Reproduction of Snake-eyed Skink Ablepharus kitaibelii (Bibron & Bory de Saint-Vincent, 1833) (Squamata: Scincidae) in Bulgaria

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1 Reproductive and Developmental Biology Research Article ACTA ZOOLOGICA BULGARICA Acta zool. bulg., 70 (4), 2018: Reproduction of Snake-eyed Skink Ablepharus kitaibelii (Bibron & Bory de Saint-Vincent, 1833) (Squamata: Scincidae) in Bulgaria Vladislav S. Vergilov 1, Victoria G. Necheva 2 & Boyan P. Zlatkov 1,3 1 National Museum of Natural History, Bulgarian Academy of Sciences, 1 Tsar Osvoboditel Blvd., 1000 Sofia, Bulgaria; vladislav8807@gmail.com 2 Faculty of Biology, Sofia University St. Kliment Ohridski, 8 Dragan Tsankov Blvd., 1164 Sofia, Bulgaria; v.necheva@abv.bg 3 Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 1 Tsar Osvoboditel Blvd., 1000 Sofia, Bulgaria; bzlatkov@gmail.com Abstract: Currently, information on the reproductive cycle of snake-eyed skink Ablepharus kitaibelii (Bibron & Bory de Saint-Vincent, 1833) in Bulgaria is lacking, though some data on egg laying were published. We performed a histological study on gametogenesis and incubation period of eggs of this species. The histology of testes demonstrated that males reached sexual maturity at snout-vent length (SVL) of 39 mm; for females, the result was not conclusive. The male reproductive cycle had four phases: quiescence in July, recrudescence in August, spermiogenesis in September October and March May and regression in June. Four phases were detected also in the females: quiescence in July October, vitellogenesis in April and May with early vitellogenesis in April, pre-ovulatory follicles in May and fertilised eggs in June. Results also revealed that females produce one clutch of eggs per year and could lay up to five eggs (usually four) in a couple of days. The species had a prolonged egg laying period: from the beginning of June to the beginning of August. The egg incubation period at room temperature was between 26 and 42 days. Considering the short lifespan of the species, A. kitaibelii reaches sexual maturity relatively early. Key words: gonads, skink, reproductive cycle, spermatogenesis, oogenesis, incubation Introduction Although there are a number of studies on the life cycle, sexual size dimorphism and reproduction in different members of the family Scincidae, data concerning spermato- and oogenesis (gametogenesis) are very few. Information on the onset of sexual maturity in family representatives is available in articles that have explored the life cycle of different species but the determination of the onset itself is not supported by histological studies of gonads, which is one of the reliable methods of establishing it (Méndez de la Cruz et al. 2014). The onset of sexual maturity in most literature sources is determined mainly using the degree of development and growth of the gonads of dissected specimens (Robertson et al. 1965, Simbotwe 1980, Vitt & Blackburn 1983, 1991, Vitt & Cooper 1986, Huang 1996, Du & Ji 2001, Taylor 2004, Clemann et al. 2004, Greenville & Dickman 2005, Vrcibradic & Rocha 2005, Ji et al. 2006, Du et al. 2012). Histology has been used to obtain more accurate results when studying the gonadal development in skinks (e.g. Towns 1975, Zug et al. 1982, Guillette 1983, Traut 1994, Vrcibradic & Rocha 2002, Goldberg 2008, 2013, Goldberg & Kraus 2010, 2012, Goldberg & Austin 2012, Sun et al. 2012, Nassar et al. 2013). Göcmen et al. (1996) provided some data on gonadal development of the congener Ablepharus 507

