1

2

Seasonality of female reproductive cycles in the Neotropical Aquatic Snake, 3

Helicops pastazae 4

5

Daniela García-Cobos 1, Andrew J. Crawford 1, Martha Patricia Ramírez-Pinilla2 6

1. Museo de Historia Natural ANDES, Department of Biological Sciences, Universidad de los 7

Andes, Bogotá, Código Postal 111711, Colombia. 8

2 Laboratorio de Biología Reproductiva de Vertebrados, Escuela de Biología, Universidad 9

Industrial de Santander, Bucaramanga, Colombia. 10

11

Abstract 12 13

Reproductive cycles of snakes have a great variability in timings and may be influenced 14

by biotic or abiotic factors. For the Neotropical region, where seasonal variation in temperature 15

is minimum, we hypothesize that the reproductive cycle of snakes is mostly influenced on the 16

precipitation pattern of the region. The reproductive cycle of the colubrid aquatic snake, 17

Helicops pastazae, was studied through monthly examination of gonadal dissections. We 18

investigated female population in a mountainous region of Colombia with seasonal precipitation 19

patterns. Other aspects of the reproductive biology such as sexual dimorphism, sexual size of 20

maturity, clutch size, and litter size was also investigated. Males and females were found during 21

all the year, with a higher encounter during the dry season. Recruitment of neonates was during 22

the low rainfall period and the dry season. Sexual dimorphism was recorded in males with larger 23

tails than females, and females with a wider midbody width than males. There was no significant 24

difference between head length and head width among both sexes. Females have a discontinuous 25

seasonal synchronic population, with a peak in reproductive activity during the period of high 26

rainfall and ovipositioning of eggs occurring during the low rainfalls or dry season. We suggest 27

the reproductive activity of the population studied is highly influenced by the rainfall patterns of 28

the region. 29

30

Introduction 31

The reproductive phenology of snakes can be influenced by biotic and abiotic factors, due 32

to their ectothermic physiology (Siegel & Fitch, 1985). Abiotic factors such as temperature, 33

photoperiod, and precipitation affect the microenvironment where incubation takes place (i.e., 34

humidity of the substrate in oviparous species) and may determine annual reproductive cycles 35

(Brown & Shine, 2006). On the other hand, biotic factors such as food availability for the mother 36

and young, as well as predation risk to eggs and juveniles can also influence these cycles 37

(Andrén & Nilson, 1983; Brown & Shine, 2006). The majority of studies on the reproductive 38

biology of snakes have focused on species in temperate regions, which exhibit very strong 39

seasonality, restricting the reproduction activity to the warmer seasons of the year (Mathies, 40

2011). The reproductive biology of tropical snakes remains poorly studied, however, despite the 41

very high diversity of species found in this region (Pizzatto et al., 2008). 42

The most common temporal patterns of reproductive cycles in tropical snakes are: 1) 43

discontinuous cyclical, the gonads become reproductively quiescent for some period during the 44

year; 2) continuous cyclical, the gonads do not become completely quiescent, but show reduced 45

activity for some period of the year; 3) acyclical, gonads exhibit essentially constant levels of 46

activity throughout the year (Mathies, 2011). The first two can be considered as seasonal or 47

aseasonal reproduction at a population level. A seasonal reproduction involves synchrony in the 48

reproductive cycles among the individuals, contrary to the aseasonal reproduction which 49

indicates asycnchrony in the reproductive cycles among the individuals. The acyclical cycle is 50

considered as aseasonal reproduction (Mathies, 2011). 51

In the tropical region, independent if species have a period of gonadal quiescence, one 52

can find population of snakes with seasonal or aseaonsal reproduction for both males and 53

females. For example, seasonal reproduction is present in some female populations of 54

Drymarchon corais, Chironius flavolineatus, Chironius bicarinatus and Helicops leopardinus; 55

and in males and females of Phylodryas patagonenesis (Avila et., 2006; Loebens et al., 2017; 56

Marques et al., 2010; Prudente et al., 2014). The lack of seasonality appears in other tropical 57

populations of snakes such as Clelia plumbea, Oxyrhopus guibei, Atractus sp. and most species 58

belonging to the Xenodontini Tribe (Pizzato & Marques, 2002; Pizzatto, 2005;Pizzatto et al., 59

