes OvaF ovarian follicles OviE oviductal eggs Ovi oviduct All scale bars equals 5 mm JanndashNov denotes from January to November

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levels and testicular masses initially increased in January peaked in April and thereafter gradually reduced from May to November (Figure 6b) The changes in testicular masses tended to correspond well to the changes in plasma testosterone levels between the 2 species in the genus Calotes (r = 0789 P = 0001 and r = 0732 P = 0001 for C emma and C versicolor respectively)34 Annual plasma estradiol and ovarian cycles in Calotes Variations in ovarian masses and the diameter of the largest follicle were observed between the 2 Calotes species

during their reproductive stages (Table 2) The ovarian weights and the diameters of the largest follicles differed significantly from the QU stage to the LV stage regarding the growth of ovarian follicles (P = 0001) in C emma It was also observed that the growth of ovarian follicles concomitantly occurred with the growth of oviductal eggs during the gestation period of the 2 species

Variations in plasma E2 levels in relation to the diameters of the largest follicles between the 2 Calotes species during reproductive stages are graphically depicted (Figures 7a and 7b) Regarding the groups of follicular sizes

Table 1 Annual changes in numbers of follicular and egg types according to Calotes species

Follicular size and eggs Jan Feb Apr May Jun Jul Aug Sep Oct Nov

C emmalt25 mm 21 - 18 - 18 22 41 33 24 2325ndash50 mm 10 - - - 4 1 - - - -gt50 mm - - - - 8 5 - - - -Oviductal eggs - - 4 - - - - - - -C versicolorlt25 mm 16 28 - - 28 - 19 16 - 1025ndash50 mm - - - - 5 - - - - -gt50 mm - - - - - - - - - -Oviductal eggs - - - - 5 - 6 5 - -

Notes JanndashNov denotes January to November respectively

Figure 4 Photomicrographs of annual changes in C emma Top left testes top right male SSK bottom left ovaries bottom right granulosa layers (GL) Notes SZ spermatozoa ST seminiferous tubules SSK sexual segments of kidney AF atretic follicle PF previtellogenic follicle VF vitellogenic follicle P pyriform cells S small cells

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of C emma plasma E2 levels were the difference between the QU and the EV stages (U = 900 P = 0090) the QU and the LV stages (U = 200 P = 0009) and the EV and the LV stages (U = 200 P = 0083) Additionally the diameters of the largest follicles were the difference between the QU and the EV stages (U = 000 P = 0004) the QU and the LV stages (U = 000 P = 0004) and the EV and the LV stages (U = 000 P = 0021) The correlation coefficient between plasma E2 and the diameter of the largest follicle was 082 (P = 0001) and 032 (P = 0365) of C emma and C versicolor respectively

4 DiscussionAnnual variations in the timing of male and female reproductive stages were encountered among populations of C emma and C versicolor even in individuals of the same population Annual changes in testicular sizes were categorized into 2 phases in both species (1) an active hypertrophied testicular phase and (2) an inactive

regressed testicular phase However the timing of those 2 events appeared asynchronously in the 2 Calotes species even in individuals of the same population (data not shown here) The testes were hypertrophied with active spermatozoal production from December to June and January to September in the representative C emma and C versicolor respectively A study by Gouder and Nadkarni (1979) showed that males of C versicolor widely distributed in India were spermatogenetically active from April to September Active vitellogenic follicles and oviductal eggs were concomitantly encountered in C emma and C versicolor Therefore these 2 Calotes species exhibited polyautochrony and multiclutches Radder et al (2001) reported that Indian garden lizards C versicolor showed polyautochrony and multiclutches The representative gravid lizards were found in April in C emma and in June and from August to September in C versicolor Gravid C versicolor whose habitat is in India was encountered from May to October (Shanbhag and Prasad 1993) We

Figure 5 Photomicrographs of annual changes in C versicolor Top left testes top right male SSK bottom left ovaries bottom right granulosa layers (GL) Notes SZ spermatozoa ST seminiferous tubules SSK sexual segments of kidney AF atretic follicle PF previtellogenic follicle VF vitellogenic follicle P pyriform cells S small cells CL corpus luteum

Figure 6 Annual profiles (mean plusmn SEM) of testosterone levels and testicular masses (a) C emma (b) C versicolor Notes JanndashDec denotes January to December The numbers (in parentheses) represent the number of analyzed samples in each month

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are likely to suggest after studying the annual alterations in the male and female reproductive morphologies of the 2 Calotes species that the active reproductive events of both males and females of the Calotes species lasted nearly 1 year with only a few months of reproductive arrest which is especially seen in C versicolor

According to our investigations on annual histological alterations in ST and male SSK timing of the 2 Calotes species both ST and SSK were in active spermatogenic and

hypertrophied stages respectively which corresponded well with the timing of the testicular hypertrophied stage mentioned above In other words the timing of arrested spermatogenesis and regressed SSK was in accordance with that of the regressed testes Likewise we confirmed that the 2 Calotes species have an active reproductive stage that is much longer than the inactive reproductive stage

SSK is present in a variety of male snakes and lizards but is absent in both turtles (Regaud and Policard 1903)

Table 2 Changes in ovarian weight and diameter of the largest follicle in Calotes during the reproduction cycle

Reproductive stages N Ovarian weights Diameter of the largest follicle

C emma (20)QU 11 002 plusmn 000a 178 plusmn 006a

EV 4 011 plusmn 009b 350 plusmn 033b

LV 4 171 plusmn 049c 802 plusmn 085c

EG 1 002 240MG - - -LG - - -C versicolor (9)QU 4 002 plusmn 000 152 plusmn 199EV 1 02 428LV - - -EG 3 002 plusmn 000 222 plusmn 069MG 2 011 plusmn 009 436 plusmn 036LG - - -

Data are presented as mean plusmn SEM The differences in superscript alphabets indicate the significant differences among the reproductive stages of nongestation at P lt 001 (N sample sizes)Notes QU quiescent EV early vitellogenic LV late vitellogenic EG early gestation MG mid-gestation and LG late gestation

Figure 7 Changes in the plasma levels of estradiol and the diameter of the largest follicle (a) C emma (b) C versicolor Notes QU quiescent EV early vitellogenic LV late vitellogenic EG early gestation MG mid-gestation LG late gestation Data are presented as mean plusmn SEM The differences in superscript alphabets (estradiol levels) and in the numbers of asterisks (diameters of the largest follicles) indicate the significant differences between the various follicular sizes at P lt 001 The number (in parentheses) represents the analyzed samples in each month

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and crocodilians (Fox 1952) Bishop (1959) found that the testes of the male garter snake Thamnophis sirtalis were spermatogenically active during the same time as the hypertrophied SSK during the active reproductive period the diameter of the SSK tubule was 5 times greater than that of the SSK tubule during the inactive reproductive period However SSK development in female lizards has been reported in the genus Cnemidophorus (Del Conte 1972 Del Conte and Tamayo 1973) and Scincella laterale (Sever and Hopkins 2005) They suggested that the females had a low level of natural androgens which caused the SSK development (Del Conte and Tamayo 1973 Sever and Hopkins 2005) In the present study we did not monitor the annual seasonal alterations in female SSK The hypertrophy of the SSK is synchronous with androgen secretion and spermatogenic activity (Sever and Hopkins 2005) Norris (2013) also stated that the SSK of sexually active squamates undergoes hypertrophy and is under the influence of androgens In this study we did not investigate any alterations in annual SSK with annual androgen secretion but we did demonstrate that the hypertrophy and the regression of SSK changed seasonally and synchronously with the active spermatogenic event and the spermatogenic arrest respectively In the Iberian rock lizard Lacerta monticola SSK secretions form a copulatory plug that adheres to the femalersquos cloaca following copulation to occlude oviductal openings however such a plug does not prevent subsequent mating nor does it reduce the femalersquos attractiveness (Moreira and Birkhead 2003)

With our investigations on annual alterations in female ovarian morphologies between 2 Calotes we found that individuals in the same Calotes species showed different timing of reproductive events throughout a 1-year period (the data are not shown here) Additionally there was quite clear evidence that QU EV and LV follicles and oviductal eggs overlapped among individuals within the same populations of both Calotes species Female reproductive status is definitely distinguishable between the 2 Calotes species Gravid lizards were encountered in 1 individual of C emma in April and in 3 individuals of C versicolor in June August and September Shanbhag et al (2000) reported that female C versicolor showed inactive reproduction from December to April and gravidity was encountered from May to October

We found that in the previtellogenic follicles (QU and EV) of females of both Calotes species the granulosa layer contained 2 types of cells pyriform and small cells Uribe et al (1996) stated that in squamates the follicular epithelium or granulosa initially consists of small cuboidal cells but differentiates during the previtellogenic phase and becomes multilayered and polymorphic by the presence of unique flask-shaped pyriform cells intermediate cells and

small cells These pyriform cells differentiate from small somatic follicular cells early in follicular development via the intermediate-cell stage to become nurse cells in direct contact with the developing oocytes (Maurizii et al 2004) Differentiation of the small cells into pyriform cells appears to be linked to the progressive appearance of glycoproteins with terminal α-N-acetylgalactosamine residues on the cell surface which may be involved in fusion between the oocyte and the follicle cell membranes as well as maintenance of the differentiated pyriform cells The pyriform cells are connected to the oocyte via intercellular bridges containing a cytoskeleton of α-tubulin and cytokeratin microtubules (Maurizii et al 2004) Tumkiratiwong et al (2012) also demonstrated that the previtellogenic follicles of the captive monocled cobra Naja kaouthia had many pyriform cells in the granulosa layer but fewer in the vitellogenic follicles In this study the pyriform cells disappeared when the follicles entered the vitellogenic stage Andreuccetti (1992) studied the differentiation of pyriform cells and their contribution to oocyte growth in 3 lizards namely Tarentola mauritanica Cordylus wittifer and Platysaurus intermedius and a colubrid snake Coluber viridiflavus and revealed that pyriform cells differentiate from small follicle cells via intermediate cells after establishing an intercellular bridge with the oocyte Once pyriform cells are differentiated they display ultrastructural features indicative of synthetic activity including abundant ribosomes Golgi membranes vacuoles mitochondria and lipid droplets These cellular components extend to the apex of the cell at the level of the intercellular bridge suggesting that constituents of pyriform cells may be transferred to the oocyte Pyriform cells and the oocytes may fulfill similar vitellogenic functions The establishment of an intercellular bridge may represent a crucial event in the development of an integrated system in which pyriform cells and oocytes cooperate Norris (2013) reported that the squamate granulosa contains the pyriform cells which are in direct contact with the developing oocyte and are apparently involved with early steps in oocyte development soon after the onset of vitellogenesis As ovulation approaches the granulosa cells as well as some thecal cells accumulate cholesterol-positive lipids and proliferate and luteinize to form corpora lutea following ovulation Follicular atresia is a common occurrence in reptilian ovaries as in other vertebrates (Norris 2013) as we found the corpora lutea in the follicle in accordance to oviductal egg appearances of a representative C versicolor collected in September Additionally as shown in Table 1 several atretic follicles lt25 mm in diameter were commonly encountered in C emma and C versicolor

Based on both morphological and histological investigations we found that testes ST and SSK were

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concomitantly active and were associated with high levels of plasma testosterone We also demonstrated that there were high correlations between levels of plasma testosterone (T) and testicular mass where annual changes occurred in the same direction as the testicular size and time of spermatogenetic events among the males of the Calotes species Radder et al (2001) reported that in the male tropical or oriental garden lizard C versicolor plasma T is highest during the breeding season which correlated with testis mass and reproductive behavior Changes in T levels are associated with high spermatogenetic activity Radder et al (2001) also stated that the changes in plasma T levels during different phases of the male reproductive cycle in C versicolor follow a reproductive pattern of a prenuptial type of spermatogenesis that is similar to that of some other species of lizards the spiny-tailed lizard Uromastix hardwicki (Arslan et al 1978a) the viviparous lizard Lacerta vivipara (Courty and Dufaure 1982) the western shingleback lizard Tiliqua (Trachydosaurus) rugosa (Bourne et al 1986) the male lizard Podarcis s sicula (Ando et al 1990) Podarcis s sicula Raf (Ando et al 1992) the white-throated savanna monitor Varanus albigularis (Phillips and Millar 1998) and the male brown anoles Anolis sagrei (Tokarz et al 1998)

We found that estradiol (E2) levels increased in vitellogenic females its high levels were associated with the presence of the largest vitellogenic follicles in the 2 Calotes species Radder et al (2001) reported that in female C versicolor with overlapping reproductive events such as vitellogenesis and gestation E2 was at low levels when the ovaries were regressed and at high levels at vitellogenic follicular recruitment reaching peak level at the time of preovulatory follicles The same patterns of E2 secretion were found again when the second set of follicles underwent vitellogenesis (Radder et al 2001) Surprisingly Amey and Whittier (2000) reported that in female bearded dragons Pagona barbata plasma E2 was low or nondetectable across all reproductive states In C versicolor E2 levels were low in nonreproductive females with small previtellogenic follicles and those in the EG phase (Radder et al 2001) We do not discuss the level of the plasma progesterone (P) during the gestation period as its level was not detectable in this study The gravid lizards in EG exhibited low plasma E2 but high P levels and the highest P levels coincided with eggshell production P levels declined after eggshell formation as reported in other gravid individuals in several species of lizards that do not possess vitellogenic follicles of the subsequent clutch including C versicolor (Radder et al 2001) Uromastix hardwicki (Arslan et al 1978b) Agama atra (Van Wyk 1984) Eumeces obsoletus Scelporus undulatus and Crotaphytus collaris (Masson and Guillette 1987) and Psammodromus algirus (Diaz et al 1994) However a

decline in P levels in MG with vitellogenic follicles did not seem to facilitate recruitment or growth of the subsequent set of vitellogenic follicles in gravid Sceloporus jarrovi (Guillette et al 1981)

In the present study there were variations in the timing of breeding between the 2 Calotes species and even within populations of the same species We could not relate the copulation timing of 2 such Calotes species to gonadal activity or sex hormonal surges as the timing of natural mating could not be observed during the times we collected data Lizard species that inhabit temperate zones have mostly exhibited seasonal reproduction (Fitch 1970 Licht 1984 Pianka and Vitt 2003) The 10 lizard species that have been studied widely to date exhibit an associated reproductive pattern (Lovern 2011) that is green anoles Anolis carolinensis (Crews 1980 Lovern et al 2004) brown anoles Anolis sagrei (Lee et al 1989 Tokarz 1998) eastern fence lizards Sceloporus undulates (Cox et al 2005) mountain spiny lizards Sceloporus jarrovi (Woodley and Moore 1999) tree lizards Urosaurus ornatus (French and Moore 2008) wall lizards Podarcis sicula (Putti et al 2009) common lizards Lacerta vivipara (Vercken and Clobert 2008) little striped whiptail lizards Cnemidophorus inornatus (Crews 2005) garden lizards Calotes versicolor (Shanbhag 2003 Lovern 2011) and leopard geckos Eublepharis macularius (Rhen et al 2005) The temperate Florida populations of the brown anole Anolis sagrei show a strong seasonality in reproduction (Lee et al 1989) while the tropical Caribbean (Licht and Gorman 1970 Sexton and Brown 1977) and Hawaiian populations of this species (Goldberg et al 2002) show a less-pronounced seasonality in which reproductively active individuals can be found throughout the year Although individuals within a population of many tropical lizard species can breed at any time no individuals within the population breed year-round (Lovern 2011) Additionally Vitt and Caldwell (2009) stated that the belief used to be that tropical squamates had continuous reproduction in aseasonal tropical environments or reproduced during the wet season in a wetndashdry seasonal tropical environment Many tropical lizard species namely the anoles Anolis acutus (Ruibal et al 1972) Anolis limifrons (Sexton et al 1971) and Anolis opalinus (Jenssen and Nunez 1994) as well as the gecko Cyrtodactylus malyanus the flying lizard Draco melanopogon (Inger and Greenberg 1966) and the parthenogenetic oviparous whiptail lizard Cnemidophorus nativo (Menezes et al 2004) showed slightly more frequent breeding during the wet season than during the dry season (Jenssen and Nunez 1994)

Reproductive patterns can be described in a variety of ways but not all species fit neatly into such categorizations However 2 general types of reproductive patterns are

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recognized in terms of prenuptial and postnuptial reproductive patterns (Lance 1998) Prenuptial reproductive pattern terms such as gonadal recrudescence sex steroid production and gametogenesis occur in advance of mating whereas postnuptial reproductive patterns occur following mating In other words in a high-elevation population of Sceloporus grammicus in Parque Nacional de Zoquiapan in central Mexico an active reproductive event occurring in the early fall is described as dissociated from testicular recrudescence in males but is associated with the initiation of ovarian recrudescence in females (Guillette and Casas-Andreu 1980 1981 Zuniga-Vega et al 2008) This is in contrast to S grammicus from Teotihuacan Mexico in which testicular recrudescence and breeding occur in the summer and fall at the onset of female ovarian recrudescence (Jimenez-Cruz et al 2005) In S mucronatus from Valle de la Cantimplora Mexico peak testicular recrudescence and mating occur during the summer prior to ovarian recrudescence which does not occur until several months later (Ortega-Leon et al 2009) This is distinct from many fall-breeding

populations elsewhere (Mendez-De La Cruz et al 1994 Villagran-Santa Cruz et al 1994) The examples above demonstrate that gonadal activity and mating behavior are clearly variable but hormone analyses have not been performed in these species and so endocrine relationships cannot be assessed at this point

In conclusion we suggest that the males and females of the 2 Calotes species have much more prolonged active reproductive phases than inactive reproductive phases The reproductive patterns of C emma and C versicolor were classified into the same reproductive pattern of continual reproduction

AcknowledgmentsWe thank the Department of Zoology of Kasetsart University for financial support We also thank the staff of Sakaerat Environmental Research Station Nakhon Ratchasima Province for devoting time for research collaboration We also thank Mrs Sureerat Sangkrut for drawing all illustrations In addition we wish to thank the anonymous referees for many helpful suggestions

References

Aldridge RD Goldberg SR Wisniewski SS Bufalino AP Dillman CB (2009) The reproductive cycle and estrus in the colubrid snakes of temperate North America Contemp Herpetol 4 1-31

Amey AP Whittier JM (2000) Seasonal patterns of plasma steroid hormones in males and females of the bearded dragon lizard Pogona barbata Gen Comp Endocrinol 117 335-342

Andreuccetti P (1992) An ultrastructural study of differentiation of pyriform cells and their contribution to oocyte growth in representative Squamata J Morphol 212 1-11

Ando S Panno ML Ciarcia G Imbrogno E Buffone M Beraldi E Sisci D Angelini F Botte V (1990) Plasma sex hormone concentrations during the reproductive cycle in the male lizard Podarcis s sicula J Reprod Fertil 90 353-360

Ando S Ciarcia G Panno ML Imbrogno E Tarantino G Buffone M Beraldi E Angelini F Botte V (1992) Sex steroids levels in the plasma and testis during the reproductive cycle of lizard Podarcis s sicula Raf Gen Comp Endocrinol 85 1-7

Arslan MJ Lobo J Zaidi AA Jalali S Qazi MH (1978a) Annual androgen rhythm in the spiny-tailed lizard Uromastix hardwicki Gen Comp Endocrinol 36 16-22

Arslan MJ Zaidi P Lobo J Zaidi AA Qazi MH (1978b) Steroid levels in preovulatory and gravid lizards (Uromastix hardwicki) Gen Comp Endocrinol 34 300-303

Avwioro G (2011) Histochemical uses of haematoxylin-a review J Pham Clin Sci 1 24-34

Bishop JE (1959) A histological and histochemical study of the kidney tubule of the common garter snake Thamnophis sirtalis with special reference to the sexual segment in the male J Morphol 104 307-358

Bourne AR Taylor JL Watson TG (1986) Annual cycles of plasma and testicular androgens in the lizard Tiliqua (Trachydosaurus) rugosa Gen Comp Endocrinol 61 278-286

Courty Y Dufaure JP (1982) Circannual testosterone dihydrotestosterone and androstenedione in the plasma and testis of Lacerta vivipara a seasonally breeding viviparous lizard Steroids 39 517-529

Cox RM Skelly SL Leo A John-Alder HB (2005) Testosterone regulates sexually dimorphic coloration in the eastern fence lizard Sceloporus undulatus Copeia 2005 597-608

Crews D (1976) Hormonal control of male courtship behavior and female attractively in the garter snake (Thamnophis sirtalis parietalis) Horm Behav 7 451-460

Crews D (1980) Interrelationships among ecological behavioral and neuroendocrine processes in the reproductive cycle of Anolis carolinensis and other reptiles Adv Stud Behav 11 1-74

Crews D (1984) Gamete production sex hormone secretion and mating behavior uncoupled Horm Behav 14 22-28

Crews D (1999) Reptilian reproduction overview In Knobil E Neil JD editors Encyclopedia of Reproduction New York NY USA Academic Press pp 239-243

Crews D Camazine B Diamond M Mason R Tokarz R Garstka WR (1984) Hormonal independence of courtship behavior in the male garter snake Horm Behav 14 29-41

Crews D (2005) Evolution of neuroendocrine mechanisms that regulate sexual behavior Trends Endocrinol Metab 16 354-361

Del Conte E (1972) Granular secretion in the kidney Rss of female lizards Cnemidophorus l lemniscatus (Sauria Teiidae) J Morphol 137 181-191

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The months that lacked samples in each species are shown in parentheses as follows males of C emma (May July and August) and C versicolor (December) females of C emma (February to March May and December) and C versicolor (March to May July October and December) We anesthetized the samples with diethyl ether and collected blood samples by puncturing the cardiac chamber however it was not possible to acquire samples from those lizards with very small cardiac chambers Blood samples were kept at 4 degC in heparinized vials and centrifuged within 2ndash3 h following blood collection The blood was centrifuged at 1600 times g for 10 min Plasma was then aspirated off and frozen at ndash79 degC for later analysis of plasma levels in male testosterone and in female estradiol22 External and internal reproductive morphologiesWe measured SVL which ranges from the snout tip to the anterior margin of the vent We sacrificed male and female lizards to investigate the general reproductive morphologies (Figure 1 bottom middle male bottom right female) and measured follicular size and testicular mass Each specimen was weighed to the nearest 001 g its SVL was measured to the nearest 005 mm23 Testicular male SSK and ovarian histologiesTestes male SSK and ovaries were excised and fixed in a 10 vv buffered neutral formalin solution processed by the paraffin technique (Avwioro 2011) The tissue was cut in a cross-section to 6 microm in thickness using a LEICA RM2145 (Nussloch Germany) Sections were stained with hematoxylin and eosin The reproductive stage of adult females was determined on the basis of the presence or absence of types of pyriform cells in the granulosa layer Follicles with the pyriform cells which appeared in the granulosa layer were considered to be previtellogenic follicles and follicles not containing the pyriform cells were considered to be vitellogenic follicles (Tumkiratiwong et al 2012) Females having vitellogenic follicles andor oviductal eggs were considered to be at the active reproductive stage while females having only previtellogenic follicles were considered to be at the QU reproductive stage Females with regressed follicles were considered to be at the postparturient stage Males with the appearance of sperm bundles andor free sperm in the seminiferous tubules (ST) were assessed as being at the active reproductive stage while males without those attributes were assessed as being at the inactive reproductive stage (Tumkiratiwong et al 2012) Males with SSK that had strongly eosinophilic-stained granules were regarded as being at the active reproductive stage (Sever et al 2002)24 Categorization of female individuals based on reproductive statusSimilar-sized follicles were organized into distinct groups The total number and diameter of follicles belonging to

the group with the largest-sized follicles were recorded from both ovaries The follicular size was measured with a Vernier caliper (0ndash150 times 002 mm) Follicles greater than or equal to 25 mm in diameter represented vitellogenic status (Shanbhag and Prasad 1993)