2 Vergilov V. S., V. G. Necheva & B. P. Zlatkov budaki Göcmen, Kumlutas & Tosunoglu, 1996 based on dissected specimens. There is only one study on the gonadal histology of A. kitaibelii but the author (Goldberg 2012) has misidentified the species. In fact, it is another representative of the genus, A. rueppellii (Gray, 1839), from Israel. Although congeners, the two skink species occupy habitats with considerably different temperature and precipitation, thus one can expect variances in the timing and duration of their reproductive cycles. The purpose of our study is to reveal some aspects of the reproduction in A. kitaibelii: the onset of sexual maturity via histology, the incubation period, egg laying, and the main events during the annual reproductive cycles. Materials and Methods For the study of gonadal development, 46 individuals (25 mature males, 18 mature females and three juvenile males; Appendix) from three localities (UTM grid squares km, zones 34T and 35T) in Bulgaria were collected: (1) Hills above Pancharevo, Sofia Region (FN91); (2) near Montana Town (FP70) and (3) the hills over Balsha Village (FN84), Sofia Region. The individuals were captured between March and October. They are deposited in the zoological collection of the Faculty of Biology (BFUS), Sofia University St. Kliment Ohridski. The snout-vent length (SVL) was measured with a plastic ruler for each specimen. Additionally, 226 specimens from the National Museum of Natural History, Bulgarian Academy of Sciences (numbers from NMNHS101 to NMNHS284 and from NMNH287 to NMNH330) were dissected to establish their sex and potential presence of eggs. The lizards were euthanized through deep inhalation with diethyl ether and dissected. Both left and right gonads were extracted and fixed with Bouin s fluid for at least 24 hours and then washed with tap water. Then the gonads were dehydrated with ethanol, cleared with xylene, embedded in paraffin and sectioned at 5 μm thickness. The sections were deparaffinised, rehydrated and stained with Harris haematoxylin and mixture of Eosin and Phloxin (10:1), then sealed with Canada balsam. The observation and photography were performed with an Amplival compound microscope (Carl Zeiss Jena) equipped with plan achromatic objectives, compensating projective and digital camera Canon EOS 700D. The incubation period of the eggs was established using gravid females specimens held in captivity in separate plastic boxes of ml (ultimately released at the point of capture). Eggs were laid by specimens originating from four localities in Bulgaria: Belovets Village (MJ45), Pchelin Village (MH84), Razdel Village (MG75) and from the tourist road Brodilovo Ahtopol (NG75). After the egg laying, each egg was carefully buried in a plastic box (with volume of 150 ml) containing soil or coconut substrate (with height of 4 5 cm) and incubated at room temperature (between 23 and 26 C). The substrate was moistened periodically with water. Thirty measurements of the diameter of the seminiferous tubules and the height of the epithelium of each specimen were performed with a compound microscope. Statistical analyses were performed with Statistica ver. 7.0 (StatSoft, Inc.) and graphs were produced using Photoshop CC ver (Adobe Sys. Inc.). Results Male reproductive cycle Similar to other amniotes, reptiles have tubular testes with permanent cell population of Sertoli cells and stable hemato-testicular barrier (Gribbins 2011). The Sertoli cells had typical pyramidal shape Fig. 1. Germ cell types during the spermatogenesis in A. kitaibelii. 1 spermatogonium, 2-5 primary spermatocytes: 2 leptotene, 3 zygotene, 4 pachytene, 5 diplotene, 6 metaphase I, 7 metaphase II, 8 round spermatid, 9-13 successive steps of spermatids, 14 spermatozoon. Scale bar: 5 μm. 508

3 Reproduction of Snake-eyed Skink Ablepharus kitaibelii (Bibron & Bory de Saint-Vincent, 1833) in Bulgaria Fig. 2. Histological sections of testes of A. kitaibelii stained with Harris haematoxylin and mixture of Eosin and Phloxin, 5 μm thickness. A General view of a testis. B Immature specimen (SVL of 36 mm). C The smallest reproductive specimen (SVL of 39 mm) in June in regression phase (reduced height of the germinative epithelium). D Quiescence in July. The diameter of the seminiferous tubule is considerably smaller, the lumen is reduced. E Recrudescence in August, note the large lumen with numerous round spermatids. F Ongoing spermiogenesis in October, large diameter of the seminiferous tubules. 