2008; Ramirez-Pinilla et al., 2017). The factors responsible for seasonal phenology in tropical 60

regions are not clear, since the tropics have complex geographic variation in climate in terms of 61

precipitation, but no marked seasonality in temperature or photoperiod compared to temperate 62

zones. 63

Colombia is a Neotropical country characterized by a remarkable variety of biomes, from 64

the everwet Chocó to the Guajira desert, plus three distinct chains of the Andean mountains, 65

resulting in an extremely high diversity of flora, fauna, and climates (Kattan et al. , 2004). 66

Regardless of elevation, temperature shows little annual variation, making precipitation the main 67

climatic factor showing seasonality. The precipitation patterns could be an important factor 68

determining the reproductive activity of tropical snakes, whether through direct, abiotic effects 69

on snake reproduction, or by affecting other biotic factors such as predators or prey. 70

Furthermore, the preliminary observations (cited above) that some tropical snakes show 71

seasonality while others do not, begs the question as to what drives seasonality in some snakes. 72

The South American snake genus, Helicops, contains 17 described species of aquatic 73

snakes with diverse ecological aspects in aquatic habitats (ranging from rivers to ponds), food 74

preference (fish, anurans, lizards) and reproductive modes. The genus includes both oviparous 75

and viviparous species (Scartozzoni, 2009). Although some species of Helicops seem to have 76

large population sizes (Avila et al., 2006) there are various species with no studies on 77

reproductive biology such as in Helicops pastazae, commonly called “mapana de agua” (Braz et 78

al., 2016). This species inhabits the the rivers of the foothills of the Cordillera Oriental of the 79

Colombian Andes. During the rainy season, these can rise tremendously during storm events, 80

potentially transforming the riparian habitat occupied by aquatic snakes. Thus, our hypothesis is 81

that given the rainfall pattern of its habitat, the reproductive phenology of H. pastazae females 82

may be markedly seasonal. In this study, first we attempt to determine the reproductive cycles of 83

females of a population of H. pastazae using both macroscopic morphology and morphometric 84

analyses of the reproductive tracts. Secondly, to test our hypothesis, we correlated the 85

reproductive cycle of the females with the rainfall patterns of this region to test this abiotic factor 86

as a principle cause of reproductive seasonality. Finally, we describe other unknown aspects of 87

the reproductive biology of the species, including sexual dimorphism, minimum size of sexual 88

maturation, gestation times, clutch size and litter. 89

90

Methods 91

Ethics statement: 92

All of the procedures for sacrificing and manipulation the individuals were approved (August 23, 93

2017) by the institutional Committee on the Care and Use of Laboratory Animals (CICUAL). 94

The individuals were collected under the permit: “Permiso Marco de Recolección de 95

Especímenes de Especies Silvestres de la Diversidad Biológica con Fines de Investigación No 96

Comercial”, resolución No 1177 to the Universidad de Los Andes, Certificated under the 97

investigation project “Diversidad criptica de anfibios y reptiles de Colombia” radicado No 98

2016057539-1-000; 20166058570-1-001; 2017015173-1-000; 2017015174-1-000; 2017055251-99

1-000; 100

101

Study site 102

All the individuals included in this study correspond to a population inhabiting the Bata 103

river located in the municipality of Santa María, Boyacá, Colombia (4.8576944, -73.26538). Our 104

study site in Santa María was located at approximately 800 meters above sea level (masl) in the 105

foothills of the Cordillera Oriental of the Colombian Andes. The sampling area experiences 106

rainfall throughout the year with a peak between April and November, and a drier period during 107

December to March. Based on six year of data of monthly precipitation, we defined the 108

following seasons during the year: dry season between January and March (mean 125 mm) high 109

rainfall period from April to July (mean 520.9 mm), and low rainfall period from August to 110

December (mean 359 mm). 111

112

Field sampling 113

We searched for individuals of Helicops pastazae (Shreve, 1934) resting under rocks 114

during the day or foraging at night in the still waters of the Bata River. We examined 43 115

specimens deposited on the reptile collection (ANDES-R) of the Museo de Historia Natural, 116

Universidad de los Andes, Bogota, Colombia that were collected during 2014-2015. In addition, 117

a total of 177 individuals were collected during fieldwork performed every month from August 118

2016 to July 2017. After six months of sampling (February through July 2017), juveniles and 119

neonates were no longer sacrificed, instead we recorded the observation along with digital 120

photographs including a ruler as scale. 121

Captured individuals were euthanized through a cardiac injection of 2% xilocaine 122

followed by tissue extraction form the liver and posteriorly were fixed in buffered formaldehyde 123