Since ovarian follicular development ovulation and gestation occur asynchronously in a population individuals with differing reproductive statuses are encountered Therefore the data from the reproductive phase were classified based on the reproductive status of individuals rather than on a monthly classification as follows (1) the QU stage previtellogenic follicles sized lt25 mm in diameter (2) the early vitellogenic (EV) stage initiation of vitellogenesis with follicles sized 25ndash50 mm in diameter and without oviductal eggs (3) the late vitellogenic (LV) stage late period of vitellogenesis with follicles sized gt50 mm in diameter (4) the early gestation (EG) stage oviductal eggs with previtellogenic follicles sized lt25 mm in diameter (5) the midgestation (MG) stage oviductal eggs with vitellogenic follicles sized 25ndash50 mm in diameter and (6) late gestation (LG) stage oviductal eggs with vitellogenic follicles sized gt50 mm in diameter (modified from Radder et al 2001)25 Measurement of testosteroneThe plasma level of testosterone was measured by a 125I radioimmunoassay (RIA) We added a 500-microL sample of plasma to 50 mL of dichloromethane in a screw-top glass extraction tube We then capped the mixture and mixed it for 60 min by gentle inversion with an end-over-end rotator and then centrifuged the sample for 5 min at 1600 times g to separate the layers The upper phase was aspirated without disturbing the interface 20 mL of the lower phase was then transferred to a clean 12 times 75 mm glass tube and evaporated to dryness under a gentle stream of nitrogen at 37 degC Finally we reconstituted the extract with 200 microL of testosterone buffer The testosterone extraction procedure was performed using a Coat A Count Testosterone RIA Kit (Diagnostic Products Los Angeles CA USA) The intra- and interassay variations expressed as coefficients of variation (CVs) were 84 and 79 respectively The approximate sensitivity of this assay was 40 pgmL The cross-reactivity with androstenedione was 05 The spiking recovery values averaged 983 plusmn 06 Dilutions of 50 25 and 125 of the undiluted concentration of 7300 pgmL were 3490 pgmL 1700 pgmL and 780 pgmL respectively26 Measurement of estradiolThe plasma level of estradiol was measured by a 125I RIA We added 250 microL of plasma to 20 mL of diethyl ether in a screw-top glass extraction tube and then capped and mixed it by gentle inversion with an end-over-end rotator for 30 min It was centrifuged for 5 min at 1600 times g to separate the layers The lower (aqueous) phase was

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frozen using dry ice the organic phase was then decanted into another vial and evaporated to dryness under a gentle stream of nitrogen at 37 degC Finally the extract was reconstituted with 250 microL of estradiol buffer The estradiol extraction procedure was performed using a Coat A Count Estradiol (TKE2) RIA kit (Diagnostic Products) The intra- and interassay variations calculated as CVs were 53 and 64 respectively The approximate sensitivity of this assay was 10 pgmL The specificity of cross-reactivity with estradiol was 032 The spiking recovery values averaged 968 plusmn 33 Dilutions of 50 25 125 and 625 of the undiluted concentration of 2309 pgmL were 1144 pgmL 600 pgmL 279 pgmL and 148 pgmL respectively27 Statistical analysisTesticular masses testosterone levels ovarian weights estradiol levels and diameters of the largest follicle were expressed as the mean plusmn standard error of the mean (SEM) The KolmogorovndashSmirnov test and Levenersquos test were used to determine if the data were normally distributed and the homogeneity of variance respectively Nonparametric tests were used as all data mentioned above were nonnormal and heteroscedastic Therefore the KruskalndashWallis H test was used to test for differences in the ovarian weights the estradiol levels and the diameters of the largest follicle among the reproductive stages of nongestation periods The MannndashWhitney U test was then used to compare differences

in the ovarian weights the estradiol levels and the diameters of the largest follicle between each reproductive stage of the various follicular growths of C emma only because of the insufficient sample size There was no statistical analysis of differences in male testosterone levels or testicular masses based on months due to the small amount of data available Spearmanrsquos correlation coefficients were used to determine relationships between the testicular mass and testosterone and the diameter of the largest follicles and estradiol levels The level of significance was set to P lt 00528 Ethical aspectsThis study was approved by the Ethics Committee of the Department of National Parks Wildlife and Plant Conservation Ministry of Natural Resources and Environment Thailand (License No 090930218344)

3 Results31 Annual alterations in male and female reproductive morphologies of Calotes Annual changes in testicular morphological events between C emma and C versicolor are depicted in Figure 2 The representative C emma testes were recrudesced in December continued to hypertrophy from January to April and in June and completely regressed from September to November In the representative C versicolor the testes became hypertrophied from January to September and regressed from October to November

Figure 2 Schematics of annual changes in testicular size Top C emma bottom C versicolor Notes T testis Vd vas deferens K kidney JanndashDec denotes from January to December All scale bars equal 5 mm

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Annual changes in ovarian morphological events (Figure 3) and annual changes in the number of follicular types and egg types (Table 1) of the representative 2 Calotes species are shown In the representative C emma we found that in January April from June to July and from August to November follicles were in the EV EG LV and QU stages respectively (Figure 3 top Table 1) In the representative C versicolor we found that in January and February the ovaries contained only QU follicles in June and August QU and EV follicles and oviductal eggs in September QU follicles and oviductal eggs in November only QU follicles (Figure 3 bottom Table 1)32 Annual histological alterations in ST SSK and ovaries of Calotes In the male representative C emma the ST and SSK were hypertrophied from January to April and in June then regressed from September to November becoming active again in December (Figure 4 top left and top right respectively) Spermatozoal masses were contained inside the hypertrophied ST Spermatogonia initially appeared in November additionally a few types of germ cells occurred but there were still no active spermatozoa in December (Figure 4 top left) In the representative C versicolor both ST and SSK were active from January to September but were inactive from November to December (Figure 5 top left and top right)

We found that in the female representative C emma active ovaries contained follicles of vitellogenic stages in January and June but inactive ovaries contained previtellogenic and atretic follicles in August (Figure 4 bottom left and bottom right) In the representative C versicolor ovaries contained previtellogenic and atretic follicles in January and both previtellogenic and EV follicles in June (Figure 5 bottom left and bottom right) Additionally we found corpus luteum in the ovaries of 1 representative in September (Figure 5 bottom left and bottom right) We also observed cellular alterations in the granulosa layer of both C emma and C versicolor It was demonstrated that inside the granulosa layer of the previtellogenic follicles both types of small and pyriform cells appeared However both such cell types disappeared in the granulosa layer of the vitellogenic follicles (Figures 4 and 5 bottom right)33 Annual plasma testosterone levels and testicular masses in Calotes Annual variations in plasma testosterone levels and testicular masses between the 2 Calotes species are depicted (Figures 6a and 6b) In the representative C emma plasma testosterone levels and testicular masses initially increased in December peaked in March and thereafter gradually reduced from April to November (Figure 6a) In the representative C versicolor plasma testosterone

Figure 3 Schematics of seasonal changes in ovarian size Top C emma bottom C versicolor Notes OvaF ovarian follicles OviE oviductal eggs Ovi oviduct All scale bars equals 5 mm JanndashNov denotes from January to November

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levels and testicular masses initially increased in January peaked in April and thereafter gradually reduced from May to November (Figure 6b) The changes in testicular masses tended to correspond well to the changes in plasma testosterone levels between the 2 species in the genus Calotes (r = 0789 P = 0001 and r = 0732 P = 0001 for C emma and C versicolor respectively)34 Annual plasma estradiol and ovarian cycles in Calotes Variations in ovarian masses and the diameter of the largest follicle were observed between the 2 Calotes species

during their reproductive stages (Table 2) The ovarian weights and the diameters of the largest follicles differed significantly from the QU stage to the LV stage regarding the growth of ovarian follicles (P = 0001) in C emma It was also observed that the growth of ovarian follicles concomitantly occurred with the growth of oviductal eggs during the gestation period of the 2 species

Variations in plasma E2 levels in relation to the diameters of the largest follicles between the 2 Calotes species during reproductive stages are graphically depicted (Figures 7a and 7b) Regarding the groups of follicular sizes

Table 1 Annual changes in numbers of follicular and egg types according to Calotes species

Follicular size and eggs Jan Feb Apr May Jun Jul Aug Sep Oct Nov

C emmalt25 mm 21 - 18 - 18 22 41 33 24 2325ndash50 mm 10 - - - 4 1 - - - -gt50 mm - - - - 8 5 - - - -Oviductal eggs - - 4 - - - - - - -C versicolorlt25 mm 16 28 - - 28 - 19 16 - 1025ndash50 mm - - - - 5 - - - - -gt50 mm - - - - - - - - - -Oviductal eggs - - - - 5 - 6 5 - -

Notes JanndashNov denotes January to November respectively

Figure 4 Photomicrographs of annual changes in C emma Top left testes top right male SSK bottom left ovaries bottom right granulosa layers (GL) Notes SZ spermatozoa ST seminiferous tubules SSK sexual segments of kidney AF atretic follicle PF previtellogenic follicle VF vitellogenic follicle P pyriform cells S small cells

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of C emma plasma E2 levels were the difference between the QU and the EV stages (U = 900 P = 0090) the QU and the LV stages (U = 200 P = 0009) and the EV and the LV stages (U = 200 P = 0083) Additionally the diameters of the largest follicles were the difference between the QU and the EV stages (U = 000 P = 0004) the QU and the LV stages (U = 000 P = 0004) and the EV and the LV stages (U = 000 P = 0021) The correlation coefficient between plasma E2 and the diameter of the largest follicle was 082 (P = 0001) and 032 (P = 0365) of C emma and C versicolor respectively

4 DiscussionAnnual variations in the timing of male and female reproductive stages were encountered among populations of C emma and C versicolor even in individuals of the same population Annual changes in testicular sizes were categorized into 2 phases in both species (1) an active hypertrophied testicular phase and (2) an inactive

regressed testicular phase However the timing of those 2 events appeared asynchronously in the 2 Calotes species even in individuals of the same population (data not shown here) The testes were hypertrophied with active spermatozoal production from December to June and January to September in the representative C emma and C versicolor respectively A study by Gouder and Nadkarni (1979) showed that males of C versicolor widely distributed in India were spermatogenetically active from April to September Active vitellogenic follicles and oviductal eggs were concomitantly encountered in C emma and C versicolor Therefore these 2 Calotes species exhibited polyautochrony and multiclutches Radder et al (2001) reported that Indian garden lizards C versicolor showed polyautochrony and multiclutches The representative gravid lizards were found in April in C emma and in June and from August to September in C versicolor Gravid C versicolor whose habitat is in India was encountered from May to October (Shanbhag and Prasad 1993) We

Figure 5 Photomicrographs of annual changes in C versicolor Top left testes top right male SSK bottom left ovaries bottom right granulosa layers (GL) Notes SZ spermatozoa ST seminiferous tubules SSK sexual segments of kidney AF atretic follicle PF previtellogenic follicle VF vitellogenic follicle P pyriform cells S small cells CL corpus luteum

Figure 6 Annual profiles (mean plusmn SEM) of testosterone levels and testicular masses (a) C emma (b) C versicolor Notes JanndashDec denotes January to December The numbers (in parentheses) represent the number of analyzed samples in each month

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are likely to suggest after studying the annual alterations in the male and female reproductive morphologies of the 2 Calotes species that the active reproductive events of both males and females of the Calotes species lasted nearly 1 year with only a few months of reproductive arrest which is especially seen in C versicolor

According to our investigations on annual histological alterations in ST and male SSK timing of the 2 Calotes species both ST and SSK were in active spermatogenic and

hypertrophied stages respectively which corresponded well with the timing of the testicular hypertrophied stage mentioned above In other words the timing of arrested spermatogenesis and regressed SSK was in accordance with that of the regressed testes Likewise we confirmed that the 2 Calotes species have an active reproductive stage that is much longer than the inactive reproductive stage

SSK is present in a variety of male snakes and lizards but is absent in both turtles (Regaud and Policard 1903)

Table 2 Changes in ovarian weight and diameter of the largest follicle in Calotes during the reproduction cycle

Reproductive stages N Ovarian weights Diameter of the largest follicle

C emma (20)QU 11 002 plusmn 000a 178 plusmn 006a

EV 4 011 plusmn 009b 350 plusmn 033b

LV 4 171 plusmn 049c 802 plusmn 085c

EG 1 002 240MG - - -LG - - -C versicolor (9)QU 4 002 plusmn 000 152 plusmn 199EV 1 02 428LV - - -EG 3 002 plusmn 000 222 plusmn 069MG 2 011 plusmn 009 436 plusmn 036LG - - -

Data are presented as mean plusmn SEM The differences in superscript alphabets indicate the significant differences among the reproductive stages of nongestation at P lt 001 (N sample sizes)Notes QU quiescent EV early vitellogenic LV late vitellogenic EG early gestation MG mid-gestation and LG late gestation

Figure 7 Changes in the plasma levels of estradiol and the diameter of the largest follicle (a) C emma (b) C versicolor Notes QU quiescent EV early vitellogenic LV late vitellogenic EG early gestation MG mid-gestation LG late gestation Data are presented as mean plusmn SEM The differences in superscript alphabets (estradiol levels) and in the numbers of asterisks (diameters of the largest follicles) indicate the significant differences between the various follicular sizes at P lt 001 The number (in parentheses) represents the analyzed samples in each month

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and crocodilians (Fox 1952) Bishop (1959) found that the testes of the male garter snake Thamnophis sirtalis were spermatogenically active during the same time as the hypertrophied SSK during the active reproductive period the diameter of the SSK tubule was 5 times greater than that of the SSK tubule during the inactive reproductive period However SSK development in female lizards has been reported in the genus Cnemidophorus (Del Conte 1972 Del Conte and Tamayo 1973) and Scincella laterale (Sever and Hopkins 2005) They suggested that the females had a low level of natural androgens which caused the SSK development (Del Conte and Tamayo 1973 Sever and Hopkins 2005) In the present study we did not monitor the annual seasonal alterations in female SSK The hypertrophy of the SSK is synchronous with androgen secretion and spermatogenic activity (Sever and Hopkins 2005) Norris (2013) also stated that the SSK of sexually active squamates undergoes hypertrophy and is under the influence of androgens In this study we did not investigate any alterations in annual SSK with annual androgen secretion but we did demonstrate that the hypertrophy and the regression of SSK changed seasonally and synchronously with the active spermatogenic event and the spermatogenic arrest respectively In the Iberian rock lizard Lacerta monticola SSK secretions form a copulatory plug that adheres to the femalersquos cloaca following copulation to occlude oviductal openings however such a plug does not prevent subsequent mating nor does it reduce the femalersquos attractiveness (Moreira and Birkhead 2003)

With our investigations on annual alterations in female ovarian morphologies between 2 Calotes we found that individuals in the same Calotes species showed different timing of reproductive events throughout a 1-year period (the data are not shown here) Additionally there was quite clear evidence that QU EV and LV follicles and oviductal eggs overlapped among individuals within the same populations of both Calotes species Female reproductive status is definitely distinguishable between the 2 Calotes species Gravid lizards were encountered in 1 individual of C emma in April and in 3 individuals of C versicolor in June August and September Shanbhag et al (2000) reported that female C versicolor showed inactive reproduction from December to April and gravidity was encountered from May to October

We found that in the previtellogenic follicles (QU and EV) of females of both Calotes species the granulosa layer contained 2 types of cells pyriform and small cells Uribe et al (1996) stated that in squamates the follicular epithelium or granulosa initially consists of small cuboidal cells but differentiates during the previtellogenic phase and becomes multilayered and polymorphic by the presence of unique flask-shaped pyriform cells intermediate cells and

small cells These pyriform cells differentiate from small somatic follicular cells early in follicular development via the intermediate-cell stage to become nurse cells in direct contact with the developing oocytes (Maurizii et al 2004) Differentiation of the small cells into pyriform cells appears to be linked to the progressive appearance of glycoproteins with terminal α-N-acetylgalactosamine residues on the cell surface which may be involved in fusion between the oocyte and the follicle cell membranes as well as maintenance of the differentiated pyriform cells The pyriform cells are connected to the oocyte via intercellular bridges containing a cytoskeleton of α-tubulin and cytokeratin microtubules (Maurizii et al 2004) Tumkiratiwong et al (2012) also demonstrated that the previtellogenic follicles of the captive monocled cobra Naja kaouthia had many pyriform cells in the granulosa layer but fewer in the vitellogenic follicles In this study the pyriform cells disappeared when the follicles entered the vitellogenic stage Andreuccetti (1992) studied the differentiation of pyriform cells and their contribution to oocyte growth in 3 lizards namely Tarentola mauritanica Cordylus wittifer and Platysaurus intermedius and a colubrid snake Coluber viridiflavus and revealed that pyriform cells differentiate from small follicle cells via intermediate cells after establishing an intercellular bridge with the oocyte Once pyriform cells are differentiated they display ultrastructural features indicative of synthetic activity including abundant ribosomes Golgi membranes vacuoles mitochondria and lipid droplets These cellular components extend to the apex of the cell at the level of the intercellular bridge suggesting that constituents of pyriform cells may be transferred to the oocyte Pyriform cells and the oocytes may fulfill similar vitellogenic functions The establishment of an intercellular bridge may represent a crucial event in the development of an integrated system in which pyriform cells and oocytes cooperate Norris (2013) reported that the squamate granulosa contains the pyriform cells which are in direct contact with the developing oocyte and are apparently involved with early steps in oocyte development soon after the onset of vitellogenesis As ovulation approaches the granulosa cells as well as some thecal cells accumulate cholesterol-positive lipids and proliferate and luteinize to form corpora lutea following ovulation Follicular atresia is a common occurrence in reptilian ovaries as in other vertebrates (Norris 2013) as we found the corpora lutea in the follicle in accordance to oviductal egg appearances of a representative C versicolor collected in September Additionally as shown in Table 1 several atretic follicles lt25 mm in diameter were commonly encountered in C emma and C versicolor

Based on both morphological and histological investigations we found that testes ST and SSK were

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700

concomitantly active and were associated with high levels of plasma testosterone We also demonstrated that there were high correlations between levels of plasma testosterone (T) and testicular mass where annual changes occurred in the same direction as the testicular size and time of spermatogenetic events among the males of the Calotes species Radder et al (2001) reported that in the male tropical or oriental garden lizard C versicolor plasma T is highest during the breeding season which correlated with testis mass and reproductive behavior Changes in T levels are associated with high spermatogenetic activity Radder et al (2001) also stated that the changes in plasma T levels during different phases of the male reproductive cycle in C versicolor follow a reproductive pattern of a prenuptial type of spermatogenesis that is similar to that of some other species of lizards the spiny-tailed lizard Uromastix hardwicki (Arslan et al 1978a) the viviparous lizard Lacerta vivipara (Courty and Dufaure 1982) the western shingleback lizard Tiliqua (Trachydosaurus) rugosa (Bourne et al 1986) the male lizard Podarcis s sicula (Ando et al 1990) Podarcis s sicula Raf (Ando et al 1992) the white-throated savanna monitor Varanus albigularis (Phillips and Millar 1998) and the male brown anoles Anolis sagrei (Tokarz et al 1998)

We found that estradiol (E2) levels increased in vitellogenic females its high levels were associated with the presence of the largest vitellogenic follicles in the 2 Calotes species Radder et al (2001) reported that in female C versicolor with overlapping reproductive events such as vitellogenesis and gestation E2 was at low levels when the ovaries were regressed and at high levels at vitellogenic follicular recruitment reaching peak level at the time of preovulatory follicles The same patterns of E2 secretion were found again when the second set of follicles underwent vitellogenesis (Radder et al 2001) Surprisingly Amey and Whittier (2000) reported that in female bearded dragons Pagona barbata plasma E2 was low or nondetectable across all reproductive states In C versicolor E2 levels were low in nonreproductive females with small previtellogenic follicles and those in the EG phase (Radder et al 2001) We do not discuss the level of the plasma progesterone (P) during the gestation period as its level was not detectable in this study The gravid lizards in EG exhibited low plasma E2 but high P levels and the highest P levels coincided with eggshell production P levels declined after eggshell formation as reported in other gravid individuals in several species of lizards that do not possess vitellogenic follicles of the subsequent clutch including C versicolor (Radder et al 2001) Uromastix hardwicki (Arslan et al 1978b) Agama atra (Van Wyk 1984) Eumeces obsoletus Scelporus undulatus and Crotaphytus collaris (Masson and Guillette 1987) and Psammodromus algirus (Diaz et al 1994) However a

decline in P levels in MG with vitellogenic follicles did not seem to facilitate recruitment or growth of the subsequent set of vitellogenic follicles in gravid Sceloporus jarrovi (Guillette et al 1981)

In the present study there were variations in the timing of breeding between the 2 Calotes species and even within populations of the same species We could not relate the copulation timing of 2 such Calotes species to gonadal activity or sex hormonal surges as the timing of natural mating could not be observed during the times we collected data Lizard species that inhabit temperate zones have mostly exhibited seasonal reproduction (Fitch 1970 Licht 1984 Pianka and Vitt 2003) The 10 lizard species that have been studied widely to date exhibit an associated reproductive pattern (Lovern 2011) that is green anoles Anolis carolinensis (Crews 1980 Lovern et al 2004) brown anoles Anolis sagrei (Lee et al 1989 Tokarz 1998) eastern fence lizards Sceloporus undulates (Cox et al 2005) mountain spiny lizards Sceloporus jarrovi (Woodley and Moore 1999) tree lizards Urosaurus ornatus (French and Moore 2008) wall lizards Podarcis sicula (Putti et al 2009) common lizards Lacerta vivipara (Vercken and Clobert 2008) little striped whiptail lizards Cnemidophorus inornatus (Crews 2005) garden lizards Calotes versicolor (Shanbhag 2003 Lovern 2011) and leopard geckos Eublepharis macularius (Rhen et al 2005) The temperate Florida populations of the brown anole Anolis sagrei show a strong seasonality in reproduction (Lee et al 1989) while the tropical Caribbean (Licht and Gorman 1970 Sexton and Brown 1977) and Hawaiian populations of this species (Goldberg et al 2002) show a less-pronounced seasonality in which reproductively active individuals can be found throughout the year Although individuals within a population of many tropical lizard species can breed at any time no individuals within the population breed year-round (Lovern 2011) Additionally Vitt and Caldwell (2009) stated that the belief used to be that tropical squamates had continuous reproduction in aseasonal tropical environments or reproduced during the wet season in a wetndashdry seasonal tropical environment Many tropical lizard species namely the anoles Anolis acutus (Ruibal et al 1972) Anolis limifrons (Sexton et al 1971) and Anolis opalinus (Jenssen and Nunez 1994) as well as the gecko Cyrtodactylus malyanus the flying lizard Draco melanopogon (Inger and Greenberg 1966) and the parthenogenetic oviparous whiptail lizard Cnemidophorus nativo (Menezes et al 2004) showed slightly more frequent breeding during the wet season than during the dry season (Jenssen and Nunez 1994)

Reproductive patterns can be described in a variety of ways but not all species fit neatly into such categorizations However 2 general types of reproductive patterns are

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recognized in terms of prenuptial and postnuptial reproductive patterns (Lance 1998) Prenuptial reproductive pattern terms such as gonadal recrudescence sex steroid production and gametogenesis occur in advance of mating whereas postnuptial reproductive patterns occur following mating In other words in a high-elevation population of Sceloporus grammicus in Parque Nacional de Zoquiapan in central Mexico an active reproductive event occurring in the early fall is described as dissociated from testicular recrudescence in males but is associated with the initiation of ovarian recrudescence in females (Guillette and Casas-Andreu 1980 1981 Zuniga-Vega et al 2008) This is in contrast to S grammicus from Teotihuacan Mexico in which testicular recrudescence and breeding occur in the summer and fall at the onset of female ovarian recrudescence (Jimenez-Cruz et al 2005) In S mucronatus from Valle de la Cantimplora Mexico peak testicular recrudescence and mating occur during the summer prior to ovarian recrudescence which does not occur until several months later (Ortega-Leon et al 2009) This is distinct from many fall-breeding

populations elsewhere (Mendez-De La Cruz et al 1994 Villagran-Santa Cruz et al 1994) The examples above demonstrate that gonadal activity and mating behavior are clearly variable but hormone analyses have not been performed in these species and so endocrine relationships cannot be assessed at this point

In conclusion we suggest that the males and females of the 2 Calotes species have much more prolonged active reproductive phases than inactive reproductive phases The reproductive patterns of C emma and C versicolor were classified into the same reproductive pattern of continual reproduction

AcknowledgmentsWe thank the Department of Zoology of Kasetsart University for financial support We also thank the staff of Sakaerat Environmental Research Station Nakhon Ratchasima Province for devoting time for research collaboration We also thank Mrs Sureerat Sangkrut for drawing all illustrations In addition we wish to thank the anonymous referees for many helpful suggestions

References

Aldridge RD Goldberg SR Wisniewski SS Bufalino AP Dillman CB (2009) The reproductive cycle and estrus in the colubrid snakes of temperate North America Contemp Herpetol 4 1-31

