ARTICLE IN PRESS

ZOOLOGY
Zoology 111 (2008) 385–400
www.elsevier.de/zool

Sexual characteristics and spermatogenesis in males of the parthenogenetic

gecko Lepidodactylus lugubris (Reptilia, Gekkonidae)
Beate Rölla,, Monika U.G. von Düringb
a
Lehrstuhl für Allgemeine Zoologie und Neurobiologie, Fakultät für Biologie, Ruhr-Universität Bochum, D-44780 Bochum,
Germany
b
Abteilung Neuroanatomie, Fakultät für Medizin, Ruhr-Universität Bochum, D-44801 Bochum, Germany

Received 16 August 2007; received in revised form 21 September 2007; accepted 24 September 2007

Abstract
Obligately parthenogenetic lizards usually are all-female populations of hybrids producing diploid oocytes by
premeiotic endomitosis and quasi-normal meiosis. In an all-female strain of the gekkonid lizard Lepidodactylus
lugubris several phenotypic males arose spontaneously. The sexual characteristics of these males were studied using
light and electron microscopy and compared with normal males of the bisexual genus Lygodactylus. Emphasis was
layed on morphology of seminiferous tubules, occurrence of spermatogenic stages and ultrastructure of spermatozoa.
The phenotypic males possessed preanal pores filled with secretions and a sexual nephric segment which were exactly
the same as in normal, reproductively active males. In the testes, density and morphology of non-spermatogenic cell
types, the Leydig and Sertoli cells, indicate a normal production of testicular testosterone and a normal function of the
blood–testis barrier, respectively. Both in the normal and the phenotypic males, all meiotic cell types of
spermatogenesis can be recognised in the seminiferous tubules and are apparently identical, indicating a normal
meiosis without impairment in the phenotypic males. In contrast, the differentiation process of spermatids is markedly
disturbed in the phenotypic males of L. lugubris. In the normal male, spermiogenesis results in mature spermatids and
spermatozoa with small elongated nuclei, an acrosomal complex, and a flagellar tail possessing one axoneme.
Spermatozoa fill both the lumen of most seminiferous tubules and the lumina of ductus epididymidis and ductus
deferens. In the phenotypic male, spermiogenesis results in seemingly normal spermatids and in spermatozoa with
large, non-elongated, deformed nuclei and/or irregular tails possessing more than one axoneme. Both the lumen of
most seminiferous tubules and the lumina of the ductus epididymidis and the ductus deferens contain relatively few
spermatozoa. We suggest that the phenotypic males inherited the ability for a premeiotic endomitosis from their all-
female ancestral lineage. While in females this leads to quasi-normal meiosis and diploid oocytes capable of
development, the small nuclei of the spermatozoa are unable to contain a diploid set of chromosomes. Because of the
high amount of deformed spermatozoa and possibly uncontrolled loss of genetic material in structurally normal, but
aneuploid spermatozoa we conclude that these otherwise perfect males are infertile, thus constituting another example
of gametic sterility.
r 2008 Elsevier GmbH. All rights reserved.

Keywords: Parthenogenesis; Male sexual characters; Spermiogenesis; Gekkonid lizards

Corresponding author.
E-mail addresses: roell@neurobiologie.rub.de, roell@neurobiologie.ruhr-uni-bochum.de (B. Röll).

0944-2006/$ - see front matter r 2008 Elsevier GmbH. All rights reserved.
doi:10.1016/j.zool.2007.09.004
 ARTICLE IN PRESS
386 B. Röll, M.U.G. von Düring / Zoology 111 (2008) 385–400