1 lumen, 2 seminiferous tubules, 3 interstitial tissue, 4 Sertoli cells, 5 primary spermatocytes, 6 round spermatids, 7, 8 later spermatids, 9 spermatozoa. Scale bars: A 200 μm, B F 20 μm. with large nucleus and centrally located nucleolus. The spermatogonia had large nuclei with diffuse chromatin (Fig. 1). Although Gribbins (2011) reports three types of spermatogonia, they are not easily distinguished with light microscopy, therefore we did not separate them. Spermatogonia were observed in all specimens. Leptotene and zygotene spermatocytes had intensively stained chromatin and visible chromosomes. They can be distinguished by the asymmetrical location of the zygotene chromosomes in the nucleus. Leptotene spermatocytes were especially numerous in immature individuals. Pachytene spermatocytes had very thick chromosomal fibres easily visible under light microscope. Diplotene spermatocytes were with large nuclei with peripherally located condensed chromosomes. As the diplotene phase in reptiles is short (Gribbins 2011), they were the least numerous prophase sper509

4 Vergilov V. S., V. G. Necheva & B. P. Zlatkov Fig. 3. Annual changes in seminiferous tubules diameter (D) and epithelium height (H) of A. kitaibelii. Cross-sections of seminiferous tubules from each phase are shown, all to scale. Phases: I quiescence, II recrudescence, III spermiogenesis, IV regression. matocyte type. Metaphase, anaphase and telophase progress very rapidly and with the exception of metaphase are hard to differentiate between one another. Secondary spermatocytes are short-living in reptiles (Gribbins 2011) and were not differentiated in our study. Metaphase I cells could be distinguished from the metaphase II cells by the size of the heterochromatin mass. After the reduction division, several steps of spermiogenesis were recognised based entirely on the morphology of spermatids (Fig. 1). The first step spermatid had spherical nucleus with diffuse chromatin and it is called round spermatid (Gribbins 2011). We considered this step as an early one. It appeared to be the most persistent spermatid step, present even in the quiescence phase. The second step was very similar to the first one but an indentation of the nucleus was present. In the next step the chromatin was more condensed and the nucleus was crescent-shaped, with visible acrosome granule. Later on, the spermatid had small Fig. 4. Changes in follicles and oocytes during oogenesis. A Oocytes in phase I (pre-vitellogenesis). The granulosa layer is thick and polymorphic. The zona pellucida is homogenous. B Oocyte (right) in phase II (early vitellogenesis). The granulosa layer is monomorphic, with reduced thickness, theca is well developed and larger yolk granules are visible in the ooplasm. The zona pellucida is with well separated hyaline band and zona radiata. C Pre-ovulatory follicle (phase III) at the last stage of vitellogenesis (bottom) sectioned near the vegetal pole. The granulosa is a single layer of small monomorphic cells (squamous). The ooplasm is occupied by yolk platelets and lipid droplets. D Shell of an egg with developing embryo in oviduct. 1 oocyte nucleus, 2 ooplasm, 3 homogenous zona pellucida, 4 granulosa cells: 4a large pyriform cells, 4b intermediate cells, 4c small basal cells, 4d small apical cells, 5 theca, 6 zona pellucida with hyaline band and zona radiata, 7 yolk platelets, 8 lipid droplets, 9 eggshell membrane, 10 eggshell, 11 oviduct wall. Scale bars: 20 μm. 510

5 Reproduction of Snake-eyed Skink Ablepharus kitaibelii (Bibron & Bory de Saint-Vincent, 1833) in Bulgaria Fig. 5. Histological sections of ovaries of A. kitaibelii in different phases stained with Harris haematoxylin and mixture of Eosin and Phloxin, 5 μm thickness. A Phase I follicles (quiescence) in October. B Ovary with phase III (left, late vitellogenesis) and several phase I follicles in May. C Embryo in oviductal egg in June. D Ovary with oviductal egg (phase IV) and functioning corpus luteum in June. E Right ovary of a female from July with phase I follicles, one of them atretic. F Left ovary of the same female with atretic follicle and degenerating corpora lutea. 