Amey AP Whittier JM (2000) Seasonal patterns of plasma steroid hormones in males and females of the bearded dragon lizard Pogona barbata Gen Comp Endocrinol 117 335-342

Andreuccetti P (1992) An ultrastructural study of differentiation of pyriform cells and their contribution to oocyte growth in representative Squamata J Morphol 212 1-11

Ando S Panno ML Ciarcia G Imbrogno E Buffone M Beraldi E Sisci D Angelini F Botte V (1990) Plasma sex hormone concentrations during the reproductive cycle in the male lizard Podarcis s sicula J Reprod Fertil 90 353-360

Ando S Ciarcia G Panno ML Imbrogno E Tarantino G Buffone M Beraldi E Angelini F Botte V (1992) Sex steroids levels in the plasma and testis during the reproductive cycle of lizard Podarcis s sicula Raf Gen Comp Endocrinol 85 1-7

Arslan MJ Lobo J Zaidi AA Jalali S Qazi MH (1978a) Annual androgen rhythm in the spiny-tailed lizard Uromastix hardwicki Gen Comp Endocrinol 36 16-22

Arslan MJ Zaidi P Lobo J Zaidi AA Qazi MH (1978b) Steroid levels in preovulatory and gravid lizards (Uromastix hardwicki) Gen Comp Endocrinol 34 300-303

Avwioro G (2011) Histochemical uses of haematoxylin-a review J Pham Clin Sci 1 24-34

Bishop JE (1959) A histological and histochemical study of the kidney tubule of the common garter snake Thamnophis sirtalis with special reference to the sexual segment in the male J Morphol 104 307-358

Bourne AR Taylor JL Watson TG (1986) Annual cycles of plasma and testicular androgens in the lizard Tiliqua (Trachydosaurus) rugosa Gen Comp Endocrinol 61 278-286

Courty Y Dufaure JP (1982) Circannual testosterone dihydrotestosterone and androstenedione in the plasma and testis of Lacerta vivipara a seasonally breeding viviparous lizard Steroids 39 517-529

Cox RM Skelly SL Leo A John-Alder HB (2005) Testosterone regulates sexually dimorphic coloration in the eastern fence lizard Sceloporus undulatus Copeia 2005 597-608

Crews D (1976) Hormonal control of male courtship behavior and female attractively in the garter snake (Thamnophis sirtalis parietalis) Horm Behav 7 451-460

Crews D (1980) Interrelationships among ecological behavioral and neuroendocrine processes in the reproductive cycle of Anolis carolinensis and other reptiles Adv Stud Behav 11 1-74

Crews D (1984) Gamete production sex hormone secretion and mating behavior uncoupled Horm Behav 14 22-28

Crews D (1999) Reptilian reproduction overview In Knobil E Neil JD editors Encyclopedia of Reproduction New York NY USA Academic Press pp 239-243

Crews D Camazine B Diamond M Mason R Tokarz R Garstka WR (1984) Hormonal independence of courtship behavior in the male garter snake Horm Behav 14 29-41

Crews D (2005) Evolution of neuroendocrine mechanisms that regulate sexual behavior Trends Endocrinol Metab 16 354-361

Del Conte E (1972) Granular secretion in the kidney Rss of female lizards Cnemidophorus l lemniscatus (Sauria Teiidae) J Morphol 137 181-191

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frozen using dry ice the organic phase was then decanted into another vial and evaporated to dryness under a gentle stream of nitrogen at 37 degC Finally the extract was reconstituted with 250 microL of estradiol buffer The estradiol extraction procedure was performed using a Coat A Count Estradiol (TKE2) RIA kit (Diagnostic Products) The intra- and interassay variations calculated as CVs were 53 and 64 respectively The approximate sensitivity of this assay was 10 pgmL The specificity of cross-reactivity with estradiol was 032 The spiking recovery values averaged 968 plusmn 33 Dilutions of 50 25 125 and 625 of the undiluted concentration of 2309 pgmL were 1144 pgmL 600 pgmL 279 pgmL and 148 pgmL respectively27 Statistical analysisTesticular masses testosterone levels ovarian weights estradiol levels and diameters of the largest follicle were expressed as the mean plusmn standard error of the mean (SEM) The KolmogorovndashSmirnov test and Levenersquos test were used to determine if the data were normally distributed and the homogeneity of variance respectively Nonparametric tests were used as all data mentioned above were nonnormal and heteroscedastic Therefore the KruskalndashWallis H test was used to test for differences in the ovarian weights the estradiol levels and the diameters of the largest follicle among the reproductive stages of nongestation periods The MannndashWhitney U test was then used to compare differences

in the ovarian weights the estradiol levels and the diameters of the largest follicle between each reproductive stage of the various follicular growths of C emma only because of the insufficient sample size There was no statistical analysis of differences in male testosterone levels or testicular masses based on months due to the small amount of data available Spearmanrsquos correlation coefficients were used to determine relationships between the testicular mass and testosterone and the diameter of the largest follicles and estradiol levels The level of significance was set to P lt 00528 Ethical aspectsThis study was approved by the Ethics Committee of the Department of National Parks Wildlife and Plant Conservation Ministry of Natural Resources and Environment Thailand (License No 090930218344)

3 Results31 Annual alterations in male and female reproductive morphologies of Calotes Annual changes in testicular morphological events between C emma and C versicolor are depicted in Figure 2 The representative C emma testes were recrudesced in December continued to hypertrophy from January to April and in June and completely regressed from September to November In the representative C versicolor the testes became hypertrophied from January to September and regressed from October to November

Figure 2 Schematics of annual changes in testicular size Top C emma bottom C versicolor Notes T testis Vd vas deferens K kidney JanndashDec denotes from January to December All scale bars equal 5 mm

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Annual changes in ovarian morphological events (Figure 3) and annual changes in the number of follicular types and egg types (Table 1) of the representative 2 Calotes species are shown In the representative C emma we found that in January April from June to July and from August to November follicles were in the EV EG LV and QU stages respectively (Figure 3 top Table 1) In the representative C versicolor we found that in January and February the ovaries contained only QU follicles in June and August QU and EV follicles and oviductal eggs in September QU follicles and oviductal eggs in November only QU follicles (Figure 3 bottom Table 1)32 Annual histological alterations in ST SSK and ovaries of Calotes In the male representative C emma the ST and SSK were hypertrophied from January to April and in June then regressed from September to November becoming active again in December (Figure 4 top left and top right respectively) Spermatozoal masses were contained inside the hypertrophied ST Spermatogonia initially appeared in November additionally a few types of germ cells occurred but there were still no active spermatozoa in December (Figure 4 top left) In the representative C versicolor both ST and SSK were active from January to September but were inactive from November to December (Figure 5 top left and top right)

We found that in the female representative C emma active ovaries contained follicles of vitellogenic stages in January and June but inactive ovaries contained previtellogenic and atretic follicles in August (Figure 4 bottom left and bottom right) In the representative C versicolor ovaries contained previtellogenic and atretic follicles in January and both previtellogenic and EV follicles in June (Figure 5 bottom left and bottom right) Additionally we found corpus luteum in the ovaries of 1 representative in September (Figure 5 bottom left and bottom right) We also observed cellular alterations in the granulosa layer of both C emma and C versicolor It was demonstrated that inside the granulosa layer of the previtellogenic follicles both types of small and pyriform cells appeared However both such cell types disappeared in the granulosa layer of the vitellogenic follicles (Figures 4 and 5 bottom right)33 Annual plasma testosterone levels and testicular masses in Calotes Annual variations in plasma testosterone levels and testicular masses between the 2 Calotes species are depicted (Figures 6a and 6b) In the representative C emma plasma testosterone levels and testicular masses initially increased in December peaked in March and thereafter gradually reduced from April to November (Figure 6a) In the representative C versicolor plasma testosterone

Figure 3 Schematics of seasonal changes in ovarian size Top C emma bottom C versicolor Notes OvaF ovarian follicles OviE oviductal eggs Ovi oviduct All scale bars equals 5 mm JanndashNov denotes from January to November

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levels and testicular masses initially increased in January peaked in April and thereafter gradually reduced from May to November (Figure 6b) The changes in testicular masses tended to correspond well to the changes in plasma testosterone levels between the 2 species in the genus Calotes (r = 0789 P = 0001 and r = 0732 P = 0001 for C emma and C versicolor respectively)34 Annual plasma estradiol and ovarian cycles in Calotes Variations in ovarian masses and the diameter of the largest follicle were observed between the 2 Calotes species

during their reproductive stages (Table 2) The ovarian weights and the diameters of the largest follicles differed significantly from the QU stage to the LV stage regarding the growth of ovarian follicles (P = 0001) in C emma It was also observed that the growth of ovarian follicles concomitantly occurred with the growth of oviductal eggs during the gestation period of the 2 species

Variations in plasma E2 levels in relation to the diameters of the largest follicles between the 2 Calotes species during reproductive stages are graphically depicted (Figures 7a and 7b) Regarding the groups of follicular sizes

Table 1 Annual changes in numbers of follicular and egg types according to Calotes species

Follicular size and eggs Jan Feb Apr May Jun Jul Aug Sep Oct Nov

C emmalt25 mm 21 - 18 - 18 22 41 33 24 2325ndash50 mm 10 - - - 4 1 - - - -gt50 mm - - - - 8 5 - - - -Oviductal eggs - - 4 - - - - - - -C versicolorlt25 mm 16 28 - - 28 - 19 16 - 1025ndash50 mm - - - - 5 - - - - -gt50 mm - - - - - - - - - -Oviductal eggs - - - - 5 - 6 5 - -

Notes JanndashNov denotes January to November respectively

Figure 4 Photomicrographs of annual changes in C emma Top left testes top right male SSK bottom left ovaries bottom right granulosa layers (GL) Notes SZ spermatozoa ST seminiferous tubules SSK sexual segments of kidney AF atretic follicle PF previtellogenic follicle VF vitellogenic follicle P pyriform cells S small cells

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of C emma plasma E2 levels were the difference between the QU and the EV stages (U = 900 P = 0090) the QU and the LV stages (U = 200 P = 0009) and the EV and the LV stages (U = 200 P = 0083) Additionally the diameters of the largest follicles were the difference between the QU and the EV stages (U = 000 P = 0004) the QU and the LV stages (U = 000 P = 0004) and the EV and the LV stages (U = 000 P = 0021) The correlation coefficient between plasma E2 and the diameter of the largest follicle was 082 (P = 0001) and 032 (P = 0365) of C emma and C versicolor respectively

4 DiscussionAnnual variations in the timing of male and female reproductive stages were encountered among populations of C emma and C versicolor even in individuals of the same population Annual changes in testicular sizes were categorized into 2 phases in both species (1) an active hypertrophied testicular phase and (2) an inactive

regressed testicular phase However the timing of those 2 events appeared asynchronously in the 2 Calotes species even in individuals of the same population (data not shown here) The testes were hypertrophied with active spermatozoal production from December to June and January to September in the representative C emma and C versicolor respectively A study by Gouder and Nadkarni (1979) showed that males of C versicolor widely distributed in India were spermatogenetically active from April to September Active vitellogenic follicles and oviductal eggs were concomitantly encountered in C emma and C versicolor Therefore these 2 Calotes species exhibited polyautochrony and multiclutches Radder et al (2001) reported that Indian garden lizards C versicolor showed polyautochrony and multiclutches The representative gravid lizards were found in April in C emma and in June and from August to September in C versicolor Gravid C versicolor whose habitat is in India was encountered from May to October (Shanbhag and Prasad 1993) We

Figure 5 Photomicrographs of annual changes in C versicolor Top left testes top right male SSK bottom left ovaries bottom right granulosa layers (GL) Notes SZ spermatozoa ST seminiferous tubules SSK sexual segments of kidney AF atretic follicle PF previtellogenic follicle VF vitellogenic follicle P pyriform cells S small cells CL corpus luteum

Figure 6 Annual profiles (mean plusmn SEM) of testosterone levels and testicular masses (a) C emma (b) C versicolor Notes JanndashDec denotes January to December The numbers (in parentheses) represent the number of analyzed samples in each month

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are likely to suggest after studying the annual alterations in the male and female reproductive morphologies of the 2 Calotes species that the active reproductive events of both males and females of the Calotes species lasted nearly 1 year with only a few months of reproductive arrest which is especially seen in C versicolor

According to our investigations on annual histological alterations in ST and male SSK timing of the 2 Calotes species both ST and SSK were in active spermatogenic and

hypertrophied stages respectively which corresponded well with the timing of the testicular hypertrophied stage mentioned above In other words the timing of arrested spermatogenesis and regressed SSK was in accordance with that of the regressed testes Likewise we confirmed that the 2 Calotes species have an active reproductive stage that is much longer than the inactive reproductive stage

SSK is present in a variety of male snakes and lizards but is absent in both turtles (Regaud and Policard 1903)

Table 2 Changes in ovarian weight and diameter of the largest follicle in Calotes during the reproduction cycle

Reproductive stages N Ovarian weights Diameter of the largest follicle

C emma (20)QU 11 002 plusmn 000a 178 plusmn 006a

EV 4 011 plusmn 009b 350 plusmn 033b

LV 4 171 plusmn 049c 802 plusmn 085c

EG 1 002 240MG - - -LG - - -C versicolor (9)QU 4 002 plusmn 000 152 plusmn 199EV 1 02 428LV - - -EG 3 002 plusmn 000 222 plusmn 069MG 2 011 plusmn 009 436 plusmn 036LG - - -

Data are presented as mean plusmn SEM The differences in superscript alphabets indicate the significant differences among the reproductive stages of nongestation at P lt 001 (N sample sizes)Notes QU quiescent EV early vitellogenic LV late vitellogenic EG early gestation MG mid-gestation and LG late gestation

Figure 7 Changes in the plasma levels of estradiol and the diameter of the largest follicle (a) C emma (b) C versicolor Notes QU quiescent EV early vitellogenic LV late vitellogenic EG early gestation MG mid-gestation LG late gestation Data are presented as mean plusmn SEM The differences in superscript alphabets (estradiol levels) and in the numbers of asterisks (diameters of the largest follicles) indicate the significant differences between the various follicular sizes at P lt 001 The number (in parentheses) represents the analyzed samples in each month

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and crocodilians (Fox 1952) Bishop (1959) found that the testes of the male garter snake Thamnophis sirtalis were spermatogenically active during the same time as the hypertrophied SSK during the active reproductive period the diameter of the SSK tubule was 5 times greater than that of the SSK tubule during the inactive reproductive period However SSK development in female lizards has been reported in the genus Cnemidophorus (Del Conte 1972 Del Conte and Tamayo 1973) and Scincella laterale (Sever and Hopkins 2005) They suggested that the females had a low level of natural androgens which caused the SSK development (Del Conte and Tamayo 1973 Sever and Hopkins 2005) In the present study we did not monitor the annual seasonal alterations in female SSK The hypertrophy of the SSK is synchronous with androgen secretion and spermatogenic activity (Sever and Hopkins 2005) Norris (2013) also stated that the SSK of sexually active squamates undergoes hypertrophy and is under the influence of androgens In this study we did not investigate any alterations in annual SSK with annual androgen secretion but we did demonstrate that the hypertrophy and the regression of SSK changed seasonally and synchronously with the active spermatogenic event and the spermatogenic arrest respectively In the Iberian rock lizard Lacerta monticola SSK secretions form a copulatory plug that adheres to the femalersquos cloaca following copulation to occlude oviductal openings however such a plug does not prevent subsequent mating nor does it reduce the femalersquos attractiveness (Moreira and Birkhead 2003)

With our investigations on annual alterations in female ovarian morphologies between 2 Calotes we found that individuals in the same Calotes species showed different timing of reproductive events throughout a 1-year period (the data are not shown here) Additionally there was quite clear evidence that QU EV and LV follicles and oviductal eggs overlapped among individuals within the same populations of both Calotes species Female reproductive status is definitely distinguishable between the 2 Calotes species Gravid lizards were encountered in 1 individual of C emma in April and in 3 individuals of C versicolor in June August and September Shanbhag et al (2000) reported that female C versicolor showed inactive reproduction from December to April and gravidity was encountered from May to October

We found that in the previtellogenic follicles (QU and EV) of females of both Calotes species the granulosa layer contained 2 types of cells pyriform and small cells Uribe et al (1996) stated that in squamates the follicular epithelium or granulosa initially consists of small cuboidal cells but differentiates during the previtellogenic phase and becomes multilayered and polymorphic by the presence of unique flask-shaped pyriform cells intermediate cells and

small cells These pyriform cells differentiate from small somatic follicular cells early in follicular development via the intermediate-cell stage to become nurse cells in direct contact with the developing oocytes (Maurizii et al 2004) Differentiation of the small cells into pyriform cells appears to be linked to the progressive appearance of glycoproteins with terminal α-N-acetylgalactosamine residues on the cell surface which may be involved in fusion between the oocyte and the follicle cell membranes as well as maintenance of the differentiated pyriform cells The pyriform cells are connected to the oocyte via intercellular bridges containing a cytoskeleton of α-tubulin and cytokeratin microtubules (Maurizii et al 2004) Tumkiratiwong et al (2012) also demonstrated that the previtellogenic follicles of the captive monocled cobra Naja kaouthia had many pyriform cells in the granulosa layer but fewer in the vitellogenic follicles In this study the pyriform cells disappeared when the follicles entered the vitellogenic stage Andreuccetti (1992) studied the differentiation of pyriform cells and their contribution to oocyte growth in 3 lizards namely Tarentola mauritanica Cordylus wittifer and Platysaurus intermedius and a colubrid snake Coluber viridiflavus and revealed that pyriform cells differentiate from small follicle cells via intermediate cells after establishing an intercellular bridge with the oocyte Once pyriform cells are differentiated they display ultrastructural features indicative of synthetic activity including abundant ribosomes Golgi membranes vacuoles mitochondria and lipid droplets These cellular components extend to the apex of the cell at the level of the intercellular bridge suggesting that constituents of pyriform cells may be transferred to the oocyte Pyriform cells and the oocytes may fulfill similar vitellogenic functions The establishment of an intercellular bridge may represent a crucial event in the development of an integrated system in which pyriform cells and oocytes cooperate Norris (2013) reported that the squamate granulosa contains the pyriform cells which are in direct contact with the developing oocyte and are apparently involved with early steps in oocyte development soon after the onset of vitellogenesis As ovulation approaches the granulosa cells as well as some thecal cells accumulate cholesterol-positive lipids and proliferate and luteinize to form corpora lutea following ovulation Follicular atresia is a common occurrence in reptilian ovaries as in other vertebrates (Norris 2013) as we found the corpora lutea in the follicle in accordance to oviductal egg appearances of a representative C versicolor collected in September Additionally as shown in Table 1 several atretic follicles lt25 mm in diameter were commonly encountered in C emma and C versicolor

Based on both morphological and histological investigations we found that testes ST and SSK were

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concomitantly active and were associated with high levels of plasma testosterone We also demonstrated that there were high correlations between levels of plasma testosterone (T) and testicular mass where annual changes occurred in the same direction as the testicular size and time of spermatogenetic events among the males of the Calotes species Radder et al (2001) reported that in the male tropical or oriental garden lizard C versicolor plasma T is highest during the breeding season which correlated with testis mass and reproductive behavior Changes in T levels are associated with high spermatogenetic activity Radder et al (2001) also stated that the changes in plasma T levels during different phases of the male reproductive cycle in C versicolor follow a reproductive pattern of a prenuptial type of spermatogenesis that is similar to that of some other species of lizards the spiny-tailed lizard Uromastix hardwicki (Arslan et al 1978a) the viviparous lizard Lacerta vivipara (Courty and Dufaure 1982) the western shingleback lizard Tiliqua (Trachydosaurus) rugosa (Bourne et al 1986) the male lizard Podarcis s sicula (Ando et al 1990) Podarcis s sicula Raf (Ando et al 1992) the white-throated savanna monitor Varanus albigularis (Phillips and Millar 1998) and the male brown anoles Anolis sagrei (Tokarz et al 1998)

We found that estradiol (E2) levels increased in vitellogenic females its high levels were associated with the presence of the largest vitellogenic follicles in the 2 Calotes species Radder et al (2001) reported that in female C versicolor with overlapping reproductive events such as vitellogenesis and gestation E2 was at low levels when the ovaries were regressed and at high levels at vitellogenic follicular recruitment reaching peak level at the time of preovulatory follicles The same patterns of E2 secretion were found again when the second set of follicles underwent vitellogenesis (Radder et al 2001) Surprisingly Amey and Whittier (2000) reported that in female bearded dragons Pagona barbata plasma E2 was low or nondetectable across all reproductive states In C versicolor E2 levels were low in nonreproductive females with small previtellogenic follicles and those in the EG phase (Radder et al 2001) We do not discuss the level of the plasma progesterone (P) during the gestation period as its level was not detectable in this study The gravid lizards in EG exhibited low plasma E2 but high P levels and the highest P levels coincided with eggshell production P levels declined after eggshell formation as reported in other gravid individuals in several species of lizards that do not possess vitellogenic follicles of the subsequent clutch including C versicolor (Radder et al 2001) Uromastix hardwicki (Arslan et al 1978b) Agama atra (Van Wyk 1984) Eumeces obsoletus Scelporus undulatus and Crotaphytus collaris (Masson and Guillette 1987) and Psammodromus algirus (Diaz et al 1994) However a

decline in P levels in MG with vitellogenic follicles did not seem to facilitate recruitment or growth of the subsequent set of vitellogenic follicles in gravid Sceloporus jarrovi (Guillette et al 1981)

In the present study there were variations in the timing of breeding between the 2 Calotes species and even within populations of the same species We could not relate the copulation timing of 2 such Calotes species to gonadal activity or sex hormonal surges as the timing of natural mating could not be observed during the times we collected data Lizard species that inhabit temperate zones have mostly exhibited seasonal reproduction (Fitch 1970 Licht 1984 Pianka and Vitt 2003) The 10 lizard species that have been studied widely to date exhibit an associated reproductive pattern (Lovern 2011) that is green anoles Anolis carolinensis (Crews 1980 Lovern et al 2004) brown anoles Anolis sagrei (Lee et al 1989 Tokarz 1998) eastern fence lizards Sceloporus undulates (Cox et al 2005) mountain spiny lizards Sceloporus jarrovi (Woodley and Moore 1999) tree lizards Urosaurus ornatus (French and Moore 2008) wall lizards Podarcis sicula (Putti et al 2009) common lizards Lacerta vivipara (Vercken and Clobert 2008) little striped whiptail lizards Cnemidophorus inornatus (Crews 2005) garden lizards Calotes versicolor (Shanbhag 2003 Lovern 2011) and leopard geckos Eublepharis macularius (Rhen et al 2005) The temperate Florida populations of the brown anole Anolis sagrei show a strong seasonality in reproduction (Lee et al 1989) while the tropical Caribbean (Licht and Gorman 1970 Sexton and Brown 1977) and Hawaiian populations of this species (Goldberg et al 2002) show a less-pronounced seasonality in which reproductively active individuals can be found throughout the year Although individuals within a population of many tropical lizard species can breed at any time no individuals within the population breed year-round (Lovern 2011) Additionally Vitt and Caldwell (2009) stated that the belief used to be that tropical squamates had continuous reproduction in aseasonal tropical environments or reproduced during the wet season in a wetndashdry seasonal tropical environment Many tropical lizard species namely the anoles Anolis acutus (Ruibal et al 1972) Anolis limifrons (Sexton et al 1971) and Anolis opalinus (Jenssen and Nunez 1994) as well as the gecko Cyrtodactylus malyanus the flying lizard Draco melanopogon (Inger and Greenberg 1966) and the parthenogenetic oviparous whiptail lizard Cnemidophorus nativo (Menezes et al 2004) showed slightly more frequent breeding during the wet season than during the dry season (Jenssen and Nunez 1994)

Reproductive patterns can be described in a variety of ways but not all species fit neatly into such categorizations However 2 general types of reproductive patterns are