10%, according to the protocols of Simmons (1987). 124

125

Laboratory procedures 126

The sex of the individuals was determined based on dissections of the reproductive tracts, 127

which were then stored in ethanol 70% for posterior measurements. The snout-vent length (SVL) 128

size of the smallest female with vitellogenic II follicles or oviductal eggs was used to define the 129

minimum size of sexual maturation, defining juveniles as individuals beneath this size. 130

Newborns (> 200 SVL) were defined as those individuals with umbilical scar. For each mature 131

female we recorded the reproductive stage according to the presence of oviductal eggs or the 132

diameter of the largest follicle and its coloration. The reproductive stages were categorized as 133

follows: (1) previtellogenic (whitish, transparent follicles with a diameter less than 4 mm), (2) 134

vitellogenic I (yellowish follicles with a diameter between 4 and 15 mm), (3) vitellogenic II 135

(yellowish follicles with a diameter greater than 15 mm) and (4) Gravid (oviductal eggs). Also, 136

to determine the approximate clutch size and its correlation with SVL, we counted oviductal 137

eggs and vitellogenic II follicles. The minimum size of newborns was established by reviewing 138

the size of neonates, and the size of hatchings from a clutch of eggs found in the field and 139

posteriorly kept in captivity until eclosion. 140

In order to establish if adults and juveniles exhibited sexual dimorphism, we took the 141

following measurements from all individuals: snout-vent length (SVL), tail length (TL), head 142

length (HL), head width (HW) and mid-body width (MBW) (Richard Shine, 1991, 1994a). Head 143

measurements were performed using a caliper, while the snout-vent length (SVL) and tail length 144

(TL) were taken using a string stretched along the ventral side of the preserved specimen which 145

was posteriorly measured with a ruler (Rivas, 2000). 146

147

Statistical analysis 148

We testes normality of the follicular diameter of females using Shapiro- Wilks, however 149

were not able to transform this data (log transformation) to be consistent with the assumptions of 150

normality and homogeneity of variances, so we did not use parametric tests. We performed a chi-151

square to evaluate significant differences in the frequency of females, males and neonates in each 152

season. We performed a Spearman rank correlation test between the follicular diameter and SVL 153

of the females to evaluate correlation among these variables. To evaluate seasonality in the 154

reproductive activity, we used two statistical approaches. First we evaluated seasonal variation in 155

the follicular diameter using a Kruskal-Wallis test. Posteriorly, we performed a post hoc paired 156

Mann-Whitney test to determine which seasons were significantly different in mean diameter of 157

eggs or follicles. The second method applied was a chi-square test to evaluate differences in the 158

occurrence and distribution of the reproductive stages of females among the seasons. 159

The relationship between the SVL and the number of oviductal eggs or vitellogenic II 160

follicles was evaluated using regression with Poisson error terms. To test for sexual dimorphism, 161

we used an analysis of covariance (ANCOVA) for the four variables measured HL, HW, MBW, 162

and TL, relative to sex (categorical variable) with SVL as the independent variable. Analyses 163

were performed with R software version 3.4.2 with a p < 0.05 as the criterion for significance. 164

165

Results 166

Recruitment of individuals 167

Helicops pastazae was very abundant in resting position under the rocks of the river. A 168

total number of 221 individuals were collected during 2013 to 2017. Males and females 169

exceeding the size of neonates (>200 mm) were captured during all the twelve months of the 170

year, however we found a higher occurrence of both sexes during the period of low rainfall 171

(males: X2=7.80, df=2, n= 71, P= 0.02 ; females: X2= 20.89, df=2, n= 111, P< 0.0001). We also 172

found a higher recruitment of neonates at the end of the low rainfall period and during the dry 173

season, compared to the period of high rainfall (Fig 1) (X2 =13.35, df=2, n=37, P=0.0012) (Fig 174

1). 175

176

Body Size and Sexual Dimorphism 177

Females were markedly larger than males in SVL. The largest female measured 715 mm 178

SVL, while the largest male found was 446 mm SVL. Only two of the four morphological 179

variables measured, showed a significant difference between males and females: TL and MBW 180