1 epithelium, 2 oocyte nucleus, 3 ooplasm, 4 yolk platelets, 5 neural tube, 6 mesenchyme, 7 optic vesicles, 8 eggshell, 9 corpus luteum, 10 atretic follicle, 11 degenerating corpora lutea. Scale bars: 200 μm. nucleus with condensed chromatin. In the following steps the head of the sperm gradually elongated. At the final step, mature spermatozoa were observed in the lumen. Presence of mature spermatozoa was regarded as a sign of maturity (Fig. 2). In the immature individuals only spermatogonia and primary spermatocytes were present (Fig. 2B). The diameter of seminiferous tubules was small ( , mean 81.49±14.43 μm, n=90). The epithelium height was also small ( , mean 37.90±7.34 μm, n=90). In mature males spermatozoa and all germ cell types were observed from March to May. Spermatids and spermatozoa were especially numerous, the diameter of seminiferous tubules and the height of the epithelium was much larger (e.g. in April the diameter was , mean ±31.86 μm, n=90; the epithelium height was , mean 72.82±12.20 μm, n=90, Fig. 3). In June, ongoing spermiogenesis was observed but the diameter of the tubules was reduced ( , mean ±24.73 μm, n=90), as well as the epithelium height ( , mean 43.48±10.65 μm, n=90). In July, spermatogonia, primary spermatocytes and round spermatids were observed; very few individual spermatozoa were detected (Fig. 2D). The diameter of the seminiferous tubules was minimal ( , 511

6 Vergilov V. S., V. G. Necheva & B. P. Zlatkov Table 1. Descriptive data of SVL (mm) at different gonadal phases in mature male and female individuals Males Females Phase n Mean Min Max SD SE I II III IV I II III IV Fig. 6. Reproductive cycles in males and females of A. kitaibelii based on own data. Copulation period is based on literature data. Phases in males (black lines): I quiescence, II recrudescence, III spermiogenesis, IV regression. Phases in females (grey lines): I quiescence, II early vitellogenesis, III pre-ovulatory (late vitellogenesis), IV oviductal eggs. mean ±36.84 μm, n=90) but the epithelium height was a little bit larger ( , mean 53.16±13.48 μm, n=90; Fig. 3). The number of round spermatids increased rapidly in August and they were the most numerous cell type at that time (Fig. 2E), which was observed in two out of four specimens. The diameter of the tubules increased significantly ( , mean ±55.24 μm, n=120), as well as the epithelium height ( , mean 70.26±18.95 μm, n=120; Fig. 3). In the other two August specimens spermiogenesis has commenced and few spermatozoa have been formed. Similarly to the spring, in September the spermatids and spermatozoa in the lumens were the most numerous cells (Fig. 2F). In this month, the seminiferous tubules diameter and epithelium height reached their maximum size (diameter: , mean ±28.04 μm, n=90; epithelium: , mean 77.89±13.89 μm, n=90). Decrease of the diameter and the epithelium height was observed in October (diameter: , mean ±30.00 μm, n=90; height: , mean 65.99±10.34 μm, n=90). The period of full rest in reptiles is characterised by the presence of spermatogonia and Sertoli cells only (Gribbins 2011). Since the population of primary spermatocytes in A. kitaibelii was always present, full rest of the spermatogenic cycle was never attained. Apparently, the spermatogenic cycle was non-continuous, with ongoing spermiogenesis from March to June and from August to October (Figs. 3, 6). In June, there was a decrease in spermiogenic activity and the height of the germinative epithelium was minimal ( , mean 43.48±10.65 μm, n=90), which was a regression phase. In July, spermatogenetic activity decreased, i.e. this was a resting period (quiescence phase). It was followed by a recrudescence phase in August (Fig. 6; Table 1). Female reproductive cycle Goldberg (2012) outlined four phases in the annual cycle for the females of A. rueppellii: I quiescence, in which there is no yolk deposition (pre-vitellogenic, Lozano 2014), II early yolk deposition, in which vitellogenic granules are accumulating within some follicles (early vitellogenic), III enlarged preovulatory ovarian follicles with yolk platelets (late vitellogenic) and IV oviductal eggs with embryo. This terminology was adopted for the description of the annual cycle of A. kitaibelii (Table 1), which demonstrated considerable similarity. The pre-vitellogenic follicles (phase I) were small with thin theca and more or less homogenous zona pellucida (Fig. 