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recognized in terms of prenuptial and postnuptial reproductive patterns (Lance 1998) Prenuptial reproductive pattern terms such as gonadal recrudescence sex steroid production and gametogenesis occur in advance of mating whereas postnuptial reproductive patterns occur following mating In other words in a high-elevation population of Sceloporus grammicus in Parque Nacional de Zoquiapan in central Mexico an active reproductive event occurring in the early fall is described as dissociated from testicular recrudescence in males but is associated with the initiation of ovarian recrudescence in females (Guillette and Casas-Andreu 1980 1981 Zuniga-Vega et al 2008) This is in contrast to S grammicus from Teotihuacan Mexico in which testicular recrudescence and breeding occur in the summer and fall at the onset of female ovarian recrudescence (Jimenez-Cruz et al 2005) In S mucronatus from Valle de la Cantimplora Mexico peak testicular recrudescence and mating occur during the summer prior to ovarian recrudescence which does not occur until several months later (Ortega-Leon et al 2009) This is distinct from many fall-breeding

populations elsewhere (Mendez-De La Cruz et al 1994 Villagran-Santa Cruz et al 1994) The examples above demonstrate that gonadal activity and mating behavior are clearly variable but hormone analyses have not been performed in these species and so endocrine relationships cannot be assessed at this point

In conclusion we suggest that the males and females of the 2 Calotes species have much more prolonged active reproductive phases than inactive reproductive phases The reproductive patterns of C emma and C versicolor were classified into the same reproductive pattern of continual reproduction

AcknowledgmentsWe thank the Department of Zoology of Kasetsart University for financial support We also thank the staff of Sakaerat Environmental Research Station Nakhon Ratchasima Province for devoting time for research collaboration We also thank Mrs Sureerat Sangkrut for drawing all illustrations In addition we wish to thank the anonymous referees for many helpful suggestions

References

Aldridge RD Goldberg SR Wisniewski SS Bufalino AP Dillman CB (2009) The reproductive cycle and estrus in the colubrid snakes of temperate North America Contemp Herpetol 4 1-31

Amey AP Whittier JM (2000) Seasonal patterns of plasma steroid hormones in males and females of the bearded dragon lizard Pogona barbata Gen Comp Endocrinol 117 335-342

Andreuccetti P (1992) An ultrastructural study of differentiation of pyriform cells and their contribution to oocyte growth in representative Squamata J Morphol 212 1-11

Ando S Panno ML Ciarcia G Imbrogno E Buffone M Beraldi E Sisci D Angelini F Botte V (1990) Plasma sex hormone concentrations during the reproductive cycle in the male lizard Podarcis s sicula J Reprod Fertil 90 353-360

Ando S Ciarcia G Panno ML Imbrogno E Tarantino G Buffone M Beraldi E Angelini F Botte V (1992) Sex steroids levels in the plasma and testis during the reproductive cycle of lizard Podarcis s sicula Raf Gen Comp Endocrinol 85 1-7

Arslan MJ Lobo J Zaidi AA Jalali S Qazi MH (1978a) Annual androgen rhythm in the spiny-tailed lizard Uromastix hardwicki Gen Comp Endocrinol 36 16-22

Arslan MJ Zaidi P Lobo J Zaidi AA Qazi MH (1978b) Steroid levels in preovulatory and gravid lizards (Uromastix hardwicki) Gen Comp Endocrinol 34 300-303

Avwioro G (2011) Histochemical uses of haematoxylin-a review J Pham Clin Sci 1 24-34

Bishop JE (1959) A histological and histochemical study of the kidney tubule of the common garter snake Thamnophis sirtalis with special reference to the sexual segment in the male J Morphol 104 307-358

Bourne AR Taylor JL Watson TG (1986) Annual cycles of plasma and testicular androgens in the lizard Tiliqua (Trachydosaurus) rugosa Gen Comp Endocrinol 61 278-286

Courty Y Dufaure JP (1982) Circannual testosterone dihydrotestosterone and androstenedione in the plasma and testis of Lacerta vivipara a seasonally breeding viviparous lizard Steroids 39 517-529

Cox RM Skelly SL Leo A John-Alder HB (2005) Testosterone regulates sexually dimorphic coloration in the eastern fence lizard Sceloporus undulatus Copeia 2005 597-608

Crews D (1976) Hormonal control of male courtship behavior and female attractively in the garter snake (Thamnophis sirtalis parietalis) Horm Behav 7 451-460

Crews D (1980) Interrelationships among ecological behavioral and neuroendocrine processes in the reproductive cycle of Anolis carolinensis and other reptiles Adv Stud Behav 11 1-74

Crews D (1984) Gamete production sex hormone secretion and mating behavior uncoupled Horm Behav 14 22-28

Crews D (1999) Reptilian reproduction overview In Knobil E Neil JD editors Encyclopedia of Reproduction New York NY USA Academic Press pp 239-243

Crews D Camazine B Diamond M Mason R Tokarz R Garstka WR (1984) Hormonal independence of courtship behavior in the male garter snake Horm Behav 14 29-41

Crews D (2005) Evolution of neuroendocrine mechanisms that regulate sexual behavior Trends Endocrinol Metab 16 354-361

Del Conte E (1972) Granular secretion in the kidney Rss of female lizards Cnemidophorus l lemniscatus (Sauria Teiidae) J Morphol 137 181-191

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Annual changes in ovarian morphological events (Figure 3) and annual changes in the number of follicular types and egg types (Table 1) of the representative 2 Calotes species are shown In the representative C emma we found that in January April from June to July and from August to November follicles were in the EV EG LV and QU stages respectively (Figure 3 top Table 1) In the representative C versicolor we found that in January and February the ovaries contained only QU follicles in June and August QU and EV follicles and oviductal eggs in September QU follicles and oviductal eggs in November only QU follicles (Figure 3 bottom Table 1)32 Annual histological alterations in ST SSK and ovaries of Calotes In the male representative C emma the ST and SSK were hypertrophied from January to April and in June then regressed from September to November becoming active again in December (Figure 4 top left and top right respectively) Spermatozoal masses were contained inside the hypertrophied ST Spermatogonia initially appeared in November additionally a few types of germ cells occurred but there were still no active spermatozoa in December (Figure 4 top left) In the representative C versicolor both ST and SSK were active from January to September but were inactive from November to December (Figure 5 top left and top right)

We found that in the female representative C emma active ovaries contained follicles of vitellogenic stages in January and June but inactive ovaries contained previtellogenic and atretic follicles in August (Figure 4 bottom left and bottom right) In the representative C versicolor ovaries contained previtellogenic and atretic follicles in January and both previtellogenic and EV follicles in June (Figure 5 bottom left and bottom right) Additionally we found corpus luteum in the ovaries of 1 representative in September (Figure 5 bottom left and bottom right) We also observed cellular alterations in the granulosa layer of both C emma and C versicolor It was demonstrated that inside the granulosa layer of the previtellogenic follicles both types of small and pyriform cells appeared However both such cell types disappeared in the granulosa layer of the vitellogenic follicles (Figures 4 and 5 bottom right)33 Annual plasma testosterone levels and testicular masses in Calotes Annual variations in plasma testosterone levels and testicular masses between the 2 Calotes species are depicted (Figures 6a and 6b) In the representative C emma plasma testosterone levels and testicular masses initially increased in December peaked in March and thereafter gradually reduced from April to November (Figure 6a) In the representative C versicolor plasma testosterone

Figure 3 Schematics of seasonal changes in ovarian size Top C emma bottom C versicolor Notes OvaF ovarian follicles OviE oviductal eggs Ovi oviduct All scale bars equals 5 mm JanndashNov denotes from January to November

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levels and testicular masses initially increased in January peaked in April and thereafter gradually reduced from May to November (Figure 6b) The changes in testicular masses tended to correspond well to the changes in plasma testosterone levels between the 2 species in the genus Calotes (r = 0789 P = 0001 and r = 0732 P = 0001 for C emma and C versicolor respectively)34 Annual plasma estradiol and ovarian cycles in Calotes Variations in ovarian masses and the diameter of the largest follicle were observed between the 2 Calotes species

during their reproductive stages (Table 2) The ovarian weights and the diameters of the largest follicles differed significantly from the QU stage to the LV stage regarding the growth of ovarian follicles (P = 0001) in C emma It was also observed that the growth of ovarian follicles concomitantly occurred with the growth of oviductal eggs during the gestation period of the 2 species

Variations in plasma E2 levels in relation to the diameters of the largest follicles between the 2 Calotes species during reproductive stages are graphically depicted (Figures 7a and 7b) Regarding the groups of follicular sizes

Table 1 Annual changes in numbers of follicular and egg types according to Calotes species

Follicular size and eggs Jan Feb Apr May Jun Jul Aug Sep Oct Nov

C emmalt25 mm 21 - 18 - 18 22 41 33 24 2325ndash50 mm 10 - - - 4 1 - - - -gt50 mm - - - - 8 5 - - - -Oviductal eggs - - 4 - - - - - - -C versicolorlt25 mm 16 28 - - 28 - 19 16 - 1025ndash50 mm - - - - 5 - - - - -gt50 mm - - - - - - - - - -Oviductal eggs - - - - 5 - 6 5 - -

Notes JanndashNov denotes January to November respectively

Figure 4 Photomicrographs of annual changes in C emma Top left testes top right male SSK bottom left ovaries bottom right granulosa layers (GL) Notes SZ spermatozoa ST seminiferous tubules SSK sexual segments of kidney AF atretic follicle PF previtellogenic follicle VF vitellogenic follicle P pyriform cells S small cells

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of C emma plasma E2 levels were the difference between the QU and the EV stages (U = 900 P = 0090) the QU and the LV stages (U = 200 P = 0009) and the EV and the LV stages (U = 200 P = 0083) Additionally the diameters of the largest follicles were the difference between the QU and the EV stages (U = 000 P = 0004) the QU and the LV stages (U = 000 P = 0004) and the EV and the LV stages (U = 000 P = 0021) The correlation coefficient between plasma E2 and the diameter of the largest follicle was 082 (P = 0001) and 032 (P = 0365) of C emma and C versicolor respectively

4 DiscussionAnnual variations in the timing of male and female reproductive stages were encountered among populations of C emma and C versicolor even in individuals of the same population Annual changes in testicular sizes were categorized into 2 phases in both species (1) an active hypertrophied testicular phase and (2) an inactive

regressed testicular phase However the timing of those 2 events appeared asynchronously in the 2 Calotes species even in individuals of the same population (data not shown here) The testes were hypertrophied with active spermatozoal production from December to June and January to September in the representative C emma and C versicolor respectively A study by Gouder and Nadkarni (1979) showed that males of C versicolor widely distributed in India were spermatogenetically active from April to September Active vitellogenic follicles and oviductal eggs were concomitantly encountered in C emma and C versicolor Therefore these 2 Calotes species exhibited polyautochrony and multiclutches Radder et al (2001) reported that Indian garden lizards C versicolor showed polyautochrony and multiclutches The representative gravid lizards were found in April in C emma and in June and from August to September in C versicolor Gravid C versicolor whose habitat is in India was encountered from May to October (Shanbhag and Prasad 1993) We

Figure 5 Photomicrographs of annual changes in C versicolor Top left testes top right male SSK bottom left ovaries bottom right granulosa layers (GL) Notes SZ spermatozoa ST seminiferous tubules SSK sexual segments of kidney AF atretic follicle PF previtellogenic follicle VF vitellogenic follicle P pyriform cells S small cells CL corpus luteum

Figure 6 Annual profiles (mean plusmn SEM) of testosterone levels and testicular masses (a) C emma (b) C versicolor Notes JanndashDec denotes January to December The numbers (in parentheses) represent the number of analyzed samples in each month

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are likely to suggest after studying the annual alterations in the male and female reproductive morphologies of the 2 Calotes species that the active reproductive events of both males and females of the Calotes species lasted nearly 1 year with only a few months of reproductive arrest which is especially seen in C versicolor

According to our investigations on annual histological alterations in ST and male SSK timing of the 2 Calotes species both ST and SSK were in active spermatogenic and

hypertrophied stages respectively which corresponded well with the timing of the testicular hypertrophied stage mentioned above In other words the timing of arrested spermatogenesis and regressed SSK was in accordance with that of the regressed testes Likewise we confirmed that the 2 Calotes species have an active reproductive stage that is much longer than the inactive reproductive stage

SSK is present in a variety of male snakes and lizards but is absent in both turtles (Regaud and Policard 1903)

Table 2 Changes in ovarian weight and diameter of the largest follicle in Calotes during the reproduction cycle

Reproductive stages N Ovarian weights Diameter of the largest follicle

C emma (20)QU 11 002 plusmn 000a 178 plusmn 006a

EV 4 011 plusmn 009b 350 plusmn 033b

LV 4 171 plusmn 049c 802 plusmn 085c

EG 1 002 240MG - - -LG - - -C versicolor (9)QU 4 002 plusmn 000 152 plusmn 199EV 1 02 428LV - - -EG 3 002 plusmn 000 222 plusmn 069MG 2 011 plusmn 009 436 plusmn 036LG - - -

Data are presented as mean plusmn SEM The differences in superscript alphabets indicate the significant differences among the reproductive stages of nongestation at P lt 001 (N sample sizes)Notes QU quiescent EV early vitellogenic LV late vitellogenic EG early gestation MG mid-gestation and LG late gestation

Figure 7 Changes in the plasma levels of estradiol and the diameter of the largest follicle (a) C emma (b) C versicolor Notes QU quiescent EV early vitellogenic LV late vitellogenic EG early gestation MG mid-gestation LG late gestation Data are presented as mean plusmn SEM The differences in superscript alphabets (estradiol levels) and in the numbers of asterisks (diameters of the largest follicles) indicate the significant differences between the various follicular sizes at P lt 001 The number (in parentheses) represents the analyzed samples in each month

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and crocodilians (Fox 1952) Bishop (1959) found that the testes of the male garter snake Thamnophis sirtalis were spermatogenically active during the same time as the hypertrophied SSK during the active reproductive period the diameter of the SSK tubule was 5 times greater than that of the SSK tubule during the inactive reproductive period However SSK development in female lizards has been reported in the genus Cnemidophorus (Del Conte 1972 Del Conte and Tamayo 1973) and Scincella laterale (Sever and Hopkins 2005) They suggested that the females had a low level of natural androgens which caused the SSK development (Del Conte and Tamayo 1973 Sever and Hopkins 2005) In the present study we did not monitor the annual seasonal alterations in female SSK The hypertrophy of the SSK is synchronous with androgen secretion and spermatogenic activity (Sever and Hopkins 2005) Norris (2013) also stated that the SSK of sexually active squamates undergoes hypertrophy and is under the influence of androgens In this study we did not investigate any alterations in annual SSK with annual androgen secretion but we did demonstrate that the hypertrophy and the regression of SSK changed seasonally and synchronously with the active spermatogenic event and the spermatogenic arrest respectively In the Iberian rock lizard Lacerta monticola SSK secretions form a copulatory plug that adheres to the femalersquos cloaca following copulation to occlude oviductal openings however such a plug does not prevent subsequent mating nor does it reduce the femalersquos attractiveness (Moreira and Birkhead 2003)

With our investigations on annual alterations in female ovarian morphologies between 2 Calotes we found that individuals in the same Calotes species showed different timing of reproductive events throughout a 1-year period (the data are not shown here) Additionally there was quite clear evidence that QU EV and LV follicles and oviductal eggs overlapped among individuals within the same populations of both Calotes species Female reproductive status is definitely distinguishable between the 2 Calotes species Gravid lizards were encountered in 1 individual of C emma in April and in 3 individuals of C versicolor in June August and September Shanbhag et al (2000) reported that female C versicolor showed inactive reproduction from December to April and gravidity was encountered from May to October

We found that in the previtellogenic follicles (QU and EV) of females of both Calotes species the granulosa layer contained 2 types of cells pyriform and small cells Uribe et al (1996) stated that in squamates the follicular epithelium or granulosa initially consists of small cuboidal cells but differentiates during the previtellogenic phase and becomes multilayered and polymorphic by the presence of unique flask-shaped pyriform cells intermediate cells and

small cells These pyriform cells differentiate from small somatic follicular cells early in follicular development via the intermediate-cell stage to become nurse cells in direct contact with the developing oocytes (Maurizii et al 2004) Differentiation of the small cells into pyriform cells appears to be linked to the progressive appearance of glycoproteins with terminal α-N-acetylgalactosamine residues on the cell surface which may be involved in fusion between the oocyte and the follicle cell membranes as well as maintenance of the differentiated pyriform cells The pyriform cells are connected to the oocyte via intercellular bridges containing a cytoskeleton of α-tubulin and cytokeratin microtubules (Maurizii et al 2004) Tumkiratiwong et al (2012) also demonstrated that the previtellogenic follicles of the captive monocled cobra Naja kaouthia had many pyriform cells in the granulosa layer but fewer in the vitellogenic follicles In this study the pyriform cells disappeared when the follicles entered the vitellogenic stage Andreuccetti (1992) studied the differentiation of pyriform cells and their contribution to oocyte growth in 3 lizards namely Tarentola mauritanica Cordylus wittifer and Platysaurus intermedius and a colubrid snake Coluber viridiflavus and revealed that pyriform cells differentiate from small follicle cells via intermediate cells after establishing an intercellular bridge with the oocyte Once pyriform cells are differentiated they display ultrastructural features indicative of synthetic activity including abundant ribosomes Golgi membranes vacuoles mitochondria and lipid droplets These cellular components extend to the apex of the cell at the level of the intercellular bridge suggesting that constituents of pyriform cells may be transferred to the oocyte Pyriform cells and the oocytes may fulfill similar vitellogenic functions The establishment of an intercellular bridge may represent a crucial event in the development of an integrated system in which pyriform cells and oocytes cooperate Norris (2013) reported that the squamate granulosa contains the pyriform cells which are in direct contact with the developing oocyte and are apparently involved with early steps in oocyte development soon after the onset of vitellogenesis As ovulation approaches the granulosa cells as well as some thecal cells accumulate cholesterol-positive lipids and proliferate and luteinize to form corpora lutea following ovulation Follicular atresia is a common occurrence in reptilian ovaries as in other vertebrates (Norris 2013) as we found the corpora lutea in the follicle in accordance to oviductal egg appearances of a representative C versicolor collected in September Additionally as shown in Table 1 several atretic follicles lt25 mm in diameter were commonly encountered in C emma and C versicolor

Based on both morphological and histological investigations we found that testes ST and SSK were

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concomitantly active and were associated with high levels of plasma testosterone We also demonstrated that there were high correlations between levels of plasma testosterone (T) and testicular mass where annual changes occurred in the same direction as the testicular size and time of spermatogenetic events among the males of the Calotes species Radder et al (2001) reported that in the male tropical or oriental garden lizard C versicolor plasma T is highest during the breeding season which correlated with testis mass and reproductive behavior Changes in T levels are associated with high spermatogenetic activity Radder et al (2001) also stated that the changes in plasma T levels during different phases of the male reproductive cycle in C versicolor follow a reproductive pattern of a prenuptial type of spermatogenesis that is similar to that of some other species of lizards the spiny-tailed lizard Uromastix hardwicki (Arslan et al 1978a) the viviparous lizard Lacerta vivipara (Courty and Dufaure 1982) the western shingleback lizard Tiliqua (Trachydosaurus) rugosa (Bourne et al 1986) the male lizard Podarcis s sicula (Ando et al 1990) Podarcis s sicula Raf (Ando et al 1992) the white-throated savanna monitor Varanus albigularis (Phillips and Millar 1998) and the male brown anoles Anolis sagrei (Tokarz et al 1998)

We found that estradiol (E2) levels increased in vitellogenic females its high levels were associated with the presence of the largest vitellogenic follicles in the 2 Calotes species Radder et al (2001) reported that in female C versicolor with overlapping reproductive events such as vitellogenesis and gestation E2 was at low levels when the ovaries were regressed and at high levels at vitellogenic follicular recruitment reaching peak level at the time of preovulatory follicles The same patterns of E2 secretion were found again when the second set of follicles underwent vitellogenesis (Radder et al 2001) Surprisingly Amey and Whittier (2000) reported that in female bearded dragons Pagona barbata plasma E2 was low or nondetectable across all reproductive states In C versicolor E2 levels were low in nonreproductive females with small previtellogenic follicles and those in the EG phase (Radder et al 2001) We do not discuss the level of the plasma progesterone (P) during the gestation period as its level was not detectable in this study The gravid lizards in EG exhibited low plasma E2 but high P levels and the highest P levels coincided with eggshell production P levels declined after eggshell formation as reported in other gravid individuals in several species of lizards that do not possess vitellogenic follicles of the subsequent clutch including C versicolor (Radder et al 2001) Uromastix hardwicki (Arslan et al 1978b) Agama atra (Van Wyk 1984) Eumeces obsoletus Scelporus undulatus and Crotaphytus collaris (Masson and Guillette 1987) and Psammodromus algirus (Diaz et al 1994) However a

decline in P levels in MG with vitellogenic follicles did not seem to facilitate recruitment or growth of the subsequent set of vitellogenic follicles in gravid Sceloporus jarrovi (Guillette et al 1981)

In the present study there were variations in the timing of breeding between the 2 Calotes species and even within populations of the same species We could not relate the copulation timing of 2 such Calotes species to gonadal activity or sex hormonal surges as the timing of natural mating could not be observed during the times we collected data Lizard species that inhabit temperate zones have mostly exhibited seasonal reproduction (Fitch 1970 Licht 1984 Pianka and Vitt 2003) The 10 lizard species that have been studied widely to date exhibit an associated reproductive pattern (Lovern 2011) that is green anoles Anolis carolinensis (Crews 1980 Lovern et al 2004) brown anoles Anolis sagrei (Lee et al 1989 Tokarz 1998) eastern fence lizards Sceloporus undulates (Cox et al 2005) mountain spiny lizards Sceloporus jarrovi (Woodley and Moore 1999) tree lizards Urosaurus ornatus (French and Moore 2008) wall lizards Podarcis sicula (Putti et al 2009) common lizards Lacerta vivipara (Vercken and Clobert 2008) little striped whiptail lizards Cnemidophorus inornatus (Crews 2005) garden lizards Calotes versicolor (Shanbhag 2003 Lovern 2011) and leopard geckos Eublepharis macularius (Rhen et al 2005) The temperate Florida populations of the brown anole Anolis sagrei show a strong seasonality in reproduction (Lee et al 1989) while the tropical Caribbean (Licht and Gorman 1970 Sexton and Brown 1977) and Hawaiian populations of this species (Goldberg et al 2002) show a less-pronounced seasonality in which reproductively active individuals can be found throughout the year Although individuals within a population of many tropical lizard species can breed at any time no individuals within the population breed year-round (Lovern 2011) Additionally Vitt and Caldwell (2009) stated that the belief used to be that tropical squamates had continuous reproduction in aseasonal tropical environments or reproduced during the wet season in a wetndashdry seasonal tropical environment Many tropical lizard species namely the anoles Anolis acutus (Ruibal et al 1972) Anolis limifrons (Sexton et al 1971) and Anolis opalinus (Jenssen and Nunez 1994) as well as the gecko Cyrtodactylus malyanus the flying lizard Draco melanopogon (Inger and Greenberg 1966) and the parthenogenetic oviparous whiptail lizard Cnemidophorus nativo (Menezes et al 2004) showed slightly more frequent breeding during the wet season than during the dry season (Jenssen and Nunez 1994)

Reproductive patterns can be described in a variety of ways but not all species fit neatly into such categorizations However 2 general types of reproductive patterns are

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recognized in terms of prenuptial and postnuptial reproductive patterns (Lance 1998) Prenuptial reproductive pattern terms such as gonadal recrudescence sex steroid production and gametogenesis occur in advance of mating whereas postnuptial reproductive patterns occur following mating In other words in a high-elevation population of Sceloporus grammicus in Parque Nacional de Zoquiapan in central Mexico an active reproductive event occurring in the early fall is described as dissociated from testicular recrudescence in males but is associated with the initiation of ovarian recrudescence in females (Guillette and Casas-Andreu 1980 1981 Zuniga-Vega et al 2008) This is in contrast to S grammicus from Teotihuacan Mexico in which testicular recrudescence and breeding occur in the summer and fall at the onset of female ovarian recrudescence (Jimenez-Cruz et al 2005) In S mucronatus from Valle de la Cantimplora Mexico peak testicular recrudescence and mating occur during the summer prior to ovarian recrudescence which does not occur until several months later (Ortega-Leon et al 2009) This is distinct from many fall-breeding

populations elsewhere (Mendez-De La Cruz et al 1994 Villagran-Santa Cruz et al 1994) The examples above demonstrate that gonadal activity and mating behavior are clearly variable but hormone analyses have not been performed in these species and so endocrine relationships cannot be assessed at this point

In conclusion we suggest that the males and females of the 2 Calotes species have much more prolonged active reproductive phases than inactive reproductive phases The reproductive patterns of C emma and C versicolor were classified into the same reproductive pattern of continual reproduction