(Fig 2). While females where larger in SVL than males, males had significantly longer tails than 181

females (F0.5, 208= 2374, R2= 0.97, n= 209, P < 0.001) (Fig 2A). Female also present a wider 182

midbody width than males (F0.5, 208= 964, R2= 0.93, n= 209, P < 0.001) (Fig 2B), however HL 183

and HW did not differ between sexes (HW: F0.5, 208= 727.1, R2= 0.96, n= 209, P =0.45; HL: F0.5, 184

208=1259, R2= 0.94, n= 209, P =0.41) (Fig 2C, D). 185

186

Females reproductive activity 187

The size of sexual maturity of females was estimated at 402 mm SVL, i.e., the length of 188

the smallest adult female with oviductal eggs. We collected a total of 142 females distributed as 189

70 adults (50%), 41 juveniles (29%) and 31 neonates (21%) (Fig 3). Reproductive status of adult 190

females was distributed as 32% previtellogenic, 32% vitellogenic I, 22% vitellogenic II and 14% 191

gravid. Gravid females were never found to simultaneously have vitellogenic II follicles. 192

Females in different reproductive stages were found during the same season (i.e., 193

previtellogenic and vitellogenic I females during the dry season (non-reproductive adult 194

females), vitellogenic I and vitellogenic II females during the period of high rainfall and all four 195

reproductive stages during the period of low rainfall (Fig 3, 4). The reproductive stages of the 196

females do not have an equitable distribution during the seasons (Fig 3). The prevalence and 197

occurrence of the reproductive stages of females is as follows: previtellogenic females found 198

principally during the dry season (January to March) (X2 =11.27, df = 2 P =0 .0035), 199

vitellogenic I females distributed during the period of high rainfall (April to July) with more than 200

60% of occurrence (X2 =18.08, df = 2, P < 0.001), and all gravid females were clustered during 201

the low rainfall period (August to December) (X2 = 20, df = 2, P < 0.001) (Fig 3). Vitellogenic II 202

females were evenly found during the periods of high and low rainfall (X2 =7.6, df = 2, P < 203

0.02), however none occurred during the dry season (Fig 3). 204

The snout-vent length of females was not correlated with the follicular diameter 205

(correlation coefficient = 0.18, P= 0.124). A significant difference in the follicular diameter 206

among months was found (Kruskal-Wallis, H= 41.02, df= 11, P < 0.0001), between the dry 207

season and period of high rainfall (Mann-Whitney, W= 396, P < 0.001) and among the dry 208

season and low rainfall period (Mann-Whitney, W=346, P < 0.001) (Fig 4). No statistical 209

difference was observed in the variation of follicular diameter between the high and low rainfall 210

period (Mann-Whitney, W=284, P = 0.24) (Fig 4). 211

Clutch size ranged from three to eighteen eggs (mean egg size= 29.6 ± 5.3, 14.3 ± 4.7 212

mm, n=36). The number of oviductal eggs was correlated with SVL (Spearman’s r = 0.80, P= 213

0.005, n = 10), however there was no significant correlation between the number of vitellogenic 214

II follicles and female body size measured as SVL (Spearman’s r = 0.344, P= 0.208, n = 15) (Fig 215

5). A clutch of eggs found in the field on December 20, 2016 was transported to the laboratory 216

and kept in captivity until the neonates were born on February 8, 2017. Neonates (n=7) born in 217

captivity (5 females and 2 males) averaged 122 mm SVL (± 10.7 mm). 218

219

Discussion 220

The frequency of males, females and neonates did not have a homogenous distribution 221

during every month of the year, indicating that the encounter rate may vary between the seasons 222

sampled. We collected males and females (>200 mm) during every month of the year, however 223

the occurrence was higher during the period of low rainfalls compared to the period of high 224

rainfall. Although no studies of habitat use of this specie have been performed, our higher 225

encounter rate during the low rainfall season may be influenced by the increase number of places 226

where the individuals may hide during the flooding of the river, making them more difficult to 227

find. However, studies on movement patterns and population abundance must be performed in 228

order to establish this hypothesis. 229

The evolution and maintenance of sexual dimorphism may be driven by sexual or natural 230

selection pressures such as: 1) intraspecific male-male competition 2) alternative reproductive 231

strategies in females to increase fecundity, and 3) reduction in intraspecific competition for food 232

resources (Fitch, 1981). Male-male combat is associated with males larger than females in a 233

given population (Shine, 1994b). However, mate competition among males can also take more 234

subtle forms, such as tail wrestling during courtship, as males search for a better grasp while 235

copulating with females (Shine, 1999). This wrestling behavior may impose a selective 236

advantage for males with longer tails. This morphological trait may also generate more space for 237

larger copulatory organs called hemipenes in squamates (Shine et al., 1999). The longer tails of 238

males of H. pastazae compared to females could suggest that males of this species may present 239

tail wrestling during copulation, though we have little data to test this hypothesis. We found only 240

one mating event in which two males and one female were exhibiting copulatory behavior under 241

a rock in the field (April 25, 2017). The presence of more than one male during the event, 242

however, implies that opportunities for this tail wrestling behavior exist in nature. 243