Introduction but only in females kept in captivity without access to

males for very long times. Facultative parthenogenesis
The majority of vertebrates reproduce sexually, with has been reported in the turkey (Meleagrididae; Olsen,
males and females contributing genetic material to their 1975), two species of Varanidae (Lenk et al., 2005; Watts
offspring. However, in a few taxa reproduction is et al., 2006), seven snake species (Pythonidae, Acro-
asexual or unisexual without genetic contribution from chordidae, Crotalidae and Colubridae; Dubach et al.,
males, i.e., hybrido-, gyno- and parthenogenesis. In the 1997; Schuett et al., 1997; Groot et al., 2003), and most
latter, the embryo develops from an oocyte in the recently in a hammerhead shark (Sphyrnidae; Chapman
absence of spermatozoa. There are two types of et al., 2007). In most cases, diploidy of the mature
parthenogenesis: obligate and facultative. Obligate oocytes is restored through fusion of the nucleus of the
parthenogenesis is restricted to squamate reptiles and ooycte with the second polar body resulting in
occurs in more than 40 species of the lizard families completely homozygous offspring. As the sex determi-
Agamidae, Chamaeleonidae, Gekkonidae, Gym- nation in snakes, varanids and birds usually follows a
nophthalmidae, Teiidae, Scincidae, Lacertidae and ZZ/ZW mechanism, homozygous offspring will be
Xantusiidae and in one snake of the family Typhlopidae either WW or ZZ. Since the WW combination is not
(Zug et al., 2001; Reeder et al., 2002; Adams et al., viable, all offspring are ZZ males.
2003). Obligate parthenogenesis is considered to be Because at best one half of the offspring (ZZ) will be
intimately associated with preceding interspecific hybri- viable and all will be completely homozygous, faculta-
disation between related bisexual species resulting in tive parthenogenesis does not seem to play an important
highly heterozygous offspring (Darevsky et al., 1985; role in the reproduction of natural populations. It does
Zug et al., 2001). Parthenogenetic squamates usually are not replace sexual reproduction in the species con-
all-female populations; hybrid males of F1 generations cerned, whereas in obligately parthenogenetic popula-
have apparently not been described. Assuming a tions reproduction is exclusively unisexual (apart from
genotypic XX/XY sex determination mechanism in the rare backcrossing events).
parental species, this could be due to the preferential The greatest variety of obligate parthenogens is found
inviability or sterility of hybrids of the heterogametic sex within the Gekkonidae, with seven parthenogenetic all-
(Haldane’s rule; Orr, 1997). The mature oocytes of female ‘‘species’’ in five genera. The most widespread
parthenogenetic females are diploid, which is achieved parthenogen is the mourning gecko Lepidodactylus
by premeiotic endomitosis. This mode can be found in lugubris occurring in Southeast Asia, Australia, most
representatives of Teiidae, Lacertidae and Gekkonidae islands of the Pacific, and Middle America. It originated
and is assumed to be the mechanism in most unisexual through hybridisation between L. moestus from Micro-
lizards (Cuellar, 1971; Suomalainen et al., 1987). nesia as the maternal ancestor and a still undescribed
Premeiotic endomitosis avoids irregular synapsis of species L. sp., which is distributed from French
homologous chromosomes at first meiotic division and Polynesia to the Marshall Islands, as the paternal
thus disruption of oogenesis as a consequence of the ancestor (Radtkey et al., 1995). The geographical
extremely high heterozygosity of hybrids. In this sense, distributions of both parental species overlap only on
parthenogenesis can be regarded as a strategy that Arno Atoll (Marshall Islands), which is thought to be
circumvents possible sterility of hybrids. The high the place where the parthenogenetic hybrid form
frequency with which F1 hybrids among squamate originated. The existence of different major diploid
lizards have established a sperm-independent reproduc- clones suggests that hybridisation events have occurred
tion suggests a cause-and-effect relationship between several times. Triploid clones originated through back-
hybridisation and the origin of parthenogenesis, perhaps crossing of the parthenogenetic L. lugubris with males of
through genetic dysfunction in the control of meiosis the parental species. Both diploid and triploid clones are
(Reeder et al., 2002). Due to the premeiotic doubling of now widespread throughout the distribution area of
the chromosomes, identical sister chromosomes form L. lugubris (Moritz et al., 1993; Yamashiro et al., 2000).
tetrads during meiosis so that neither crossing-over nor There are reports on bisexual populations of
segregation results in genetic recombination. Thus, the L. lugubris or on the occasional emergence of pheno-
offspring is – except for rare mutations – genetically typic males in female assemblages of L. lugubris (Ineich,
identical to its mother and forms a diploid clone with 1988; Volobouev et al., 1993). Actually, males of the
her. In most unisexual lizards there are also triploid ‘bisexual populations’ belonged to other species, either
clones which originated through backcrossing of a L. moestus or L. sp. or other still undescribed sexual
parthenogenetic female with a male of one of the species of Lepidodactylus (Radtkey et al., 1995).
parental or of another related species (Darevsky et al., Furthermore, most of the phenotypic males are hybrids
1985; Zug et al., 2001; Reeder et al., 2002). between the ancestral sexual species and L. lugubris,
In contrast to obligate parthenogenesis, the faculta- which lack functional gonads and associated organs,
tive type has not yet been found in natural populations, thus indicating sterility on a very basic level (Ineich,
 ARTICLE IN PRESS
B. Röll, M.U.G. von Düring / Zoology 111 (2008) 385–400 387