AcknowledgmentsWe thank the Department of Zoology of Kasetsart University for financial support We also thank the staff of Sakaerat Environmental Research Station Nakhon Ratchasima Province for devoting time for research collaboration We also thank Mrs Sureerat Sangkrut for drawing all illustrations In addition we wish to thank the anonymous referees for many helpful suggestions

References

Aldridge RD Goldberg SR Wisniewski SS Bufalino AP Dillman CB (2009) The reproductive cycle and estrus in the colubrid snakes of temperate North America Contemp Herpetol 4 1-31

Amey AP Whittier JM (2000) Seasonal patterns of plasma steroid hormones in males and females of the bearded dragon lizard Pogona barbata Gen Comp Endocrinol 117 335-342

Andreuccetti P (1992) An ultrastructural study of differentiation of pyriform cells and their contribution to oocyte growth in representative Squamata J Morphol 212 1-11

Ando S Panno ML Ciarcia G Imbrogno E Buffone M Beraldi E Sisci D Angelini F Botte V (1990) Plasma sex hormone concentrations during the reproductive cycle in the male lizard Podarcis s sicula J Reprod Fertil 90 353-360

Ando S Ciarcia G Panno ML Imbrogno E Tarantino G Buffone M Beraldi E Angelini F Botte V (1992) Sex steroids levels in the plasma and testis during the reproductive cycle of lizard Podarcis s sicula Raf Gen Comp Endocrinol 85 1-7

Arslan MJ Lobo J Zaidi AA Jalali S Qazi MH (1978a) Annual androgen rhythm in the spiny-tailed lizard Uromastix hardwicki Gen Comp Endocrinol 36 16-22

Arslan MJ Zaidi P Lobo J Zaidi AA Qazi MH (1978b) Steroid levels in preovulatory and gravid lizards (Uromastix hardwicki) Gen Comp Endocrinol 34 300-303

Avwioro G (2011) Histochemical uses of haematoxylin-a review J Pham Clin Sci 1 24-34

Bishop JE (1959) A histological and histochemical study of the kidney tubule of the common garter snake Thamnophis sirtalis with special reference to the sexual segment in the male J Morphol 104 307-358

Bourne AR Taylor JL Watson TG (1986) Annual cycles of plasma and testicular androgens in the lizard Tiliqua (Trachydosaurus) rugosa Gen Comp Endocrinol 61 278-286

Courty Y Dufaure JP (1982) Circannual testosterone dihydrotestosterone and androstenedione in the plasma and testis of Lacerta vivipara a seasonally breeding viviparous lizard Steroids 39 517-529

Cox RM Skelly SL Leo A John-Alder HB (2005) Testosterone regulates sexually dimorphic coloration in the eastern fence lizard Sceloporus undulatus Copeia 2005 597-608

Crews D (1976) Hormonal control of male courtship behavior and female attractively in the garter snake (Thamnophis sirtalis parietalis) Horm Behav 7 451-460

Crews D (1980) Interrelationships among ecological behavioral and neuroendocrine processes in the reproductive cycle of Anolis carolinensis and other reptiles Adv Stud Behav 11 1-74

Crews D (1984) Gamete production sex hormone secretion and mating behavior uncoupled Horm Behav 14 22-28

Crews D (1999) Reptilian reproduction overview In Knobil E Neil JD editors Encyclopedia of Reproduction New York NY USA Academic Press pp 239-243

Crews D Camazine B Diamond M Mason R Tokarz R Garstka WR (1984) Hormonal independence of courtship behavior in the male garter snake Horm Behav 14 29-41

Crews D (2005) Evolution of neuroendocrine mechanisms that regulate sexual behavior Trends Endocrinol Metab 16 354-361

Del Conte E (1972) Granular secretion in the kidney Rss of female lizards Cnemidophorus l lemniscatus (Sauria Teiidae) J Morphol 137 181-191

MEESOOK et al Turk J Zool

696

levels and testicular masses initially increased in January peaked in April and thereafter gradually reduced from May to November (Figure 6b) The changes in testicular masses tended to correspond well to the changes in plasma testosterone levels between the 2 species in the genus Calotes (r = 0789 P = 0001 and r = 0732 P = 0001 for C emma and C versicolor respectively)34 Annual plasma estradiol and ovarian cycles in Calotes Variations in ovarian masses and the diameter of the largest follicle were observed between the 2 Calotes species

during their reproductive stages (Table 2) The ovarian weights and the diameters of the largest follicles differed significantly from the QU stage to the LV stage regarding the growth of ovarian follicles (P = 0001) in C emma It was also observed that the growth of ovarian follicles concomitantly occurred with the growth of oviductal eggs during the gestation period of the 2 species

Variations in plasma E2 levels in relation to the diameters of the largest follicles between the 2 Calotes species during reproductive stages are graphically depicted (Figures 7a and 7b) Regarding the groups of follicular sizes

Table 1 Annual changes in numbers of follicular and egg types according to Calotes species

Follicular size and eggs Jan Feb Apr May Jun Jul Aug Sep Oct Nov

C emmalt25 mm 21 - 18 - 18 22 41 33 24 2325ndash50 mm 10 - - - 4 1 - - - -gt50 mm - - - - 8 5 - - - -Oviductal eggs - - 4 - - - - - - -C versicolorlt25 mm 16 28 - - 28 - 19 16 - 1025ndash50 mm - - - - 5 - - - - -gt50 mm - - - - - - - - - -Oviductal eggs - - - - 5 - 6 5 - -

Notes JanndashNov denotes January to November respectively

Figure 4 Photomicrographs of annual changes in C emma Top left testes top right male SSK bottom left ovaries bottom right granulosa layers (GL) Notes SZ spermatozoa ST seminiferous tubules SSK sexual segments of kidney AF atretic follicle PF previtellogenic follicle VF vitellogenic follicle P pyriform cells S small cells

MEESOOK et al Turk J Zool

697

of C emma plasma E2 levels were the difference between the QU and the EV stages (U = 900 P = 0090) the QU and the LV stages (U = 200 P = 0009) and the EV and the LV stages (U = 200 P = 0083) Additionally the diameters of the largest follicles were the difference between the QU and the EV stages (U = 000 P = 0004) the QU and the LV stages (U = 000 P = 0004) and the EV and the LV stages (U = 000 P = 0021) The correlation coefficient between plasma E2 and the diameter of the largest follicle was 082 (P = 0001) and 032 (P = 0365) of C emma and C versicolor respectively

4 DiscussionAnnual variations in the timing of male and female reproductive stages were encountered among populations of C emma and C versicolor even in individuals of the same population Annual changes in testicular sizes were categorized into 2 phases in both species (1) an active hypertrophied testicular phase and (2) an inactive

regressed testicular phase However the timing of those 2 events appeared asynchronously in the 2 Calotes species even in individuals of the same population (data not shown here) The testes were hypertrophied with active spermatozoal production from December to June and January to September in the representative C emma and C versicolor respectively A study by Gouder and Nadkarni (1979) showed that males of C versicolor widely distributed in India were spermatogenetically active from April to September Active vitellogenic follicles and oviductal eggs were concomitantly encountered in C emma and C versicolor Therefore these 2 Calotes species exhibited polyautochrony and multiclutches Radder et al (2001) reported that Indian garden lizards C versicolor showed polyautochrony and multiclutches The representative gravid lizards were found in April in C emma and in June and from August to September in C versicolor Gravid C versicolor whose habitat is in India was encountered from May to October (Shanbhag and Prasad 1993) We

Figure 5 Photomicrographs of annual changes in C versicolor Top left testes top right male SSK bottom left ovaries bottom right granulosa layers (GL) Notes SZ spermatozoa ST seminiferous tubules SSK sexual segments of kidney AF atretic follicle PF previtellogenic follicle VF vitellogenic follicle P pyriform cells S small cells CL corpus luteum

Figure 6 Annual profiles (mean plusmn SEM) of testosterone levels and testicular masses (a) C emma (b) C versicolor Notes JanndashDec denotes January to December The numbers (in parentheses) represent the number of analyzed samples in each month

MEESOOK et al Turk J Zool

698

are likely to suggest after studying the annual alterations in the male and female reproductive morphologies of the 2 Calotes species that the active reproductive events of both males and females of the Calotes species lasted nearly 1 year with only a few months of reproductive arrest which is especially seen in C versicolor

According to our investigations on annual histological alterations in ST and male SSK timing of the 2 Calotes species both ST and SSK were in active spermatogenic and

hypertrophied stages respectively which corresponded well with the timing of the testicular hypertrophied stage mentioned above In other words the timing of arrested spermatogenesis and regressed SSK was in accordance with that of the regressed testes Likewise we confirmed that the 2 Calotes species have an active reproductive stage that is much longer than the inactive reproductive stage

SSK is present in a variety of male snakes and lizards but is absent in both turtles (Regaud and Policard 1903)

Table 2 Changes in ovarian weight and diameter of the largest follicle in Calotes during the reproduction cycle

Reproductive stages N Ovarian weights Diameter of the largest follicle

C emma (20)QU 11 002 plusmn 000a 178 plusmn 006a

EV 4 011 plusmn 009b 350 plusmn 033b

LV 4 171 plusmn 049c 802 plusmn 085c

EG 1 002 240MG - - -LG - - -C versicolor (9)QU 4 002 plusmn 000 152 plusmn 199EV 1 02 428LV - - -EG 3 002 plusmn 000 222 plusmn 069MG 2 011 plusmn 009 436 plusmn 036LG - - -

Data are presented as mean plusmn SEM The differences in superscript alphabets indicate the significant differences among the reproductive stages of nongestation at P lt 001 (N sample sizes)Notes QU quiescent EV early vitellogenic LV late vitellogenic EG early gestation MG mid-gestation and LG late gestation

Figure 7 Changes in the plasma levels of estradiol and the diameter of the largest follicle (a) C emma (b) C versicolor Notes QU quiescent EV early vitellogenic LV late vitellogenic EG early gestation MG mid-gestation LG late gestation Data are presented as mean plusmn SEM The differences in superscript alphabets (estradiol levels) and in the numbers of asterisks (diameters of the largest follicles) indicate the significant differences between the various follicular sizes at P lt 001 The number (in parentheses) represents the analyzed samples in each month

MEESOOK et al Turk J Zool

699

and crocodilians (Fox 1952) Bishop (1959) found that the testes of the male garter snake Thamnophis sirtalis were spermatogenically active during the same time as the hypertrophied SSK during the active reproductive period the diameter of the SSK tubule was 5 times greater than that of the SSK tubule during the inactive reproductive period However SSK development in female lizards has been reported in the genus Cnemidophorus (Del Conte 1972 Del Conte and Tamayo 1973) and Scincella laterale (Sever and Hopkins 2005) They suggested that the females had a low level of natural androgens which caused the SSK development (Del Conte and Tamayo 1973 Sever and Hopkins 2005) In the present study we did not monitor the annual seasonal alterations in female SSK The hypertrophy of the SSK is synchronous with androgen secretion and spermatogenic activity (Sever and Hopkins 2005) Norris (2013) also stated that the SSK of sexually active squamates undergoes hypertrophy and is under the influence of androgens In this study we did not investigate any alterations in annual SSK with annual androgen secretion but we did demonstrate that the hypertrophy and the regression of SSK changed seasonally and synchronously with the active spermatogenic event and the spermatogenic arrest respectively In the Iberian rock lizard Lacerta monticola SSK secretions form a copulatory plug that adheres to the femalersquos cloaca following copulation to occlude oviductal openings however such a plug does not prevent subsequent mating nor does it reduce the femalersquos attractiveness (Moreira and Birkhead 2003)

With our investigations on annual alterations in female ovarian morphologies between 2 Calotes we found that individuals in the same Calotes species showed different timing of reproductive events throughout a 1-year period (the data are not shown here) Additionally there was quite clear evidence that QU EV and LV follicles and oviductal eggs overlapped among individuals within the same populations of both Calotes species Female reproductive status is definitely distinguishable between the 2 Calotes species Gravid lizards were encountered in 1 individual of C emma in April and in 3 individuals of C versicolor in June August and September Shanbhag et al (2000) reported that female C versicolor showed inactive reproduction from December to April and gravidity was encountered from May to October

We found that in the previtellogenic follicles (QU and EV) of females of both Calotes species the granulosa layer contained 2 types of cells pyriform and small cells Uribe et al (1996) stated that in squamates the follicular epithelium or granulosa initially consists of small cuboidal cells but differentiates during the previtellogenic phase and becomes multilayered and polymorphic by the presence of unique flask-shaped pyriform cells intermediate cells and

small cells These pyriform cells differentiate from small somatic follicular cells early in follicular development via the intermediate-cell stage to become nurse cells in direct contact with the developing oocytes (Maurizii et al 2004) Differentiation of the small cells into pyriform cells appears to be linked to the progressive appearance of glycoproteins with terminal α-N-acetylgalactosamine residues on the cell surface which may be involved in fusion between the oocyte and the follicle cell membranes as well as maintenance of the differentiated pyriform cells The pyriform cells are connected to the oocyte via intercellular bridges containing a cytoskeleton of α-tubulin and cytokeratin microtubules (Maurizii et al 2004) Tumkiratiwong et al (2012) also demonstrated that the previtellogenic follicles of the captive monocled cobra Naja kaouthia had many pyriform cells in the granulosa layer but fewer in the vitellogenic follicles In this study the pyriform cells disappeared when the follicles entered the vitellogenic stage Andreuccetti (1992) studied the differentiation of pyriform cells and their contribution to oocyte growth in 3 lizards namely Tarentola mauritanica Cordylus wittifer and Platysaurus intermedius and a colubrid snake Coluber viridiflavus and revealed that pyriform cells differentiate from small follicle cells via intermediate cells after establishing an intercellular bridge with the oocyte Once pyriform cells are differentiated they display ultrastructural features indicative of synthetic activity including abundant ribosomes Golgi membranes vacuoles mitochondria and lipid droplets These cellular components extend to the apex of the cell at the level of the intercellular bridge suggesting that constituents of pyriform cells may be transferred to the oocyte Pyriform cells and the oocytes may fulfill similar vitellogenic functions The establishment of an intercellular bridge may represent a crucial event in the development of an integrated system in which pyriform cells and oocytes cooperate Norris (2013) reported that the squamate granulosa contains the pyriform cells which are in direct contact with the developing oocyte and are apparently involved with early steps in oocyte development soon after the onset of vitellogenesis As ovulation approaches the granulosa cells as well as some thecal cells accumulate cholesterol-positive lipids and proliferate and luteinize to form corpora lutea following ovulation Follicular atresia is a common occurrence in reptilian ovaries as in other vertebrates (Norris 2013) as we found the corpora lutea in the follicle in accordance to oviductal egg appearances of a representative C versicolor collected in September Additionally as shown in Table 1 several atretic follicles lt25 mm in diameter were commonly encountered in C emma and C versicolor

Based on both morphological and histological investigations we found that testes ST and SSK were

MEESOOK et al Turk J Zool

700

concomitantly active and were associated with high levels of plasma testosterone We also demonstrated that there were high correlations between levels of plasma testosterone (T) and testicular mass where annual changes occurred in the same direction as the testicular size and time of spermatogenetic events among the males of the Calotes species Radder et al (2001) reported that in the male tropical or oriental garden lizard C versicolor plasma T is highest during the breeding season which correlated with testis mass and reproductive behavior Changes in T levels are associated with high spermatogenetic activity Radder et al (2001) also stated that the changes in plasma T levels during different phases of the male reproductive cycle in C versicolor follow a reproductive pattern of a prenuptial type of spermatogenesis that is similar to that of some other species of lizards the spiny-tailed lizard Uromastix hardwicki (Arslan et al 1978a) the viviparous lizard Lacerta vivipara (Courty and Dufaure 1982) the western shingleback lizard Tiliqua (Trachydosaurus) rugosa (Bourne et al 1986) the male lizard Podarcis s sicula (Ando et al 1990) Podarcis s sicula Raf (Ando et al 1992) the white-throated savanna monitor Varanus albigularis (Phillips and Millar 1998) and the male brown anoles Anolis sagrei (Tokarz et al 1998)

We found that estradiol (E2) levels increased in vitellogenic females its high levels were associated with the presence of the largest vitellogenic follicles in the 2 Calotes species Radder et al (2001) reported that in female C versicolor with overlapping reproductive events such as vitellogenesis and gestation E2 was at low levels when the ovaries were regressed and at high levels at vitellogenic follicular recruitment reaching peak level at the time of preovulatory follicles The same patterns of E2 secretion were found again when the second set of follicles underwent vitellogenesis (Radder et al 2001) Surprisingly Amey and Whittier (2000) reported that in female bearded dragons Pagona barbata plasma E2 was low or nondetectable across all reproductive states In C versicolor E2 levels were low in nonreproductive females with small previtellogenic follicles and those in the EG phase (Radder et al 2001) We do not discuss the level of the plasma progesterone (P) during the gestation period as its level was not detectable in this study The gravid lizards in EG exhibited low plasma E2 but high P levels and the highest P levels coincided with eggshell production P levels declined after eggshell formation as reported in other gravid individuals in several species of lizards that do not possess vitellogenic follicles of the subsequent clutch including C versicolor (Radder et al 2001) Uromastix hardwicki (Arslan et al 1978b) Agama atra (Van Wyk 1984) Eumeces obsoletus Scelporus undulatus and Crotaphytus collaris (Masson and Guillette 1987) and Psammodromus algirus (Diaz et al 1994) However a

decline in P levels in MG with vitellogenic follicles did not seem to facilitate recruitment or growth of the subsequent set of vitellogenic follicles in gravid Sceloporus jarrovi (Guillette et al 1981)

In the present study there were variations in the timing of breeding between the 2 Calotes species and even within populations of the same species We could not relate the copulation timing of 2 such Calotes species to gonadal activity or sex hormonal surges as the timing of natural mating could not be observed during the times we collected data Lizard species that inhabit temperate zones have mostly exhibited seasonal reproduction (Fitch 1970 Licht 1984 Pianka and Vitt 2003) The 10 lizard species that have been studied widely to date exhibit an associated reproductive pattern (Lovern 2011) that is green anoles Anolis carolinensis (Crews 1980 Lovern et al 2004) brown anoles Anolis sagrei (Lee et al 1989 Tokarz 1998) eastern fence lizards Sceloporus undulates (Cox et al 2005) mountain spiny lizards Sceloporus jarrovi (Woodley and Moore 1999) tree lizards Urosaurus ornatus (French and Moore 2008) wall lizards Podarcis sicula (Putti et al 2009) common lizards Lacerta vivipara (Vercken and Clobert 2008) little striped whiptail lizards Cnemidophorus inornatus (Crews 2005) garden lizards Calotes versicolor (Shanbhag 2003 Lovern 2011) and leopard geckos Eublepharis macularius (Rhen et al 2005) The temperate Florida populations of the brown anole Anolis sagrei show a strong seasonality in reproduction (Lee et al 1989) while the tropical Caribbean (Licht and Gorman 1970 Sexton and Brown 1977) and Hawaiian populations of this species (Goldberg et al 2002) show a less-pronounced seasonality in which reproductively active individuals can be found throughout the year Although individuals within a population of many tropical lizard species can breed at any time no individuals within the population breed year-round (Lovern 2011) Additionally Vitt and Caldwell (2009) stated that the belief used to be that tropical squamates had continuous reproduction in aseasonal tropical environments or reproduced during the wet season in a wetndashdry seasonal tropical environment Many tropical lizard species namely the anoles Anolis acutus (Ruibal et al 1972) Anolis limifrons (Sexton et al 1971) and Anolis opalinus (Jenssen and Nunez 1994) as well as the gecko Cyrtodactylus malyanus the flying lizard Draco melanopogon (Inger and Greenberg 1966) and the parthenogenetic oviparous whiptail lizard Cnemidophorus nativo (Menezes et al 2004) showed slightly more frequent breeding during the wet season than during the dry season (Jenssen and Nunez 1994)

Reproductive patterns can be described in a variety of ways but not all species fit neatly into such categorizations However 2 general types of reproductive patterns are

MEESOOK et al Turk J Zool

701

recognized in terms of prenuptial and postnuptial reproductive patterns (Lance 1998) Prenuptial reproductive pattern terms such as gonadal recrudescence sex steroid production and gametogenesis occur in advance of mating whereas postnuptial reproductive patterns occur following mating In other words in a high-elevation population of Sceloporus grammicus in Parque Nacional de Zoquiapan in central Mexico an active reproductive event occurring in the early fall is described as dissociated from testicular recrudescence in males but is associated with the initiation of ovarian recrudescence in females (Guillette and Casas-Andreu 1980 1981 Zuniga-Vega et al 2008) This is in contrast to S grammicus from Teotihuacan Mexico in which testicular recrudescence and breeding occur in the summer and fall at the onset of female ovarian recrudescence (Jimenez-Cruz et al 2005) In S mucronatus from Valle de la Cantimplora Mexico peak testicular recrudescence and mating occur during the summer prior to ovarian recrudescence which does not occur until several months later (Ortega-Leon et al 2009) This is distinct from many fall-breeding

populations elsewhere (Mendez-De La Cruz et al 1994 Villagran-Santa Cruz et al 1994) The examples above demonstrate that gonadal activity and mating behavior are clearly variable but hormone analyses have not been performed in these species and so endocrine relationships cannot be assessed at this point

In conclusion we suggest that the males and females of the 2 Calotes species have much more prolonged active reproductive phases than inactive reproductive phases The reproductive patterns of C emma and C versicolor were classified into the same reproductive pattern of continual reproduction

AcknowledgmentsWe thank the Department of Zoology of Kasetsart University for financial support We also thank the staff of Sakaerat Environmental Research Station Nakhon Ratchasima Province for devoting time for research collaboration We also thank Mrs Sureerat Sangkrut for drawing all illustrations In addition we wish to thank the anonymous referees for many helpful suggestions

References

Aldridge RD Goldberg SR Wisniewski SS Bufalino AP Dillman CB (2009) The reproductive cycle and estrus in the colubrid snakes of temperate North America Contemp Herpetol 4 1-31

Amey AP Whittier JM (2000) Seasonal patterns of plasma steroid hormones in males and females of the bearded dragon lizard Pogona barbata Gen Comp Endocrinol 117 335-342

Andreuccetti P (1992) An ultrastructural study of differentiation of pyriform cells and their contribution to oocyte growth in representative Squamata J Morphol 212 1-11

Ando S Panno ML Ciarcia G Imbrogno E Buffone M Beraldi E Sisci D Angelini F Botte V (1990) Plasma sex hormone concentrations during the reproductive cycle in the male lizard Podarcis s sicula J Reprod Fertil 90 353-360

Ando S Ciarcia G Panno ML Imbrogno E Tarantino G Buffone M Beraldi E Angelini F Botte V (1992) Sex steroids levels in the plasma and testis during the reproductive cycle of lizard Podarcis s sicula Raf Gen Comp Endocrinol 85 1-7

Arslan MJ Lobo J Zaidi AA Jalali S Qazi MH (1978a) Annual androgen rhythm in the spiny-tailed lizard Uromastix hardwicki Gen Comp Endocrinol 36 16-22

Arslan MJ Zaidi P Lobo J Zaidi AA Qazi MH (1978b) Steroid levels in preovulatory and gravid lizards (Uromastix hardwicki) Gen Comp Endocrinol 34 300-303

Avwioro G (2011) Histochemical uses of haematoxylin-a review J Pham Clin Sci 1 24-34

Bishop JE (1959) A histological and histochemical study of the kidney tubule of the common garter snake Thamnophis sirtalis with special reference to the sexual segment in the male J Morphol 104 307-358

Bourne AR Taylor JL Watson TG (1986) Annual cycles of plasma and testicular androgens in the lizard Tiliqua (Trachydosaurus) rugosa Gen Comp Endocrinol 61 278-286

Courty Y Dufaure JP (1982) Circannual testosterone dihydrotestosterone and androstenedione in the plasma and testis of Lacerta vivipara a seasonally breeding viviparous lizard Steroids 39 517-529

Cox RM Skelly SL Leo A John-Alder HB (2005) Testosterone regulates sexually dimorphic coloration in the eastern fence lizard Sceloporus undulatus Copeia 2005 597-608

Crews D (1976) Hormonal control of male courtship behavior and female attractively in the garter snake (Thamnophis sirtalis parietalis) Horm Behav 7 451-460

Crews D (1980) Interrelationships among ecological behavioral and neuroendocrine processes in the reproductive cycle of Anolis carolinensis and other reptiles Adv Stud Behav 11 1-74

Crews D (1984) Gamete production sex hormone secretion and mating behavior uncoupled Horm Behav 14 22-28