Females of H. pastazae were significantly larger in body size (SVL) than males. Sexual 244

dimorphism in body size is common in snakes and could be driven by fecundity selection on 245

females to increase clutch size (Shine, 1994b). Here we showed that, indeed, clutch size was 246

significantly and positively correlated with females SVL in our study population of H. pasaztae. 247

On the other hand, smaller SVL in males is also associated with improved locomotor 248

performance in other snakes, especially among species where male show more active foraging 249

strategies (Shine & Shetty, 2001). For example, in the aquatic snake, Acrochordus arafurae, 250

females are more sedentary and act as ambush predators, while males are active searchers 251

(Vincent et al., 2005). Different foraging behavior could cause intraspecific dietary divergence as 252

has been seen in various aquatic snakes such as American water snakes (Natricinae), file snakes 253

(Achrochordidae) and marine elapids (Shetty & Shine, 2002). Larger females (SVL) and dietary 254

divergence between sexes can drive head size dimorphism, as females can ingest larger prey 255

items than males, resulting in having larger jaws (Shetty & Shine, 2002). However, we did not 256

find this type of sexual dimorphism in the HL and HD of H. pastazae, relative to body size. 257

The reproductive cycles of females of H. pastazae are highly seasonal, and we proposed 258

that this seasonality is driven by local rainfall patterns. From April to July, the period of high 259

rainfall, females present a peak in reproductive activity, as measured by the development of 260

vitellogenic follicles. Moreover, gravid females (with oviductal eggs) were found during the low 261

rainfall period from August to December, suggesting that oviposition occurs during the dry 262

season (January to March) that follows. After ovipositing their eggs, females are found with 263

previtellogenic follicles as they enter into a period of quiescence and reproductive inactivity. As 264

all females showed this quiescence, our data demonstrate a discontinuous reproductive cycle at 265

the individual level and for the population as a whole. Furthermore, the prevalence of distinct 266

reproductive stages of females in certain seasons (i.e., previtellogenic during dry season, 267

vitellogenic I in the period of high rainfall, and gravid during the period of low rainfall; Fig 3), 268

defines this population as synchronic in its reproduction. The previtellogenic females found 269

simultaneously with vitellogenic II or gravid females, were post-gravid (post- gravid (distended 270

uterus and occurrence of corpus luteum). Therefore, according to the classification of Mathies 271

(2011), H. pastazae has a discontinuous seasonal synchronic reproductive cycle. 272

The chronology of the vitellogenisis cycle indicates that an increase in precipitation may 273

be the factor that triggers the start of reproductive activity. This is evidenced as females start to 274

increase their follicular diameter as the monthly precipitation increases (Fig 4). It is unlikely that 275

variation in other abiotic factors such as annual temperature, influences the phenology of this 276

population of snakes, since the temperature regime in the neotropics is uniform at a monthly 277

basis (Janzen, 1967). Although a decrease in the temperature may occur during the night in this 278

mountainous region, this is a constant factor during the whole year that should not directly affect 279

the reproductive cycles of the species. 280

The flood pulse of the rivers may be a main factor preventing aquatic snakes from 281

reproducing year round. Females of the viviparous species, H. leopardinus, also show seasonal 282

reproduction, similar to that reported here for H. pastazae, however, with parturition of the 283

newborns occurring late in the rainy season (Avila et al., 2006). However, another viviparous 284

species of Helicops, H. infrataeniatus, does not show marked seasonality in the reproductive 285

cycle since it has embryos during nine months (Aguiar & Di-Bernardo, 2005), compared to H. 286

pastazae, which has embryos only four months of the year. In the aquatic boid (Eunectes 287

murinus) in the Venezuelan llanos, the recruitment of newborn is observed during the dry season 288