4A). The granulosa consisted of well differentiated cells (Fig. 4A): small (basal and apical) with small nuclei; pyriform cells with large nuclei, central nucleolus and compact heterochromatic clumps and intermediate cells with smaller nuclei, compact nucleoli and more diffuse heterochromatic clumps. This complex granulosa is typical for the follicle wall of Squamata (Guraya 1989). In the larger pre-vitellogenic follicles the two components of the zona pellucida (hyaline band and zona radiata) could be distinguished, though the zona radiata had not reached its maximal development. The early vitellogenic follicles (phase II) were characterised by active deposition of yolk granules. The zona pellucida was thick, with well separated hyaline band and zona radiata (Fig. 4B). The granulosa was squamous, consisting of a single layer of small monomorphic cells, but the theca was thickened. The late vitellogenic (pre-ovulatory, phase III) follicles (Figs. 4C; 5B) were larger than 3 mm in diameter, contain large yolk platelets and lipid droplets. The hyaline band 512

7 Reproduction of Snake-eyed Skink Ablepharus kitaibelii (Bibron & Bory de Saint-Vincent, 1833) in Bulgaria Table 2. Periods of egg laying by each female and number of hatched juveniles Locality female # n eggs Period of laying 1 st Belovets Pchelin Brodilovo Ahtopol 2 nd rd st nd rd st 5 2 nd 5 n juv. hatched Period of hatching Razdel 1 st of zona pellucida was very thin at this stage but zona radiata was thick. The eggs in the oviducts (phase IV) contained developing embryos and had fully developed shell (Fig. 4D). Considerable changes in the oocytes and follicular cells were observed in the ovary during the year (Fig. 5). Pre-vitellogenic follicles were found in all females, vitellogenic follicles were found only in April and May. In this way, the ovaries remained in quiescence phase of the oogenic cycle during most of the year, from July to October (Fig. 5A). Most probably quiescence phase continued in the winter months but material from that period was lacking. Early vitellogenesis was observed in April in some follicles manifested as small yolk deposits. Pre-ovulatory follicles were observed only in the specimens from May (Fig. 5B), i.e. this was the third phase of the annual cycle. In June, the fourth phase with oviductal eggs containing developing embryos was observed (Fig. 5C). Corpora lutea (Fig. 5D) were formed in the two female specimens from this month. In July, the ovaries contained normal and atretic pre-vitellogenic follicles, as well as degenerating corpora lutea. Atretic follicles (Figs. 5E, F) were observed in two of three females from this month. Atretic follicles were observed also in a female from April (early vitellogenic) and another one from May (pre-ovulatory). Follicular atresia is a common phenomenon after the end of the breeding season in seasonally breeding lizards but can occur at any time of the cycle (Guraya 1989). In the next months were observed only pre-vitellogenic follicles. All these observations lead to the conclusion that the females of A. kitaibelii expressed type I vitellogenesis, initiating in spring and continuing without interruption until ovulation. After ovulation the ovaries enter quiescence until the next spring (Guraya 1989). Egg laying Copulation occurs in April May (Rotter 1962). Fertilisation in May was supported by the presence of pre-ovulatory follicles in females in this period. Ovulated eggs with embryos were observed in June, at that time the males were still in active spermiogenesis phase. The males were in a period of relative rest in July, which coincided with the onset of quiescence in the females. In August, the males were again in active phase, but the females did not show signs of vitellogenesis until next spring. Absence of vitellogenesis in the autumn months precluded fertilisation in this period. Despite ongoing spermiogenesis in late summer, no signs (bite marks) of autumn copulations were detected (Fig. 6). The smallest number of eggs laid from a single female was two and the largest was five. Three females from Belovets laid twelve eggs, two females from Brodilovo