Crews D (1999) Reptilian reproduction overview In Knobil E Neil JD editors Encyclopedia of Reproduction New York NY USA Academic Press pp 239-243

Crews D Camazine B Diamond M Mason R Tokarz R Garstka WR (1984) Hormonal independence of courtship behavior in the male garter snake Horm Behav 14 29-41

Crews D (2005) Evolution of neuroendocrine mechanisms that regulate sexual behavior Trends Endocrinol Metab 16 354-361

Del Conte E (1972) Granular secretion in the kidney Rss of female lizards Cnemidophorus l lemniscatus (Sauria Teiidae) J Morphol 137 181-191

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of C emma plasma E2 levels were the difference between the QU and the EV stages (U = 900 P = 0090) the QU and the LV stages (U = 200 P = 0009) and the EV and the LV stages (U = 200 P = 0083) Additionally the diameters of the largest follicles were the difference between the QU and the EV stages (U = 000 P = 0004) the QU and the LV stages (U = 000 P = 0004) and the EV and the LV stages (U = 000 P = 0021) The correlation coefficient between plasma E2 and the diameter of the largest follicle was 082 (P = 0001) and 032 (P = 0365) of C emma and C versicolor respectively

4 DiscussionAnnual variations in the timing of male and female reproductive stages were encountered among populations of C emma and C versicolor even in individuals of the same population Annual changes in testicular sizes were categorized into 2 phases in both species (1) an active hypertrophied testicular phase and (2) an inactive

regressed testicular phase However the timing of those 2 events appeared asynchronously in the 2 Calotes species even in individuals of the same population (data not shown here) The testes were hypertrophied with active spermatozoal production from December to June and January to September in the representative C emma and C versicolor respectively A study by Gouder and Nadkarni (1979) showed that males of C versicolor widely distributed in India were spermatogenetically active from April to September Active vitellogenic follicles and oviductal eggs were concomitantly encountered in C emma and C versicolor Therefore these 2 Calotes species exhibited polyautochrony and multiclutches Radder et al (2001) reported that Indian garden lizards C versicolor showed polyautochrony and multiclutches The representative gravid lizards were found in April in C emma and in June and from August to September in C versicolor Gravid C versicolor whose habitat is in India was encountered from May to October (Shanbhag and Prasad 1993) We

Figure 5 Photomicrographs of annual changes in C versicolor Top left testes top right male SSK bottom left ovaries bottom right granulosa layers (GL) Notes SZ spermatozoa ST seminiferous tubules SSK sexual segments of kidney AF atretic follicle PF previtellogenic follicle VF vitellogenic follicle P pyriform cells S small cells CL corpus luteum

Figure 6 Annual profiles (mean plusmn SEM) of testosterone levels and testicular masses (a) C emma (b) C versicolor Notes JanndashDec denotes January to December The numbers (in parentheses) represent the number of analyzed samples in each month

MEESOOK et al Turk J Zool

698

are likely to suggest after studying the annual alterations in the male and female reproductive morphologies of the 2 Calotes species that the active reproductive events of both males and females of the Calotes species lasted nearly 1 year with only a few months of reproductive arrest which is especially seen in C versicolor

According to our investigations on annual histological alterations in ST and male SSK timing of the 2 Calotes species both ST and SSK were in active spermatogenic and

hypertrophied stages respectively which corresponded well with the timing of the testicular hypertrophied stage mentioned above In other words the timing of arrested spermatogenesis and regressed SSK was in accordance with that of the regressed testes Likewise we confirmed that the 2 Calotes species have an active reproductive stage that is much longer than the inactive reproductive stage

SSK is present in a variety of male snakes and lizards but is absent in both turtles (Regaud and Policard 1903)

Table 2 Changes in ovarian weight and diameter of the largest follicle in Calotes during the reproduction cycle

Reproductive stages N Ovarian weights Diameter of the largest follicle

C emma (20)QU 11 002 plusmn 000a 178 plusmn 006a

EV 4 011 plusmn 009b 350 plusmn 033b

LV 4 171 plusmn 049c 802 plusmn 085c

EG 1 002 240MG - - -LG - - -C versicolor (9)QU 4 002 plusmn 000 152 plusmn 199EV 1 02 428LV - - -EG 3 002 plusmn 000 222 plusmn 069MG 2 011 plusmn 009 436 plusmn 036LG - - -

Data are presented as mean plusmn SEM The differences in superscript alphabets indicate the significant differences among the reproductive stages of nongestation at P lt 001 (N sample sizes)Notes QU quiescent EV early vitellogenic LV late vitellogenic EG early gestation MG mid-gestation and LG late gestation

Figure 7 Changes in the plasma levels of estradiol and the diameter of the largest follicle (a) C emma (b) C versicolor Notes QU quiescent EV early vitellogenic LV late vitellogenic EG early gestation MG mid-gestation LG late gestation Data are presented as mean plusmn SEM The differences in superscript alphabets (estradiol levels) and in the numbers of asterisks (diameters of the largest follicles) indicate the significant differences between the various follicular sizes at P lt 001 The number (in parentheses) represents the analyzed samples in each month

MEESOOK et al Turk J Zool

699

and crocodilians (Fox 1952) Bishop (1959) found that the testes of the male garter snake Thamnophis sirtalis were spermatogenically active during the same time as the hypertrophied SSK during the active reproductive period the diameter of the SSK tubule was 5 times greater than that of the SSK tubule during the inactive reproductive period However SSK development in female lizards has been reported in the genus Cnemidophorus (Del Conte 1972 Del Conte and Tamayo 1973) and Scincella laterale (Sever and Hopkins 2005) They suggested that the females had a low level of natural androgens which caused the SSK development (Del Conte and Tamayo 1973 Sever and Hopkins 2005) In the present study we did not monitor the annual seasonal alterations in female SSK The hypertrophy of the SSK is synchronous with androgen secretion and spermatogenic activity (Sever and Hopkins 2005) Norris (2013) also stated that the SSK of sexually active squamates undergoes hypertrophy and is under the influence of androgens In this study we did not investigate any alterations in annual SSK with annual androgen secretion but we did demonstrate that the hypertrophy and the regression of SSK changed seasonally and synchronously with the active spermatogenic event and the spermatogenic arrest respectively In the Iberian rock lizard Lacerta monticola SSK secretions form a copulatory plug that adheres to the femalersquos cloaca following copulation to occlude oviductal openings however such a plug does not prevent subsequent mating nor does it reduce the femalersquos attractiveness (Moreira and Birkhead 2003)

With our investigations on annual alterations in female ovarian morphologies between 2 Calotes we found that individuals in the same Calotes species showed different timing of reproductive events throughout a 1-year period (the data are not shown here) Additionally there was quite clear evidence that QU EV and LV follicles and oviductal eggs overlapped among individuals within the same populations of both Calotes species Female reproductive status is definitely distinguishable between the 2 Calotes species Gravid lizards were encountered in 1 individual of C emma in April and in 3 individuals of C versicolor in June August and September Shanbhag et al (2000) reported that female C versicolor showed inactive reproduction from December to April and gravidity was encountered from May to October

We found that in the previtellogenic follicles (QU and EV) of females of both Calotes species the granulosa layer contained 2 types of cells pyriform and small cells Uribe et al (1996) stated that in squamates the follicular epithelium or granulosa initially consists of small cuboidal cells but differentiates during the previtellogenic phase and becomes multilayered and polymorphic by the presence of unique flask-shaped pyriform cells intermediate cells and

small cells These pyriform cells differentiate from small somatic follicular cells early in follicular development via the intermediate-cell stage to become nurse cells in direct contact with the developing oocytes (Maurizii et al 2004) Differentiation of the small cells into pyriform cells appears to be linked to the progressive appearance of glycoproteins with terminal α-N-acetylgalactosamine residues on the cell surface which may be involved in fusion between the oocyte and the follicle cell membranes as well as maintenance of the differentiated pyriform cells The pyriform cells are connected to the oocyte via intercellular bridges containing a cytoskeleton of α-tubulin and cytokeratin microtubules (Maurizii et al 2004) Tumkiratiwong et al (2012) also demonstrated that the previtellogenic follicles of the captive monocled cobra Naja kaouthia had many pyriform cells in the granulosa layer but fewer in the vitellogenic follicles In this study the pyriform cells disappeared when the follicles entered the vitellogenic stage Andreuccetti (1992) studied the differentiation of pyriform cells and their contribution to oocyte growth in 3 lizards namely Tarentola mauritanica Cordylus wittifer and Platysaurus intermedius and a colubrid snake Coluber viridiflavus and revealed that pyriform cells differentiate from small follicle cells via intermediate cells after establishing an intercellular bridge with the oocyte Once pyriform cells are differentiated they display ultrastructural features indicative of synthetic activity including abundant ribosomes Golgi membranes vacuoles mitochondria and lipid droplets These cellular components extend to the apex of the cell at the level of the intercellular bridge suggesting that constituents of pyriform cells may be transferred to the oocyte Pyriform cells and the oocytes may fulfill similar vitellogenic functions The establishment of an intercellular bridge may represent a crucial event in the development of an integrated system in which pyriform cells and oocytes cooperate Norris (2013) reported that the squamate granulosa contains the pyriform cells which are in direct contact with the developing oocyte and are apparently involved with early steps in oocyte development soon after the onset of vitellogenesis As ovulation approaches the granulosa cells as well as some thecal cells accumulate cholesterol-positive lipids and proliferate and luteinize to form corpora lutea following ovulation Follicular atresia is a common occurrence in reptilian ovaries as in other vertebrates (Norris 2013) as we found the corpora lutea in the follicle in accordance to oviductal egg appearances of a representative C versicolor collected in September Additionally as shown in Table 1 several atretic follicles lt25 mm in diameter were commonly encountered in C emma and C versicolor

Based on both morphological and histological investigations we found that testes ST and SSK were

MEESOOK et al Turk J Zool

700

concomitantly active and were associated with high levels of plasma testosterone We also demonstrated that there were high correlations between levels of plasma testosterone (T) and testicular mass where annual changes occurred in the same direction as the testicular size and time of spermatogenetic events among the males of the Calotes species Radder et al (2001) reported that in the male tropical or oriental garden lizard C versicolor plasma T is highest during the breeding season which correlated with testis mass and reproductive behavior Changes in T levels are associated with high spermatogenetic activity Radder et al (2001) also stated that the changes in plasma T levels during different phases of the male reproductive cycle in C versicolor follow a reproductive pattern of a prenuptial type of spermatogenesis that is similar to that of some other species of lizards the spiny-tailed lizard Uromastix hardwicki (Arslan et al 1978a) the viviparous lizard Lacerta vivipara (Courty and Dufaure 1982) the western shingleback lizard Tiliqua (Trachydosaurus) rugosa (Bourne et al 1986) the male lizard Podarcis s sicula (Ando et al 1990) Podarcis s sicula Raf (Ando et al 1992) the white-throated savanna monitor Varanus albigularis (Phillips and Millar 1998) and the male brown anoles Anolis sagrei (Tokarz et al 1998)

We found that estradiol (E2) levels increased in vitellogenic females its high levels were associated with the presence of the largest vitellogenic follicles in the 2 Calotes species Radder et al (2001) reported that in female C versicolor with overlapping reproductive events such as vitellogenesis and gestation E2 was at low levels when the ovaries were regressed and at high levels at vitellogenic follicular recruitment reaching peak level at the time of preovulatory follicles The same patterns of E2 secretion were found again when the second set of follicles underwent vitellogenesis (Radder et al 2001) Surprisingly Amey and Whittier (2000) reported that in female bearded dragons Pagona barbata plasma E2 was low or nondetectable across all reproductive states In C versicolor E2 levels were low in nonreproductive females with small previtellogenic follicles and those in the EG phase (Radder et al 2001) We do not discuss the level of the plasma progesterone (P) during the gestation period as its level was not detectable in this study The gravid lizards in EG exhibited low plasma E2 but high P levels and the highest P levels coincided with eggshell production P levels declined after eggshell formation as reported in other gravid individuals in several species of lizards that do not possess vitellogenic follicles of the subsequent clutch including C versicolor (Radder et al 2001) Uromastix hardwicki (Arslan et al 1978b) Agama atra (Van Wyk 1984) Eumeces obsoletus Scelporus undulatus and Crotaphytus collaris (Masson and Guillette 1987) and Psammodromus algirus (Diaz et al 1994) However a

decline in P levels in MG with vitellogenic follicles did not seem to facilitate recruitment or growth of the subsequent set of vitellogenic follicles in gravid Sceloporus jarrovi (Guillette et al 1981)

In the present study there were variations in the timing of breeding between the 2 Calotes species and even within populations of the same species We could not relate the copulation timing of 2 such Calotes species to gonadal activity or sex hormonal surges as the timing of natural mating could not be observed during the times we collected data Lizard species that inhabit temperate zones have mostly exhibited seasonal reproduction (Fitch 1970 Licht 1984 Pianka and Vitt 2003) The 10 lizard species that have been studied widely to date exhibit an associated reproductive pattern (Lovern 2011) that is green anoles Anolis carolinensis (Crews 1980 Lovern et al 2004) brown anoles Anolis sagrei (Lee et al 1989 Tokarz 1998) eastern fence lizards Sceloporus undulates (Cox et al 2005) mountain spiny lizards Sceloporus jarrovi (Woodley and Moore 1999) tree lizards Urosaurus ornatus (French and Moore 2008) wall lizards Podarcis sicula (Putti et al 2009) common lizards Lacerta vivipara (Vercken and Clobert 2008) little striped whiptail lizards Cnemidophorus inornatus (Crews 2005) garden lizards Calotes versicolor (Shanbhag 2003 Lovern 2011) and leopard geckos Eublepharis macularius (Rhen et al 2005) The temperate Florida populations of the brown anole Anolis sagrei show a strong seasonality in reproduction (Lee et al 1989) while the tropical Caribbean (Licht and Gorman 1970 Sexton and Brown 1977) and Hawaiian populations of this species (Goldberg et al 2002) show a less-pronounced seasonality in which reproductively active individuals can be found throughout the year Although individuals within a population of many tropical lizard species can breed at any time no individuals within the population breed year-round (Lovern 2011) Additionally Vitt and Caldwell (2009) stated that the belief used to be that tropical squamates had continuous reproduction in aseasonal tropical environments or reproduced during the wet season in a wetndashdry seasonal tropical environment Many tropical lizard species namely the anoles Anolis acutus (Ruibal et al 1972) Anolis limifrons (Sexton et al 1971) and Anolis opalinus (Jenssen and Nunez 1994) as well as the gecko Cyrtodactylus malyanus the flying lizard Draco melanopogon (Inger and Greenberg 1966) and the parthenogenetic oviparous whiptail lizard Cnemidophorus nativo (Menezes et al 2004) showed slightly more frequent breeding during the wet season than during the dry season (Jenssen and Nunez 1994)

Reproductive patterns can be described in a variety of ways but not all species fit neatly into such categorizations However 2 general types of reproductive patterns are

MEESOOK et al Turk J Zool

701

recognized in terms of prenuptial and postnuptial reproductive patterns (Lance 1998) Prenuptial reproductive pattern terms such as gonadal recrudescence sex steroid production and gametogenesis occur in advance of mating whereas postnuptial reproductive patterns occur following mating In other words in a high-elevation population of Sceloporus grammicus in Parque Nacional de Zoquiapan in central Mexico an active reproductive event occurring in the early fall is described as dissociated from testicular recrudescence in males but is associated with the initiation of ovarian recrudescence in females (Guillette and Casas-Andreu 1980 1981 Zuniga-Vega et al 2008) This is in contrast to S grammicus from Teotihuacan Mexico in which testicular recrudescence and breeding occur in the summer and fall at the onset of female ovarian recrudescence (Jimenez-Cruz et al 2005) In S mucronatus from Valle de la Cantimplora Mexico peak testicular recrudescence and mating occur during the summer prior to ovarian recrudescence which does not occur until several months later (Ortega-Leon et al 2009) This is distinct from many fall-breeding

populations elsewhere (Mendez-De La Cruz et al 1994 Villagran-Santa Cruz et al 1994) The examples above demonstrate that gonadal activity and mating behavior are clearly variable but hormone analyses have not been performed in these species and so endocrine relationships cannot be assessed at this point

In conclusion we suggest that the males and females of the 2 Calotes species have much more prolonged active reproductive phases than inactive reproductive phases The reproductive patterns of C emma and C versicolor were classified into the same reproductive pattern of continual reproduction

AcknowledgmentsWe thank the Department of Zoology of Kasetsart University for financial support We also thank the staff of Sakaerat Environmental Research Station Nakhon Ratchasima Province for devoting time for research collaboration We also thank Mrs Sureerat Sangkrut for drawing all illustrations In addition we wish to thank the anonymous referees for many helpful suggestions

References

Aldridge RD Goldberg SR Wisniewski SS Bufalino AP Dillman CB (2009) The reproductive cycle and estrus in the colubrid snakes of temperate North America Contemp Herpetol 4 1-31

Amey AP Whittier JM (2000) Seasonal patterns of plasma steroid hormones in males and females of the bearded dragon lizard Pogona barbata Gen Comp Endocrinol 117 335-342

Andreuccetti P (1992) An ultrastructural study of differentiation of pyriform cells and their contribution to oocyte growth in representative Squamata J Morphol 212 1-11

Ando S Panno ML Ciarcia G Imbrogno E Buffone M Beraldi E Sisci D Angelini F Botte V (1990) Plasma sex hormone concentrations during the reproductive cycle in the male lizard Podarcis s sicula J Reprod Fertil 90 353-360

Ando S Ciarcia G Panno ML Imbrogno E Tarantino G Buffone M Beraldi E Angelini F Botte V (1992) Sex steroids levels in the plasma and testis during the reproductive cycle of lizard Podarcis s sicula Raf Gen Comp Endocrinol 85 1-7

Arslan MJ Lobo J Zaidi AA Jalali S Qazi MH (1978a) Annual androgen rhythm in the spiny-tailed lizard Uromastix hardwicki Gen Comp Endocrinol 36 16-22

Arslan MJ Zaidi P Lobo J Zaidi AA Qazi MH (1978b) Steroid levels in preovulatory and gravid lizards (Uromastix hardwicki) Gen Comp Endocrinol 34 300-303

Avwioro G (2011) Histochemical uses of haematoxylin-a review J Pham Clin Sci 1 24-34

Bishop JE (1959) A histological and histochemical study of the kidney tubule of the common garter snake Thamnophis sirtalis with special reference to the sexual segment in the male J Morphol 104 307-358

Bourne AR Taylor JL Watson TG (1986) Annual cycles of plasma and testicular androgens in the lizard Tiliqua (Trachydosaurus) rugosa Gen Comp Endocrinol 61 278-286

Courty Y Dufaure JP (1982) Circannual testosterone dihydrotestosterone and androstenedione in the plasma and testis of Lacerta vivipara a seasonally breeding viviparous lizard Steroids 39 517-529

Cox RM Skelly SL Leo A John-Alder HB (2005) Testosterone regulates sexually dimorphic coloration in the eastern fence lizard Sceloporus undulatus Copeia 2005 597-608

Crews D (1976) Hormonal control of male courtship behavior and female attractively in the garter snake (Thamnophis sirtalis parietalis) Horm Behav 7 451-460

Crews D (1980) Interrelationships among ecological behavioral and neuroendocrine processes in the reproductive cycle of Anolis carolinensis and other reptiles Adv Stud Behav 11 1-74

Crews D (1984) Gamete production sex hormone secretion and mating behavior uncoupled Horm Behav 14 22-28

Crews D (1999) Reptilian reproduction overview In Knobil E Neil JD editors Encyclopedia of Reproduction New York NY USA Academic Press pp 239-243

Crews D Camazine B Diamond M Mason R Tokarz R Garstka WR (1984) Hormonal independence of courtship behavior in the male garter snake Horm Behav 14 29-41

Crews D (2005) Evolution of neuroendocrine mechanisms that regulate sexual behavior Trends Endocrinol Metab 16 354-361

Del Conte E (1972) Granular secretion in the kidney Rss of female lizards Cnemidophorus l lemniscatus (Sauria Teiidae) J Morphol 137 181-191

MEESOOK et al Turk J Zool

698

are likely to suggest after studying the annual alterations in the male and female reproductive morphologies of the 2 Calotes species that the active reproductive events of both males and females of the Calotes species lasted nearly 1 year with only a few months of reproductive arrest which is especially seen in C versicolor

According to our investigations on annual histological alterations in ST and male SSK timing of the 2 Calotes species both ST and SSK were in active spermatogenic and

hypertrophied stages respectively which corresponded well with the timing of the testicular hypertrophied stage mentioned above In other words the timing of arrested spermatogenesis and regressed SSK was in accordance with that of the regressed testes Likewise we confirmed that the 2 Calotes species have an active reproductive stage that is much longer than the inactive reproductive stage

SSK is present in a variety of male snakes and lizards but is absent in both turtles (Regaud and Policard 1903)

Table 2 Changes in ovarian weight and diameter of the largest follicle in Calotes during the reproduction cycle

Reproductive stages N Ovarian weights Diameter of the largest follicle

C emma (20)QU 11 002 plusmn 000a 178 plusmn 006a

EV 4 011 plusmn 009b 350 plusmn 033b

LV 4 171 plusmn 049c 802 plusmn 085c

EG 1 002 240MG - - -LG - - -C versicolor (9)QU 4 002 plusmn 000 152 plusmn 199EV 1 02 428LV - - -EG 3 002 plusmn 000 222 plusmn 069MG 2 011 plusmn 009 436 plusmn 036LG - - -

Data are presented as mean plusmn SEM The differences in superscript alphabets indicate the significant differences among the reproductive stages of nongestation at P lt 001 (N sample sizes)Notes QU quiescent EV early vitellogenic LV late vitellogenic EG early gestation MG mid-gestation and LG late gestation

Figure 7 Changes in the plasma levels of estradiol and the diameter of the largest follicle (a) C emma (b) C versicolor Notes QU quiescent EV early vitellogenic LV late vitellogenic EG early gestation MG mid-gestation LG late gestation Data are presented as mean plusmn SEM The differences in superscript alphabets (estradiol levels) and in the numbers of asterisks (diameters of the largest follicles) indicate the significant differences between the various follicular sizes at P lt 001 The number (in parentheses) represents the analyzed samples in each month

MEESOOK et al Turk J Zool

699

and crocodilians (Fox 1952) Bishop (1959) found that the testes of the male garter snake Thamnophis sirtalis were spermatogenically active during the same time as the hypertrophied SSK during the active reproductive period the diameter of the SSK tubule was 5 times greater than that of the SSK tubule during the inactive reproductive period However SSK development in female lizards has been reported in the genus Cnemidophorus (Del Conte 1972 Del Conte and Tamayo 1973) and Scincella laterale (Sever and Hopkins 2005) They suggested that the females had a low level of natural androgens which caused the SSK development (Del Conte and Tamayo 1973 Sever and Hopkins 2005) In the present study we did not monitor the annual seasonal alterations in female SSK The hypertrophy of the SSK is synchronous with androgen secretion and spermatogenic activity (Sever and Hopkins 2005) Norris (2013) also stated that the SSK of sexually active squamates undergoes hypertrophy and is under the influence of androgens In this study we did not investigate any alterations in annual SSK with annual androgen secretion but we did demonstrate that the hypertrophy and the regression of SSK changed seasonally and synchronously with the active spermatogenic event and the spermatogenic arrest respectively In the Iberian rock lizard Lacerta monticola SSK secretions form a copulatory plug that adheres to the femalersquos cloaca following copulation to occlude oviductal openings however such a plug does not prevent subsequent mating nor does it reduce the femalersquos attractiveness (Moreira and Birkhead 2003)

With our investigations on annual alterations in female ovarian morphologies between 2 Calotes we found that individuals in the same Calotes species showed different timing of reproductive events throughout a 1-year period (the data are not shown here) Additionally there was quite clear evidence that QU EV and LV follicles and oviductal eggs overlapped among individuals within the same populations of both Calotes species Female reproductive status is definitely distinguishable between the 2 Calotes species Gravid lizards were encountered in 1 individual of C emma in April and in 3 individuals of C versicolor in June August and September Shanbhag et al (2000) reported that female C versicolor showed inactive reproduction from December to April and gravidity was encountered from May to October