1988; Radtkey et al., 1995; Ota et al., 1995). However, compliance with the current laws of the Federal
rare males were also reported from other locations Republic of Germany.
(Hawaiian Islands, Mariana Islands, Ryukyu Archipe-
lago) where no other bisexual species of Lepidodactylus Light microscopy
occurs (Brown and Murphy-Walker, 1996; Yamashiro
and Ota, 1998; Yamashiro et al., 2000). It is assumed For light microscopy, the whole urogenital system up
that a hormonal sex inversion causes the emergence of to the tail base of a male Lygodactylus picturatus and of
such males (Darevsky et al., 1985, for parthenogenetic a phenotypic male of L. lugubris were fixed in situ in
lacertids). Light microscopical investigation of the Bouin’s fluid and decalcified in a trichloracetic acid/
gonads of two such males revealed an aberrant formaldehyde solution (5 g trichloracetic acid in 100 ml
spermatogenesis compared with that in normal lizard distilled water mixed with 20 ml of 35% formaldehyde).
males (Brown and Murphy-Walker, 1996; Yamashiro After washing for 2 or 3 days in 70% ethanol, specimens
and Ota, 1998). However, it remains unclear in which were dehydrated in ethanol, cleared in iso-propanol and
phase of spermatogenesis the dysfunction occurs and embedded in paraffin. Each preparation was sectioned
what the reason for it may be. serially at 10 mm in the transverse plane. The sections
In the present study, the sexual characteristics of were stained with aldehyde fuchsin after Goldner and
phenotypic males occurring spontaneously in an investigated with a Zeiss axioplane photomicroscope.
exclusively parthenogenetically reproducing strain of
the gecko L. lugubris were investigated with emphasis Electron microscopy
on the morphology of seminiferous tubules and
ultrastructure of spermatozoa. Most temperate-zone For transmission electron microscopy, testes and
lizards and many lizards from tropical regions are epididymes including associated ducts were fixed for
seasonal breeders depending on seasonal patterns of 12 h in 2.5% glutaraldehyde and 1% paraformaldehyde
temperature or rainfall. Thus, in order to ensure that the in phosphate buffered saline at pH 7.4. After rinsing, the
phenotypic males were not out of their reproductive preparations were postfixed in 2% osmium tetroxide for
phase, the morphology of the nephric sexual segment 2 h and dehydrated through a graded ethanol series. The
and the activity of the preanal glands were examined as samples were passed through propylene oxide, em-
well. For comparison, the sexual characteristics bedded in araldite and cut in series of alternate semithin
and spermatozoal ultrastructure of normal males of and ultrathin sections. Semithin sections were stained
the bisexual gekkonid genus Lygodactylus were also with 1% toluidene blue, pH 9.0, and examined with a
determined. Zeiss axioplane photomicroscope. Ultrathin sections
were double-stained with uranyl acetate and lead citrate,
examined with a Philips EM 420 and photographed
Materials and methods using a digital camera.
Animals
Identification of cell types in the seminiferous
Animals were housed in glass terrariums under a epithelium
12:12 h light/dark cycle; temperatures ranged from
28–34 1C during the day to 21–23 1C at night. The For identification of the different cell types, especially
geckos were fed a variety of insects and their larvae; the meiotic stages of primary spermatocytes, and
drinking water was enriched with calcium, phosphate measurements of selected cell types, semithin sections
and vitamins. The two males of bisexual species have been used. The identification has been based on the
investigated here belong to African species of the genus description of germ cells of the European wall lizard
Lygodactylus (L. capensis, L. picturatus) which were Podarcis muralis provided by Gribbins and Gist (2003).
bred in the laboratory. According to the dorsal colour
patterns, the strain of L. lugubris from which the
phenotypic males originated belongs to the major clone Results
2 nA, which is the most widespread diploid clone on
Pacific islands (Ineich, 1988; Moritz et al., 1993). External characteristics
Altogether, five phenotypic males occurred within 3
months among approximately 2000 geckos which had The phenotypic males of L. lugubris measure 47 mm
been bred for two decades; two of these males were used in snout-to-vent length and 44 mm in tail length and are
in this investigation. Animals were anaesthetised and approximately 5 mm larger in total length than females
killed by an overdose of pentobarbital (0.2 ml nembutal (measurements of male character averages based on the
intraperitoneally). The experiments were performed in five specimens mentioned above). Their dorsal colour
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388 B. Röll, M.U.G. von Düring / Zoology 111 (2008) 385–400