(Rivas, 2000). In oviparous species, egg-laying during the dry season may match optimal timing 289

for food availability for hatchlings at the beginning of the wet season (Brooks et al., 2009), and 290

can optimize the embryonic development of the clutch (Marques et al., 2006). The only clutch of 291

eggs of H. pastazae recorded in the field was found under a large rock during the dry season 292

(December 21, 2017), approximately two meters away from the river. If females of this 293

population lay the eggs during the period of high rainfall, the increase water levels of the river 294

could wash away or drown the eggs. 295

This study of the reproductive cycle of females enlightens other aspects of the 296

reproductive biology not reported previously. The size of sexual maturity (SVL =400 mm) in the 297

studied population is larger than that reported for H. infraenatus (SVL= 353 mm) (Aguiar et al., 298

2005). Clutch size ranged from three to eighteen eggs and was highly correlated with SVL. The 299

number of vitellogenic II follicles varied from one to nineteen and did not correlate to the SVL 300

of females. This absence of correlation may be explained by the presence of two large females 301

that were probably just entering the cycle of vitelogenesis II. The occurrence of previtellogenic 302

females (period of quiescence in the reproduction), coupled with the fact that we never found a 303

female containing simultaneously vitellogenic II follicles and oviductal eggs, means that a 304

maximum of one clutch is produced during the year. Also, given that gravid females where 305

found only from August to December, we approximate that gestation may last around four 306

months. 307

In conclusion, our study indicates that females of the Santa María, Boyacá population 308

display discontinuous seasonal reproductive cycle that correlates with the rainfall regimen of the 309

region. While many biotic and abiotic favors vary seasonally, we hypothesize that aquatic snakes 310

often lay their eggs when seasonal precipitation is low in order to guarantee the survival of the 311

newborn due to the direct, abiotic effects of the microenviroment during incubation, or due to 312

indirect, biotic effects of resource availability. Understanding components of the natural history 313

such as the reproductive phenology of the species is extremely important in establishing 314

conservation strategies, as it informs us the seasons of recruitment and vulnerability of a 315

population. 316

317

Acknowledgements 318

We thank John D. Lynch and Diego Gómez Sanchez for developing some of the initial 319

ideas for this study. We thank Diego Gómez Sanchez, and members of the snake group from 320

Museo de Historia Natural ANDES for the enthusiasm and substantial help during fieldwork. 321

The Institutional Committee on the animal care and Use of Laboratory Animals (CICUAL) of 322

the Universidad de los Andes for the advice and approval of procedures the performed. The 323

Autoridad Nacional de Licencias Ambientales (ANLA) de Colombia for approval of collecting 324

permits (see ethic statements). 325

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412

413

Figures: 414 415 Figure 1: 416

417 418

Fig 1: Monthly distribution of individuals found during the months sampled. White bars: 419

females. Gray bars: males. Black bars: neonates. 420

Figure 2: 421

422 423 Fig 2: Sexual dimorphisms in four morphological variables: tail length, midbody width, head 424

length and head width. Black circles: Females. Open circles: Males. Asterisks: variables that are 425

significantly different between males and females 426

427

428

429

430

431

432

433

434 Figure3: 435

436 Fig 3: Monthly distribution and sizes (SVL) of females in different stages of maturity. Black 437

circles: adults. White triangles: juveniles. Gray squares: neonates. The arrow indicate the 438

minimum size at sexual maturity for females 439

440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455

456 Figure 4: 457

458

459

Fig 4: Seasonal and monthly occurrence of females in different reproductive stages. Light gray 460

bars: previtellogenic. Dark gray bars: vitellogenic I. White bars: vitellogenic II. Black bars: 461

gravid. One asterisk (*) indicates copulatory events, two asterisk (**) clutch of eggs found 462

463 464 465 466 467 468 469 470 471 472 473 474 475

476 477 478 479 Figure 5: 480

481 482 Fig 5. Seasonal and monthly variation in the follicular diameter. Grey triangles: Previtellogenic 483

follicles. Squares: Vitellogenic I follicles. Black circles: Vitellogenic II follicles. Open circles: 484

Females with oviductal eggs. Slash line represents the annual precipitation. Asterisks: season that 485

differed significantly in the follicular diameter. 486

Figure 6 487

488 Fig 6: Correlation of number of oviductal eggs or vitellogenic II follicles with the SVL of the 489

females. Black circles: oviductal eggs. Open circles: Vitellogenic II follicles. 490