We found that in the previtellogenic follicles (QU and EV) of females of both Calotes species the granulosa layer contained 2 types of cells pyriform and small cells Uribe et al (1996) stated that in squamates the follicular epithelium or granulosa initially consists of small cuboidal cells but differentiates during the previtellogenic phase and becomes multilayered and polymorphic by the presence of unique flask-shaped pyriform cells intermediate cells and

small cells These pyriform cells differentiate from small somatic follicular cells early in follicular development via the intermediate-cell stage to become nurse cells in direct contact with the developing oocytes (Maurizii et al 2004) Differentiation of the small cells into pyriform cells appears to be linked to the progressive appearance of glycoproteins with terminal α-N-acetylgalactosamine residues on the cell surface which may be involved in fusion between the oocyte and the follicle cell membranes as well as maintenance of the differentiated pyriform cells The pyriform cells are connected to the oocyte via intercellular bridges containing a cytoskeleton of α-tubulin and cytokeratin microtubules (Maurizii et al 2004) Tumkiratiwong et al (2012) also demonstrated that the previtellogenic follicles of the captive monocled cobra Naja kaouthia had many pyriform cells in the granulosa layer but fewer in the vitellogenic follicles In this study the pyriform cells disappeared when the follicles entered the vitellogenic stage Andreuccetti (1992) studied the differentiation of pyriform cells and their contribution to oocyte growth in 3 lizards namely Tarentola mauritanica Cordylus wittifer and Platysaurus intermedius and a colubrid snake Coluber viridiflavus and revealed that pyriform cells differentiate from small follicle cells via intermediate cells after establishing an intercellular bridge with the oocyte Once pyriform cells are differentiated they display ultrastructural features indicative of synthetic activity including abundant ribosomes Golgi membranes vacuoles mitochondria and lipid droplets These cellular components extend to the apex of the cell at the level of the intercellular bridge suggesting that constituents of pyriform cells may be transferred to the oocyte Pyriform cells and the oocytes may fulfill similar vitellogenic functions The establishment of an intercellular bridge may represent a crucial event in the development of an integrated system in which pyriform cells and oocytes cooperate Norris (2013) reported that the squamate granulosa contains the pyriform cells which are in direct contact with the developing oocyte and are apparently involved with early steps in oocyte development soon after the onset of vitellogenesis As ovulation approaches the granulosa cells as well as some thecal cells accumulate cholesterol-positive lipids and proliferate and luteinize to form corpora lutea following ovulation Follicular atresia is a common occurrence in reptilian ovaries as in other vertebrates (Norris 2013) as we found the corpora lutea in the follicle in accordance to oviductal egg appearances of a representative C versicolor collected in September Additionally as shown in Table 1 several atretic follicles lt25 mm in diameter were commonly encountered in C emma and C versicolor

Based on both morphological and histological investigations we found that testes ST and SSK were

MEESOOK et al Turk J Zool

700

concomitantly active and were associated with high levels of plasma testosterone We also demonstrated that there were high correlations between levels of plasma testosterone (T) and testicular mass where annual changes occurred in the same direction as the testicular size and time of spermatogenetic events among the males of the Calotes species Radder et al (2001) reported that in the male tropical or oriental garden lizard C versicolor plasma T is highest during the breeding season which correlated with testis mass and reproductive behavior Changes in T levels are associated with high spermatogenetic activity Radder et al (2001) also stated that the changes in plasma T levels during different phases of the male reproductive cycle in C versicolor follow a reproductive pattern of a prenuptial type of spermatogenesis that is similar to that of some other species of lizards the spiny-tailed lizard Uromastix hardwicki (Arslan et al 1978a) the viviparous lizard Lacerta vivipara (Courty and Dufaure 1982) the western shingleback lizard Tiliqua (Trachydosaurus) rugosa (Bourne et al 1986) the male lizard Podarcis s sicula (Ando et al 1990) Podarcis s sicula Raf (Ando et al 1992) the white-throated savanna monitor Varanus albigularis (Phillips and Millar 1998) and the male brown anoles Anolis sagrei (Tokarz et al 1998)

We found that estradiol (E2) levels increased in vitellogenic females its high levels were associated with the presence of the largest vitellogenic follicles in the 2 Calotes species Radder et al (2001) reported that in female C versicolor with overlapping reproductive events such as vitellogenesis and gestation E2 was at low levels when the ovaries were regressed and at high levels at vitellogenic follicular recruitment reaching peak level at the time of preovulatory follicles The same patterns of E2 secretion were found again when the second set of follicles underwent vitellogenesis (Radder et al 2001) Surprisingly Amey and Whittier (2000) reported that in female bearded dragons Pagona barbata plasma E2 was low or nondetectable across all reproductive states In C versicolor E2 levels were low in nonreproductive females with small previtellogenic follicles and those in the EG phase (Radder et al 2001) We do not discuss the level of the plasma progesterone (P) during the gestation period as its level was not detectable in this study The gravid lizards in EG exhibited low plasma E2 but high P levels and the highest P levels coincided with eggshell production P levels declined after eggshell formation as reported in other gravid individuals in several species of lizards that do not possess vitellogenic follicles of the subsequent clutch including C versicolor (Radder et al 2001) Uromastix hardwicki (Arslan et al 1978b) Agama atra (Van Wyk 1984) Eumeces obsoletus Scelporus undulatus and Crotaphytus collaris (Masson and Guillette 1987) and Psammodromus algirus (Diaz et al 1994) However a

decline in P levels in MG with vitellogenic follicles did not seem to facilitate recruitment or growth of the subsequent set of vitellogenic follicles in gravid Sceloporus jarrovi (Guillette et al 1981)

In the present study there were variations in the timing of breeding between the 2 Calotes species and even within populations of the same species We could not relate the copulation timing of 2 such Calotes species to gonadal activity or sex hormonal surges as the timing of natural mating could not be observed during the times we collected data Lizard species that inhabit temperate zones have mostly exhibited seasonal reproduction (Fitch 1970 Licht 1984 Pianka and Vitt 2003) The 10 lizard species that have been studied widely to date exhibit an associated reproductive pattern (Lovern 2011) that is green anoles Anolis carolinensis (Crews 1980 Lovern et al 2004) brown anoles Anolis sagrei (Lee et al 1989 Tokarz 1998) eastern fence lizards Sceloporus undulates (Cox et al 2005) mountain spiny lizards Sceloporus jarrovi (Woodley and Moore 1999) tree lizards Urosaurus ornatus (French and Moore 2008) wall lizards Podarcis sicula (Putti et al 2009) common lizards Lacerta vivipara (Vercken and Clobert 2008) little striped whiptail lizards Cnemidophorus inornatus (Crews 2005) garden lizards Calotes versicolor (Shanbhag 2003 Lovern 2011) and leopard geckos Eublepharis macularius (Rhen et al 2005) The temperate Florida populations of the brown anole Anolis sagrei show a strong seasonality in reproduction (Lee et al 1989) while the tropical Caribbean (Licht and Gorman 1970 Sexton and Brown 1977) and Hawaiian populations of this species (Goldberg et al 2002) show a less-pronounced seasonality in which reproductively active individuals can be found throughout the year Although individuals within a population of many tropical lizard species can breed at any time no individuals within the population breed year-round (Lovern 2011) Additionally Vitt and Caldwell (2009) stated that the belief used to be that tropical squamates had continuous reproduction in aseasonal tropical environments or reproduced during the wet season in a wetndashdry seasonal tropical environment Many tropical lizard species namely the anoles Anolis acutus (Ruibal et al 1972) Anolis limifrons (Sexton et al 1971) and Anolis opalinus (Jenssen and Nunez 1994) as well as the gecko Cyrtodactylus malyanus the flying lizard Draco melanopogon (Inger and Greenberg 1966) and the parthenogenetic oviparous whiptail lizard Cnemidophorus nativo (Menezes et al 2004) showed slightly more frequent breeding during the wet season than during the dry season (Jenssen and Nunez 1994)

Reproductive patterns can be described in a variety of ways but not all species fit neatly into such categorizations However 2 general types of reproductive patterns are

MEESOOK et al Turk J Zool

701

recognized in terms of prenuptial and postnuptial reproductive patterns (Lance 1998) Prenuptial reproductive pattern terms such as gonadal recrudescence sex steroid production and gametogenesis occur in advance of mating whereas postnuptial reproductive patterns occur following mating In other words in a high-elevation population of Sceloporus grammicus in Parque Nacional de Zoquiapan in central Mexico an active reproductive event occurring in the early fall is described as dissociated from testicular recrudescence in males but is associated with the initiation of ovarian recrudescence in females (Guillette and Casas-Andreu 1980 1981 Zuniga-Vega et al 2008) This is in contrast to S grammicus from Teotihuacan Mexico in which testicular recrudescence and breeding occur in the summer and fall at the onset of female ovarian recrudescence (Jimenez-Cruz et al 2005) In S mucronatus from Valle de la Cantimplora Mexico peak testicular recrudescence and mating occur during the summer prior to ovarian recrudescence which does not occur until several months later (Ortega-Leon et al 2009) This is distinct from many fall-breeding

populations elsewhere (Mendez-De La Cruz et al 1994 Villagran-Santa Cruz et al 1994) The examples above demonstrate that gonadal activity and mating behavior are clearly variable but hormone analyses have not been performed in these species and so endocrine relationships cannot be assessed at this point

In conclusion we suggest that the males and females of the 2 Calotes species have much more prolonged active reproductive phases than inactive reproductive phases The reproductive patterns of C emma and C versicolor were classified into the same reproductive pattern of continual reproduction

AcknowledgmentsWe thank the Department of Zoology of Kasetsart University for financial support We also thank the staff of Sakaerat Environmental Research Station Nakhon Ratchasima Province for devoting time for research collaboration We also thank Mrs Sureerat Sangkrut for drawing all illustrations In addition we wish to thank the anonymous referees for many helpful suggestions

References

Aldridge RD Goldberg SR Wisniewski SS Bufalino AP Dillman CB (2009) The reproductive cycle and estrus in the colubrid snakes of temperate North America Contemp Herpetol 4 1-31

Amey AP Whittier JM (2000) Seasonal patterns of plasma steroid hormones in males and females of the bearded dragon lizard Pogona barbata Gen Comp Endocrinol 117 335-342

Andreuccetti P (1992) An ultrastructural study of differentiation of pyriform cells and their contribution to oocyte growth in representative Squamata J Morphol 212 1-11

Ando S Panno ML Ciarcia G Imbrogno E Buffone M Beraldi E Sisci D Angelini F Botte V (1990) Plasma sex hormone concentrations during the reproductive cycle in the male lizard Podarcis s sicula J Reprod Fertil 90 353-360

Ando S Ciarcia G Panno ML Imbrogno E Tarantino G Buffone M Beraldi E Angelini F Botte V (1992) Sex steroids levels in the plasma and testis during the reproductive cycle of lizard Podarcis s sicula Raf Gen Comp Endocrinol 85 1-7

Arslan MJ Lobo J Zaidi AA Jalali S Qazi MH (1978a) Annual androgen rhythm in the spiny-tailed lizard Uromastix hardwicki Gen Comp Endocrinol 36 16-22

Arslan MJ Zaidi P Lobo J Zaidi AA Qazi MH (1978b) Steroid levels in preovulatory and gravid lizards (Uromastix hardwicki) Gen Comp Endocrinol 34 300-303

Avwioro G (2011) Histochemical uses of haematoxylin-a review J Pham Clin Sci 1 24-34

Bishop JE (1959) A histological and histochemical study of the kidney tubule of the common garter snake Thamnophis sirtalis with special reference to the sexual segment in the male J Morphol 104 307-358

Bourne AR Taylor JL Watson TG (1986) Annual cycles of plasma and testicular androgens in the lizard Tiliqua (Trachydosaurus) rugosa Gen Comp Endocrinol 61 278-286

Courty Y Dufaure JP (1982) Circannual testosterone dihydrotestosterone and androstenedione in the plasma and testis of Lacerta vivipara a seasonally breeding viviparous lizard Steroids 39 517-529

Cox RM Skelly SL Leo A John-Alder HB (2005) Testosterone regulates sexually dimorphic coloration in the eastern fence lizard Sceloporus undulatus Copeia 2005 597-608

Crews D (1976) Hormonal control of male courtship behavior and female attractively in the garter snake (Thamnophis sirtalis parietalis) Horm Behav 7 451-460

Crews D (1980) Interrelationships among ecological behavioral and neuroendocrine processes in the reproductive cycle of Anolis carolinensis and other reptiles Adv Stud Behav 11 1-74

Crews D (1984) Gamete production sex hormone secretion and mating behavior uncoupled Horm Behav 14 22-28

Crews D (1999) Reptilian reproduction overview In Knobil E Neil JD editors Encyclopedia of Reproduction New York NY USA Academic Press pp 239-243

Crews D Camazine B Diamond M Mason R Tokarz R Garstka WR (1984) Hormonal independence of courtship behavior in the male garter snake Horm Behav 14 29-41

Crews D (2005) Evolution of neuroendocrine mechanisms that regulate sexual behavior Trends Endocrinol Metab 16 354-361

Del Conte E (1972) Granular secretion in the kidney Rss of female lizards Cnemidophorus l lemniscatus (Sauria Teiidae) J Morphol 137 181-191

MEESOOK et al Turk J Zool

699

and crocodilians (Fox 1952) Bishop (1959) found that the testes of the male garter snake Thamnophis sirtalis were spermatogenically active during the same time as the hypertrophied SSK during the active reproductive period the diameter of the SSK tubule was 5 times greater than that of the SSK tubule during the inactive reproductive period However SSK development in female lizards has been reported in the genus Cnemidophorus (Del Conte 1972 Del Conte and Tamayo 1973) and Scincella laterale (Sever and Hopkins 2005) They suggested that the females had a low level of natural androgens which caused the SSK development (Del Conte and Tamayo 1973 Sever and Hopkins 2005) In the present study we did not monitor the annual seasonal alterations in female SSK The hypertrophy of the SSK is synchronous with androgen secretion and spermatogenic activity (Sever and Hopkins 2005) Norris (2013) also stated that the SSK of sexually active squamates undergoes hypertrophy and is under the influence of androgens In this study we did not investigate any alterations in annual SSK with annual androgen secretion but we did demonstrate that the hypertrophy and the regression of SSK changed seasonally and synchronously with the active spermatogenic event and the spermatogenic arrest respectively In the Iberian rock lizard Lacerta monticola SSK secretions form a copulatory plug that adheres to the femalersquos cloaca following copulation to occlude oviductal openings however such a plug does not prevent subsequent mating nor does it reduce the femalersquos attractiveness (Moreira and Birkhead 2003)

With our investigations on annual alterations in female ovarian morphologies between 2 Calotes we found that individuals in the same Calotes species showed different timing of reproductive events throughout a 1-year period (the data are not shown here) Additionally there was quite clear evidence that QU EV and LV follicles and oviductal eggs overlapped among individuals within the same populations of both Calotes species Female reproductive status is definitely distinguishable between the 2 Calotes species Gravid lizards were encountered in 1 individual of C emma in April and in 3 individuals of C versicolor in June August and September Shanbhag et al (2000) reported that female C versicolor showed inactive reproduction from December to April and gravidity was encountered from May to October

We found that in the previtellogenic follicles (QU and EV) of females of both Calotes species the granulosa layer contained 2 types of cells pyriform and small cells Uribe et al (1996) stated that in squamates the follicular epithelium or granulosa initially consists of small cuboidal cells but differentiates during the previtellogenic phase and becomes multilayered and polymorphic by the presence of unique flask-shaped pyriform cells intermediate cells and

small cells These pyriform cells differentiate from small somatic follicular cells early in follicular development via the intermediate-cell stage to become nurse cells in direct contact with the developing oocytes (Maurizii et al 2004) Differentiation of the small cells into pyriform cells appears to be linked to the progressive appearance of glycoproteins with terminal α-N-acetylgalactosamine residues on the cell surface which may be involved in fusion between the oocyte and the follicle cell membranes as well as maintenance of the differentiated pyriform cells The pyriform cells are connected to the oocyte via intercellular bridges containing a cytoskeleton of α-tubulin and cytokeratin microtubules (Maurizii et al 2004) Tumkiratiwong et al (2012) also demonstrated that the previtellogenic follicles of the captive monocled cobra Naja kaouthia had many pyriform cells in the granulosa layer but fewer in the vitellogenic follicles In this study the pyriform cells disappeared when the follicles entered the vitellogenic stage Andreuccetti (1992) studied the differentiation of pyriform cells and their contribution to oocyte growth in 3 lizards namely Tarentola mauritanica Cordylus wittifer and Platysaurus intermedius and a colubrid snake Coluber viridiflavus and revealed that pyriform cells differentiate from small follicle cells via intermediate cells after establishing an intercellular bridge with the oocyte Once pyriform cells are differentiated they display ultrastructural features indicative of synthetic activity including abundant ribosomes Golgi membranes vacuoles mitochondria and lipid droplets These cellular components extend to the apex of the cell at the level of the intercellular bridge suggesting that constituents of pyriform cells may be transferred to the oocyte Pyriform cells and the oocytes may fulfill similar vitellogenic functions The establishment of an intercellular bridge may represent a crucial event in the development of an integrated system in which pyriform cells and oocytes cooperate Norris (2013) reported that the squamate granulosa contains the pyriform cells which are in direct contact with the developing oocyte and are apparently involved with early steps in oocyte development soon after the onset of vitellogenesis As ovulation approaches the granulosa cells as well as some thecal cells accumulate cholesterol-positive lipids and proliferate and luteinize to form corpora lutea following ovulation Follicular atresia is a common occurrence in reptilian ovaries as in other vertebrates (Norris 2013) as we found the corpora lutea in the follicle in accordance to oviductal egg appearances of a representative C versicolor collected in September Additionally as shown in Table 1 several atretic follicles lt25 mm in diameter were commonly encountered in C emma and C versicolor

Based on both morphological and histological investigations we found that testes ST and SSK were

MEESOOK et al Turk J Zool

700

concomitantly active and were associated with high levels of plasma testosterone We also demonstrated that there were high correlations between levels of plasma testosterone (T) and testicular mass where annual changes occurred in the same direction as the testicular size and time of spermatogenetic events among the males of the Calotes species Radder et al (2001) reported that in the male tropical or oriental garden lizard C versicolor plasma T is highest during the breeding season which correlated with testis mass and reproductive behavior Changes in T levels are associated with high spermatogenetic activity Radder et al (2001) also stated that the changes in plasma T levels during different phases of the male reproductive cycle in C versicolor follow a reproductive pattern of a prenuptial type of spermatogenesis that is similar to that of some other species of lizards the spiny-tailed lizard Uromastix hardwicki (Arslan et al 1978a) the viviparous lizard Lacerta vivipara (Courty and Dufaure 1982) the western shingleback lizard Tiliqua (Trachydosaurus) rugosa (Bourne et al 1986) the male lizard Podarcis s sicula (Ando et al 1990) Podarcis s sicula Raf (Ando et al 1992) the white-throated savanna monitor Varanus albigularis (Phillips and Millar 1998) and the male brown anoles Anolis sagrei (Tokarz et al 1998)

We found that estradiol (E2) levels increased in vitellogenic females its high levels were associated with the presence of the largest vitellogenic follicles in the 2 Calotes species Radder et al (2001) reported that in female C versicolor with overlapping reproductive events such as vitellogenesis and gestation E2 was at low levels when the ovaries were regressed and at high levels at vitellogenic follicular recruitment reaching peak level at the time of preovulatory follicles The same patterns of E2 secretion were found again when the second set of follicles underwent vitellogenesis (Radder et al 2001) Surprisingly Amey and Whittier (2000) reported that in female bearded dragons Pagona barbata plasma E2 was low or nondetectable across all reproductive states In C versicolor E2 levels were low in nonreproductive females with small previtellogenic follicles and those in the EG phase (Radder et al 2001) We do not discuss the level of the plasma progesterone (P) during the gestation period as its level was not detectable in this study The gravid lizards in EG exhibited low plasma E2 but high P levels and the highest P levels coincided with eggshell production P levels declined after eggshell formation as reported in other gravid individuals in several species of lizards that do not possess vitellogenic follicles of the subsequent clutch including C versicolor (Radder et al 2001) Uromastix hardwicki (Arslan et al 1978b) Agama atra (Van Wyk 1984) Eumeces obsoletus Scelporus undulatus and Crotaphytus collaris (Masson and Guillette 1987) and Psammodromus algirus (Diaz et al 1994) However a

decline in P levels in MG with vitellogenic follicles did not seem to facilitate recruitment or growth of the subsequent set of vitellogenic follicles in gravid Sceloporus jarrovi (Guillette et al 1981)

In the present study there were variations in the timing of breeding between the 2 Calotes species and even within populations of the same species We could not relate the copulation timing of 2 such Calotes species to gonadal activity or sex hormonal surges as the timing of natural mating could not be observed during the times we collected data Lizard species that inhabit temperate zones have mostly exhibited seasonal reproduction (Fitch 1970 Licht 1984 Pianka and Vitt 2003) The 10 lizard species that have been studied widely to date exhibit an associated reproductive pattern (Lovern 2011) that is green anoles Anolis carolinensis (Crews 1980 Lovern et al 2004) brown anoles Anolis sagrei (Lee et al 1989 Tokarz 1998) eastern fence lizards Sceloporus undulates (Cox et al 2005) mountain spiny lizards Sceloporus jarrovi (Woodley and Moore 1999) tree lizards Urosaurus ornatus (French and Moore 2008) wall lizards Podarcis sicula (Putti et al 2009) common lizards Lacerta vivipara (Vercken and Clobert 2008) little striped whiptail lizards Cnemidophorus inornatus (Crews 2005) garden lizards Calotes versicolor (Shanbhag 2003 Lovern 2011) and leopard geckos Eublepharis macularius (Rhen et al 2005) The temperate Florida populations of the brown anole Anolis sagrei show a strong seasonality in reproduction (Lee et al 1989) while the tropical Caribbean (Licht and Gorman 1970 Sexton and Brown 1977) and Hawaiian populations of this species (Goldberg et al 2002) show a less-pronounced seasonality in which reproductively active individuals can be found throughout the year Although individuals within a population of many tropical lizard species can breed at any time no individuals within the population breed year-round (Lovern 2011) Additionally Vitt and Caldwell (2009) stated that the belief used to be that tropical squamates had continuous reproduction in aseasonal tropical environments or reproduced during the wet season in a wetndashdry seasonal tropical environment Many tropical lizard species namely the anoles Anolis acutus (Ruibal et al 1972) Anolis limifrons (Sexton et al 1971) and Anolis opalinus (Jenssen and Nunez 1994) as well as the gecko Cyrtodactylus malyanus the flying lizard Draco melanopogon (Inger and Greenberg 1966) and the parthenogenetic oviparous whiptail lizard Cnemidophorus nativo (Menezes et al 2004) showed slightly more frequent breeding during the wet season than during the dry season (Jenssen and Nunez 1994)

Reproductive patterns can be described in a variety of ways but not all species fit neatly into such categorizations However 2 general types of reproductive patterns are

MEESOOK et al Turk J Zool

701

recognized in terms of prenuptial and postnuptial reproductive patterns (Lance 1998) Prenuptial reproductive pattern terms such as gonadal recrudescence sex steroid production and gametogenesis occur in advance of mating whereas postnuptial reproductive patterns occur following mating In other words in a high-elevation population of Sceloporus grammicus in Parque Nacional de Zoquiapan in central Mexico an active reproductive event occurring in the early fall is described as dissociated from testicular recrudescence in males but is associated with the initiation of ovarian recrudescence in females (Guillette and Casas-Andreu 1980 1981 Zuniga-Vega et al 2008) This is in contrast to S grammicus from Teotihuacan Mexico in which testicular recrudescence and breeding occur in the summer and fall at the onset of female ovarian recrudescence (Jimenez-Cruz et al 2005) In S mucronatus from Valle de la Cantimplora Mexico peak testicular recrudescence and mating occur during the summer prior to ovarian recrudescence which does not occur until several months later (Ortega-Leon et al 2009) This is distinct from many fall-breeding

populations elsewhere (Mendez-De La Cruz et al 1994 Villagran-Santa Cruz et al 1994) The examples above demonstrate that gonadal activity and mating behavior are clearly variable but hormone analyses have not been performed in these species and so endocrine relationships cannot be assessed at this point

In conclusion we suggest that the males and females of the 2 Calotes species have much more prolonged active reproductive phases than inactive reproductive phases The reproductive patterns of C emma and C versicolor were classified into the same reproductive pattern of continual reproduction