Ahtopol laid ten eggs, one female from Razdel laid three eggs and three females from Pchelin laid ten eggs together in one box with coconut substrate (three were found smashed on the next day). Two of the females from Pchelin laid four eggs each and one laid only two. Not all females laid their eggs at once (Table 2). No juveniles hatched from the eggs from the females from Razdel Village. Discussion Data on spermatogenesis are not available in November February, therefore the classification of the cycle type of A. kitaibelii is uncertain. Our observations are in accordance with those by Angelini et al. (1983) for Tarentola mauritanica (L., 1758). The authors described stasis in the spermatogenetic cycle during the warmest and coldest months (thermorigostatic cycle). Occurrence of a second period of stasis in the winter months could be expected also 513

8 Vergilov V. S., V. G. Necheva & B. P. Zlatkov for A. kitaibelii, which was suggested by the decreasing of the diameter of the seminiferous tubules and the epithelium height (Fig. 3). Despite the small temporal differences with the spermatogenic cycle of A. rueppellii (Goldberg 2012; wrongly identified as A. kitaibelii), in general, the cycles of both species appear similar. Goldberg (2012) has not studied specimens from June to October nor has provided information on a summer stasis, but it could not be dismissed as it is probably related to the high summer temperatures. He also stated that the formation of spermatozoa begins in late autumn, with ongoing spermatogenesis observed in a single specimen from November. The histological study shows that the youngest reproductive male (with spermatozoa in the lumina of the testes, Fig. 2C; Table 1) has a SVL of 39 mm. This contradicts with previous observations about the onset of sexual maturity in A. kitaibelii 32 mm SVL according to Schmidtler (1997) and Ljubisavljević et al. (2002) but the method for establishing sexual maturity in these studies has not been specified. Vergilov & Tzankov (2014) stated that the smallest male specimen with fully developed reproductive organs, according to the size of the testes, had a SVL of 30.7 mm. That observation was established by dissection. It is clear however, that the visual observation of the gonads via dissection, without a histological approach, cannot provide correct information on the onset of sexual maturity. Rotter (1962) observed that A. kitaibelii reached sexual maturity in the beginning of its third year. Presence of immature specimens with SVL of 35 and 36 mm in July (Appendix) suggests that most probably they have hatched later in the previous year (in the autumn). These individuals could become sexually mature after their first hibernation in late summer or autumn of next year. They can copulate with females in the spring after their second hibernation. However, the presence of mature individual with SVL of 39 mm in June reveals that some individuals could become mature and capable of copulation sooner after their first hibernation, in case they have hatched earlier in the previous year (in the summer). This is consistent with the skeletochronological data (Vergilov et al. 2018), revealing that this short-lived species reaches sexual maturity earlier in its lifespan. According to Vergilov et al. (2018), A. kitaibelii lives up to four years in Bulgaria. In comparison, the smallest reproductively active male A. rueppellii is much smaller: only 23 mm SVL (Goldberg 2012). A possibly useful external trait for separation of sexually mature and immature individuals is the colour of the tail, as the juveniles usually hatch with reddish tail (Vergilov 2017). The studied non-reproductive male individuals (SVL of 35 and 36 mm) had the same tail colour as the body. On the other hand, one of the newly hatched individuals also had a tail as dark as the body. These observations revealed that the tail colour cannot be used as evidence for sexual maturity. In the current study, it has not been determined at what body size females become sexually mature, but it can be assumed that the size is similar to that of males, because of the same lifespan in both sexes (Vergilov et al. 2018). According to the data on the growth of A. kitaibelii, the onset of maturity in females most likely corresponds to that in males: early hatched females could become fit for copulation and fertilisation after the first hibernation; females hatched later could grow enough and become fit for copulation and fertilisation in the spring after their second hibernation. Despite the more rapid growth in females, Vergilov et al. (2018) stated that statistical significant difference could be observed at the age of three hibernations. According to Goldberg (2012), there is no indication that A. rueppelii produces multiple clutches of eggs. Ablepharus kitaibelii females also lay eggs once a year, during a few days. However, that period is extended in time and the egg laying in different females occurred from June until the beginning of August (Table 2; Fig. 6). From our data of formation and egg laying, we could assume that the copulation and fertilisation in A. kitaibelii, at least in Bulgaria, occurs in April May (Fig. 6). The southern populations (from Razdel Village and Brodilovo Ahtopol) tend to lay eggs earlier than the northern ones (Belovets and Pchelin Villages; Table 2). According to Valakos et al. (2008), A. kitaibelii produces a clutch of eggs earlier (in May), which could be explained with the more southern location of the studied population. Göcmen et al. (1996) provided information on the breeding biology of the related species A. budaki. According to the authors data, it can be suggested that the egg laying occurs at the end of May; newly hatched individuals were observed in the middle of July. Goldberg (2012) revealed that reproductively active females of A. rueppellii were present even earlier, in March, April and June. The body length in females is greater than in males, because of the development of relatively long eggs in their abdomen. Dissection of fixed museum specimens revealed that some of the largest females (with longer bodies) could hold inside up to five eggs, but usually held four. This was also supported by observations of individuals held in captivity that laid a maximum of five eggs. It can be assumed that younger and respectively smaller-sized females could 514

9 Reproduction of Snake-eyed Skink Ablepharus kitaibelii (Bibron & Bory de Saint-Vincent, 1833) in Bulgaria have developed a smaller number of eggs in their abdomens (from 1 to 4). The observations of egg laying support previous data about the number of eggs in a clutch: 2 4 (Garbov 1990, Beshkov & Nanev 2006, Valakos et al. 2008, Stojanov et al. 2011), 2 5 (Fuhn 1970, Baran & Atatür 1998 after Goldberg 2012). The maximum number of eggs in the congener A. rueppellii is lower 3 (Goldberg 2012). The incubation period of the eggs (at room temperature during the summer) was between 26 and 42 days (Table 2). The hatching of juveniles is rather fast (Vergilov & Natchev 2018). According to Stojanov et al. (2011), the incubation period is much longer and could take up to three months, depending on the climatic conditions, but this probably is an overestimation because the data are not based on direct observation of eggs. Valakos et al. (2008) state that the incubation period in the species is two months. Regarding the differences in the periods of egg laying between the northern and southern populations in Bulgaria, it can be assumed that differences and small temporal shifts in the phases of the reproductive cycles could also be expected in other populations from the northernmost and southernmost range of the species. Acknowledgments: We would like to thank G. Popgeorgiev for the advice and the statistical analyses and Y. Kornilev and S. Lukanov for the valuable advice and corrections. The specimens were collected under permits No. 411/ and No. 413/ for scientific purposes issued by the Ministry of Environment and Water of Bulgaria, for which we are grateful. References Angelini F., Ciarcia G., Picariello O. & D Alterio E The Annual Spermatogenetic Cycle of Tarentola mauritanica L. (Reptilia, Gekkonidae). I The Spermatogenetic Cycle in Nature. 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