AcknowledgmentsWe thank the Department of Zoology of Kasetsart University for financial support We also thank the staff of Sakaerat Environmental Research Station Nakhon Ratchasima Province for devoting time for research collaboration We also thank Mrs Sureerat Sangkrut for drawing all illustrations In addition we wish to thank the anonymous referees for many helpful suggestions

References

Aldridge RD Goldberg SR Wisniewski SS Bufalino AP Dillman CB (2009) The reproductive cycle and estrus in the colubrid snakes of temperate North America Contemp Herpetol 4 1-31

Amey AP Whittier JM (2000) Seasonal patterns of plasma steroid hormones in males and females of the bearded dragon lizard Pogona barbata Gen Comp Endocrinol 117 335-342

Andreuccetti P (1992) An ultrastructural study of differentiation of pyriform cells and their contribution to oocyte growth in representative Squamata J Morphol 212 1-11

Ando S Panno ML Ciarcia G Imbrogno E Buffone M Beraldi E Sisci D Angelini F Botte V (1990) Plasma sex hormone concentrations during the reproductive cycle in the male lizard Podarcis s sicula J Reprod Fertil 90 353-360

Ando S Ciarcia G Panno ML Imbrogno E Tarantino G Buffone M Beraldi E Angelini F Botte V (1992) Sex steroids levels in the plasma and testis during the reproductive cycle of lizard Podarcis s sicula Raf Gen Comp Endocrinol 85 1-7

Arslan MJ Lobo J Zaidi AA Jalali S Qazi MH (1978a) Annual androgen rhythm in the spiny-tailed lizard Uromastix hardwicki Gen Comp Endocrinol 36 16-22

Arslan MJ Zaidi P Lobo J Zaidi AA Qazi MH (1978b) Steroid levels in preovulatory and gravid lizards (Uromastix hardwicki) Gen Comp Endocrinol 34 300-303

Avwioro G (2011) Histochemical uses of haematoxylin-a review J Pham Clin Sci 1 24-34

Bishop JE (1959) A histological and histochemical study of the kidney tubule of the common garter snake Thamnophis sirtalis with special reference to the sexual segment in the male J Morphol 104 307-358

Bourne AR Taylor JL Watson TG (1986) Annual cycles of plasma and testicular androgens in the lizard Tiliqua (Trachydosaurus) rugosa Gen Comp Endocrinol 61 278-286

Courty Y Dufaure JP (1982) Circannual testosterone dihydrotestosterone and androstenedione in the plasma and testis of Lacerta vivipara a seasonally breeding viviparous lizard Steroids 39 517-529

Cox RM Skelly SL Leo A John-Alder HB (2005) Testosterone regulates sexually dimorphic coloration in the eastern fence lizard Sceloporus undulatus Copeia 2005 597-608

Crews D (1976) Hormonal control of male courtship behavior and female attractively in the garter snake (Thamnophis sirtalis parietalis) Horm Behav 7 451-460

Crews D (1980) Interrelationships among ecological behavioral and neuroendocrine processes in the reproductive cycle of Anolis carolinensis and other reptiles Adv Stud Behav 11 1-74

Crews D (1984) Gamete production sex hormone secretion and mating behavior uncoupled Horm Behav 14 22-28

Crews D (1999) Reptilian reproduction overview In Knobil E Neil JD editors Encyclopedia of Reproduction New York NY USA Academic Press pp 239-243

Crews D Camazine B Diamond M Mason R Tokarz R Garstka WR (1984) Hormonal independence of courtship behavior in the male garter snake Horm Behav 14 29-41

Crews D (2005) Evolution of neuroendocrine mechanisms that regulate sexual behavior Trends Endocrinol Metab 16 354-361

Del Conte E (1972) Granular secretion in the kidney Rss of female lizards Cnemidophorus l lemniscatus (Sauria Teiidae) J Morphol 137 181-191

MEESOOK et al Turk J Zool

700

concomitantly active and were associated with high levels of plasma testosterone We also demonstrated that there were high correlations between levels of plasma testosterone (T) and testicular mass where annual changes occurred in the same direction as the testicular size and time of spermatogenetic events among the males of the Calotes species Radder et al (2001) reported that in the male tropical or oriental garden lizard C versicolor plasma T is highest during the breeding season which correlated with testis mass and reproductive behavior Changes in T levels are associated with high spermatogenetic activity Radder et al (2001) also stated that the changes in plasma T levels during different phases of the male reproductive cycle in C versicolor follow a reproductive pattern of a prenuptial type of spermatogenesis that is similar to that of some other species of lizards the spiny-tailed lizard Uromastix hardwicki (Arslan et al 1978a) the viviparous lizard Lacerta vivipara (Courty and Dufaure 1982) the western shingleback lizard Tiliqua (Trachydosaurus) rugosa (Bourne et al 1986) the male lizard Podarcis s sicula (Ando et al 1990) Podarcis s sicula Raf (Ando et al 1992) the white-throated savanna monitor Varanus albigularis (Phillips and Millar 1998) and the male brown anoles Anolis sagrei (Tokarz et al 1998)

We found that estradiol (E2) levels increased in vitellogenic females its high levels were associated with the presence of the largest vitellogenic follicles in the 2 Calotes species Radder et al (2001) reported that in female C versicolor with overlapping reproductive events such as vitellogenesis and gestation E2 was at low levels when the ovaries were regressed and at high levels at vitellogenic follicular recruitment reaching peak level at the time of preovulatory follicles The same patterns of E2 secretion were found again when the second set of follicles underwent vitellogenesis (Radder et al 2001) Surprisingly Amey and Whittier (2000) reported that in female bearded dragons Pagona barbata plasma E2 was low or nondetectable across all reproductive states In C versicolor E2 levels were low in nonreproductive females with small previtellogenic follicles and those in the EG phase (Radder et al 2001) We do not discuss the level of the plasma progesterone (P) during the gestation period as its level was not detectable in this study The gravid lizards in EG exhibited low plasma E2 but high P levels and the highest P levels coincided with eggshell production P levels declined after eggshell formation as reported in other gravid individuals in several species of lizards that do not possess vitellogenic follicles of the subsequent clutch including C versicolor (Radder et al 2001) Uromastix hardwicki (Arslan et al 1978b) Agama atra (Van Wyk 1984) Eumeces obsoletus Scelporus undulatus and Crotaphytus collaris (Masson and Guillette 1987) and Psammodromus algirus (Diaz et al 1994) However a

decline in P levels in MG with vitellogenic follicles did not seem to facilitate recruitment or growth of the subsequent set of vitellogenic follicles in gravid Sceloporus jarrovi (Guillette et al 1981)

In the present study there were variations in the timing of breeding between the 2 Calotes species and even within populations of the same species We could not relate the copulation timing of 2 such Calotes species to gonadal activity or sex hormonal surges as the timing of natural mating could not be observed during the times we collected data Lizard species that inhabit temperate zones have mostly exhibited seasonal reproduction (Fitch 1970 Licht 1984 Pianka and Vitt 2003) The 10 lizard species that have been studied widely to date exhibit an associated reproductive pattern (Lovern 2011) that is green anoles Anolis carolinensis (Crews 1980 Lovern et al 2004) brown anoles Anolis sagrei (Lee et al 1989 Tokarz 1998) eastern fence lizards Sceloporus undulates (Cox et al 2005) mountain spiny lizards Sceloporus jarrovi (Woodley and Moore 1999) tree lizards Urosaurus ornatus (French and Moore 2008) wall lizards Podarcis sicula (Putti et al 2009) common lizards Lacerta vivipara (Vercken and Clobert 2008) little striped whiptail lizards Cnemidophorus inornatus (Crews 2005) garden lizards Calotes versicolor (Shanbhag 2003 Lovern 2011) and leopard geckos Eublepharis macularius (Rhen et al 2005) The temperate Florida populations of the brown anole Anolis sagrei show a strong seasonality in reproduction (Lee et al 1989) while the tropical Caribbean (Licht and Gorman 1970 Sexton and Brown 1977) and Hawaiian populations of this species (Goldberg et al 2002) show a less-pronounced seasonality in which reproductively active individuals can be found throughout the year Although individuals within a population of many tropical lizard species can breed at any time no individuals within the population breed year-round (Lovern 2011) Additionally Vitt and Caldwell (2009) stated that the belief used to be that tropical squamates had continuous reproduction in aseasonal tropical environments or reproduced during the wet season in a wetndashdry seasonal tropical environment Many tropical lizard species namely the anoles Anolis acutus (Ruibal et al 1972) Anolis limifrons (Sexton et al 1971) and Anolis opalinus (Jenssen and Nunez 1994) as well as the gecko Cyrtodactylus malyanus the flying lizard Draco melanopogon (Inger and Greenberg 1966) and the parthenogenetic oviparous whiptail lizard Cnemidophorus nativo (Menezes et al 2004) showed slightly more frequent breeding during the wet season than during the dry season (Jenssen and Nunez 1994)

Reproductive patterns can be described in a variety of ways but not all species fit neatly into such categorizations However 2 general types of reproductive patterns are

MEESOOK et al Turk J Zool

701

recognized in terms of prenuptial and postnuptial reproductive patterns (Lance 1998) Prenuptial reproductive pattern terms such as gonadal recrudescence sex steroid production and gametogenesis occur in advance of mating whereas postnuptial reproductive patterns occur following mating In other words in a high-elevation population of Sceloporus grammicus in Parque Nacional de Zoquiapan in central Mexico an active reproductive event occurring in the early fall is described as dissociated from testicular recrudescence in males but is associated with the initiation of ovarian recrudescence in females (Guillette and Casas-Andreu 1980 1981 Zuniga-Vega et al 2008) This is in contrast to S grammicus from Teotihuacan Mexico in which testicular recrudescence and breeding occur in the summer and fall at the onset of female ovarian recrudescence (Jimenez-Cruz et al 2005) In S mucronatus from Valle de la Cantimplora Mexico peak testicular recrudescence and mating occur during the summer prior to ovarian recrudescence which does not occur until several months later (Ortega-Leon et al 2009) This is distinct from many fall-breeding

populations elsewhere (Mendez-De La Cruz et al 1994 Villagran-Santa Cruz et al 1994) The examples above demonstrate that gonadal activity and mating behavior are clearly variable but hormone analyses have not been performed in these species and so endocrine relationships cannot be assessed at this point

In conclusion we suggest that the males and females of the 2 Calotes species have much more prolonged active reproductive phases than inactive reproductive phases The reproductive patterns of C emma and C versicolor were classified into the same reproductive pattern of continual reproduction

AcknowledgmentsWe thank the Department of Zoology of Kasetsart University for financial support We also thank the staff of Sakaerat Environmental Research Station Nakhon Ratchasima Province for devoting time for research collaboration We also thank Mrs Sureerat Sangkrut for drawing all illustrations In addition we wish to thank the anonymous referees for many helpful suggestions

References

Aldridge RD Goldberg SR Wisniewski SS Bufalino AP Dillman CB (2009) The reproductive cycle and estrus in the colubrid snakes of temperate North America Contemp Herpetol 4 1-31

Amey AP Whittier JM (2000) Seasonal patterns of plasma steroid hormones in males and females of the bearded dragon lizard Pogona barbata Gen Comp Endocrinol 117 335-342

Andreuccetti P (1992) An ultrastructural study of differentiation of pyriform cells and their contribution to oocyte growth in representative Squamata J Morphol 212 1-11

Ando S Panno ML Ciarcia G Imbrogno E Buffone M Beraldi E Sisci D Angelini F Botte V (1990) Plasma sex hormone concentrations during the reproductive cycle in the male lizard Podarcis s sicula J Reprod Fertil 90 353-360

Ando S Ciarcia G Panno ML Imbrogno E Tarantino G Buffone M Beraldi E Angelini F Botte V (1992) Sex steroids levels in the plasma and testis during the reproductive cycle of lizard Podarcis s sicula Raf Gen Comp Endocrinol 85 1-7

Arslan MJ Lobo J Zaidi AA Jalali S Qazi MH (1978a) Annual androgen rhythm in the spiny-tailed lizard Uromastix hardwicki Gen Comp Endocrinol 36 16-22

Arslan MJ Zaidi P Lobo J Zaidi AA Qazi MH (1978b) Steroid levels in preovulatory and gravid lizards (Uromastix hardwicki) Gen Comp Endocrinol 34 300-303

Avwioro G (2011) Histochemical uses of haematoxylin-a review J Pham Clin Sci 1 24-34

Bishop JE (1959) A histological and histochemical study of the kidney tubule of the common garter snake Thamnophis sirtalis with special reference to the sexual segment in the male J Morphol 104 307-358

Bourne AR Taylor JL Watson TG (1986) Annual cycles of plasma and testicular androgens in the lizard Tiliqua (Trachydosaurus) rugosa Gen Comp Endocrinol 61 278-286

Courty Y Dufaure JP (1982) Circannual testosterone dihydrotestosterone and androstenedione in the plasma and testis of Lacerta vivipara a seasonally breeding viviparous lizard Steroids 39 517-529

Cox RM Skelly SL Leo A John-Alder HB (2005) Testosterone regulates sexually dimorphic coloration in the eastern fence lizard Sceloporus undulatus Copeia 2005 597-608

Crews D (1976) Hormonal control of male courtship behavior and female attractively in the garter snake (Thamnophis sirtalis parietalis) Horm Behav 7 451-460

Crews D (1980) Interrelationships among ecological behavioral and neuroendocrine processes in the reproductive cycle of Anolis carolinensis and other reptiles Adv Stud Behav 11 1-74

Crews D (1984) Gamete production sex hormone secretion and mating behavior uncoupled Horm Behav 14 22-28

Crews D (1999) Reptilian reproduction overview In Knobil E Neil JD editors Encyclopedia of Reproduction New York NY USA Academic Press pp 239-243

Crews D Camazine B Diamond M Mason R Tokarz R Garstka WR (1984) Hormonal independence of courtship behavior in the male garter snake Horm Behav 14 29-41

Crews D (2005) Evolution of neuroendocrine mechanisms that regulate sexual behavior Trends Endocrinol Metab 16 354-361

Del Conte E (1972) Granular secretion in the kidney Rss of female lizards Cnemidophorus l lemniscatus (Sauria Teiidae) J Morphol 137 181-191

MEESOOK et al Turk J Zool

701

recognized in terms of prenuptial and postnuptial reproductive patterns (Lance 1998) Prenuptial reproductive pattern terms such as gonadal recrudescence sex steroid production and gametogenesis occur in advance of mating whereas postnuptial reproductive patterns occur following mating In other words in a high-elevation population of Sceloporus grammicus in Parque Nacional de Zoquiapan in central Mexico an active reproductive event occurring in the early fall is described as dissociated from testicular recrudescence in males but is associated with the initiation of ovarian recrudescence in females (Guillette and Casas-Andreu 1980 1981 Zuniga-Vega et al 2008) This is in contrast to S grammicus from Teotihuacan Mexico in which testicular recrudescence and breeding occur in the summer and fall at the onset of female ovarian recrudescence (Jimenez-Cruz et al 2005) In S mucronatus from Valle de la Cantimplora Mexico peak testicular recrudescence and mating occur during the summer prior to ovarian recrudescence which does not occur until several months later (Ortega-Leon et al 2009) This is distinct from many fall-breeding

populations elsewhere (Mendez-De La Cruz et al 1994 Villagran-Santa Cruz et al 1994) The examples above demonstrate that gonadal activity and mating behavior are clearly variable but hormone analyses have not been performed in these species and so endocrine relationships cannot be assessed at this point

In conclusion we suggest that the males and females of the 2 Calotes species have much more prolonged active reproductive phases than inactive reproductive phases The reproductive patterns of C emma and C versicolor were classified into the same reproductive pattern of continual reproduction

AcknowledgmentsWe thank the Department of Zoology of Kasetsart University for financial support We also thank the staff of Sakaerat Environmental Research Station Nakhon Ratchasima Province for devoting time for research collaboration We also thank Mrs Sureerat Sangkrut for drawing all illustrations In addition we wish to thank the anonymous referees for many helpful suggestions

References

Aldridge RD Goldberg SR Wisniewski SS Bufalino AP Dillman CB (2009) The reproductive cycle and estrus in the colubrid snakes of temperate North America Contemp Herpetol 4 1-31

Amey AP Whittier JM (2000) Seasonal patterns of plasma steroid hormones in males and females of the bearded dragon lizard Pogona barbata Gen Comp Endocrinol 117 335-342

Andreuccetti P (1992) An ultrastructural study of differentiation of pyriform cells and their contribution to oocyte growth in representative Squamata J Morphol 212 1-11

Ando S Panno ML Ciarcia G Imbrogno E Buffone M Beraldi E Sisci D Angelini F Botte V (1990) Plasma sex hormone concentrations during the reproductive cycle in the male lizard Podarcis s sicula J Reprod Fertil 90 353-360

Ando S Ciarcia G Panno ML Imbrogno E Tarantino G Buffone M Beraldi E Angelini F Botte V (1992) Sex steroids levels in the plasma and testis during the reproductive cycle of lizard Podarcis s sicula Raf Gen Comp Endocrinol 85 1-7

Arslan MJ Lobo J Zaidi AA Jalali S Qazi MH (1978a) Annual androgen rhythm in the spiny-tailed lizard Uromastix hardwicki Gen Comp Endocrinol 36 16-22

Arslan MJ Zaidi P Lobo J Zaidi AA Qazi MH (1978b) Steroid levels in preovulatory and gravid lizards (Uromastix hardwicki) Gen Comp Endocrinol 34 300-303

Avwioro G (2011) Histochemical uses of haematoxylin-a review J Pham Clin Sci 1 24-34

Bishop JE (1959) A histological and histochemical study of the kidney tubule of the common garter snake Thamnophis sirtalis with special reference to the sexual segment in the male J Morphol 104 307-358

Bourne AR Taylor JL Watson TG (1986) Annual cycles of plasma and testicular androgens in the lizard Tiliqua (Trachydosaurus) rugosa Gen Comp Endocrinol 61 278-286

Courty Y Dufaure JP (1982) Circannual testosterone dihydrotestosterone and androstenedione in the plasma and testis of Lacerta vivipara a seasonally breeding viviparous lizard Steroids 39 517-529

Cox RM Skelly SL Leo A John-Alder HB (2005) Testosterone regulates sexually dimorphic coloration in the eastern fence lizard Sceloporus undulatus Copeia 2005 597-608

Crews D (1976) Hormonal control of male courtship behavior and female attractively in the garter snake (Thamnophis sirtalis parietalis) Horm Behav 7 451-460

Crews D (1980) Interrelationships among ecological behavioral and neuroendocrine processes in the reproductive cycle of Anolis carolinensis and other reptiles Adv Stud Behav 11 1-74

Crews D (1984) Gamete production sex hormone secretion and mating behavior uncoupled Horm Behav 14 22-28

Crews D (1999) Reptilian reproduction overview In Knobil E Neil JD editors Encyclopedia of Reproduction New York NY USA Academic Press pp 239-243

Crews D Camazine B Diamond M Mason R Tokarz R Garstka WR (1984) Hormonal independence of courtship behavior in the male garter snake Horm Behav 14 29-41

Crews D (2005) Evolution of neuroendocrine mechanisms that regulate sexual behavior Trends Endocrinol Metab 16 354-361

Del Conte E (1972) Granular secretion in the kidney Rss of female lizards Cnemidophorus l lemniscatus (Sauria Teiidae) J Morphol 137 181-191

MEESOOK et al Turk J Zool

702

Del Conte E Tamayo JG (1973) Ultrastructure of the Rss of the kidneys in male and female lizards Cnemidophorus l lemniscatus (L) Z Zellforsch 144 325-327

Diaz JA Alonso-Gomez AL Delgado MJ (1994) Seasonal variation of gonadal development sexual steroids and lipid reserves in a population of the lizard Psammodromus algirus J Herpetol 28 199-205

Fitch HS (1970) Reproductive Cycles in Lizards and Snakes 2nd ed Lawrence KS USA The University of Kansas Museum of Natural History

Fox W (1952) Seasonal variation in the male reproductive system of Pacific coast garter snakes J Morphol 90 481-553

French SS Moore MC (2008) Immune function varies with reproductive stage and context in female and male tree lizards Urosaurus ornatus Gen Comp Endocrinol 155 148-156

Garstka WR Camazine B Crews D (1982) Interactions of behavior and physiology during the annual reproductive cycle of the red-garter snake (Thamnophissirtalis parietalis) Herpetologica 38 104-123

Goldberg SR Kraus F Bursey CR (2002) Reproduction in an introduced population of the brown anole Anolis sagrei from Oahu Hawaii Pac Sci 56 163-168

Gouder BYM Nadkarni VB (1979) Histometric and histochemical changes in the seminiferous epithelium Leydig cells and Sertoli cells in the testis of Calotes versicolor Biol Bull India 1 15-22

Guillette LJ Jr Casas-Andreu G (1980) Fall reproductive activity in the high altitude Mexican lizard Sceloporus grammicus microlepidotus J Herpetol 14 143-147

Guillette LJ Jr Casas-Andreu G (1981) Seasonal variation in fat body weights of the Mexican high elevation lizard Sceloporus grammicus microlepidotus J Herpetol 15 366-371

Guillette LJ Jr Spielvogel S Moore FL (1981) Luteal development placentation and plasma progesterone concentration in the viviparous lizard Sceloporus jarrovi Gen Comp Endocrinol 27 389-400

Hartmann T Geissler P Poyarkov AN Jr Ihlow F Galoyan AE Roumldder D Boumlhme W (2013) A new species of the genus Calotes Cuvier 1817 (Squamata Agamidae) from southern Vietnam Zootaxa 3599 246-260

Inger RF Greenberg B (1966) Annual reproductive patterns of lizards from a Bornean rainforest Ecology 47 1007-1021

Jimenez-Cruz E Ramırez-Bautista A Marshall JC Lizana-Avia M Nieto-Montes De Oca A (2005) Reproductive cycle of Sceloporus grammicus (Squamata Phrynosomatidae) from Teotihuacan Mexico Southwest Natur 50 178-187

Jenssen TA Nunez SC (1994) Male and female reproductive cycles of the Jamaican lizard Anolis opalinus Copeia 1994 767-780

Lance VA (1998) Female reproductive system reptiles In Knobil E Neill JD editors Encyclopedia of Reproduction New York NY USA Academic Press pp 239-243

Lee JC Clayton D Eisenstein S Perez I (1989) The reproductive cycle of Anolis sagrei in southern Florida Copeia 1989 930-937

Laohachinda W (2009) Herpetology Bangkok Thailand Kasetsart University Press

Licht P (1984) Seasonal cycles in reptilian reproductive physiology In Lamming GE editor Marshallrsquos Physiology of Reproduction New York NY USA Churchill-Livingstone

Licht P Gorman GC (1970) Reproductive and fat cycles in Caribbean Anolis lizards Univ Calif Publ Zool 95 1-52

Lofts B (1977) Patterns of spermatogenesis and steroidogenesis in male reptiles In Calaby JH Tyndale-Biscoe CH editors Reproduction and Evolution Canberra Australia Australian Academic Science pp 127-136

Lovern MB Holmes MM Wade J (2004) The green anole (Anolis carolinensis) a reptilian model for laboratory studies of reproductive morphology and behavior ILAR J 45 54-64

Lovern MB (2011) Hormones and reproductive cycles in lizards In Norris DO Lopez KH editors Hormones and Reproduction of Vertebrates Vol 3 Reptiles New York Academic Press pp 321-353

Masson GR Guillette LJ Jr (1987) Changes in oviductal vascularity during the reproductive cycle of three oviparous lizards (Eumeces obsoletus Sceloporus undulatus and Crotaphytus collaris) J Reprod Fertil 80 361-371

Maurizii MG Alibardi L Taddei C (2004) Alpha-tubulin and acetylated alpha-tubulin during ovarian follicle differentiation in the lizard Podarcis sicula Raf J Exp Zool 301 532-541

Mendez-De La Cruz FR Villagran-Santa Cruz M Cuellar O (1994) Geographic variation of spermatogenesis in the Mexican viviparous lizard Sceloporus mucronatus Biogeographica 70 59-67

Menezes VA Rocha CFD Dutra GF (2004) Reproductive ecology of the parthenogenetic whiptail lizard Cnemidophorus nativo in a Brazilian restinga habitat J Herpetol 38 280-282

Moreira PL Birkhead TR (2003) Copulatory plugs in the Iberian rock lizard do not prevent insemination by rival males Funct Ecol 17 796-802

Norris DO (2013) Vertebrate Endocrinology 5th ed Tokyo Japan Elsevier Academic Press

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