patterns consisting of two longitudinal rows of more or tissue, blood vessels and lymphatic vessels. Superficially,
less black spots beside the mid-dorsal line are identical the seminiferous epithelia of both types of males appear
to that of the female strain. The phenotypic males to be normal as there are all main spermatogenic cell
possess well-developed hemipenis pockets in the post- types as expected in sexually active males. In paraffin
cloacal region (Fig. 1b) containing the inverted hemi- sections, spermatogonia, spermatocytes, early sperma-
penes as revealed in histological sections. The tids, late spermatids with tails and luminal spermatozoa
postcloacal swelling in the normal male of L. capensis can be recognised (Fig. 2c, d), even though most
is less distinct (Fig. 1a). The phenotypic males have seminiferous tubules of the phenotypic male contain
12–14 preanal and about 10 femoral pores which are relatively few spermatozoa. However, the arrangement
completely absent in females of L. lugubris. The preanal of cell types within the seminiferous epithelium differs
pores are filled with wax-like secretions comparable to between the two types of males. In the normal males, the
the five preanal pores of the normal male of L. capensis cell types are well-arranged in successive layers from the
(L. capensis lacks femoral pores; Fig. 1a, b). The basement membrane towards the lumen (Fig. 2e). Here,
secretions originate from holocrine epidermal glands, the tails of mature spermatids are oriented towards the
accumulate in short ducts and project from the pore as lumen. In contrast, in the phenotypic male spermato-
little waxy cones (Fig. 1c, d). The phenotypic males also cytes, early and especially late spermatids are not
possess cloacal spurs lacking or appearing much smaller arranged in clear layers (Fig. 2f). Here, several
in females of L. lugubris and lacking in the normal male spermatids with darkly stained condensed nuclei and
of L. capensis (Fig. 1a, b). some with tails are located deeply within the seminifer-
ous epithelium, between spermatocytes and even sper-
matogonia, but not in the luminal part. The nuclei of
Sexual segment of the kidney both early and late spermatids of the phenotypic males
appear to be larger and somewhat deformed compared
In the kidneys (metanephros) of both the phenotypic
with the nuclei in the normal males. Some spermatids
males and the normal males (L. picturatus, L. capensis),
appear to have their tails oriented towards the basement
a normally developed segment and a sexual segment can
membrane (Fig. 2f).
be clearly distinguished. The normal renal segment
Between the seminiferous tubules some Leydig cells
consists of uriniferous tubules possessing cylindrical
can be recognised in the interstitial tissue of the normal
cells with central nuclei. In contrast, the sexual renal
and of the phenotypic males both in paraffin and in
segment has larger tubules which are characterised by
semithin sections (Fig. 3a, m). The density of the Leydig
hypertrophied columnar cells with basal nuclei and
cells appears to be similar in all preparations. They
numerous granules (Fig. 1e, f). It consists mainly of the
occur singly or in small groups of 2–3 cells near blood
terminal portion of the collecting ducts lying in the renal
vessels, and in semithin sections they are characterised
medulla. It cannot be excluded that parts of the ureter
by an elongated shape, large oval nuclei with a
are also hypertrophied as they are in other lizard species;
prominent nucleolus and a vacuolated cytoplasm which
however, the ureter was not cut in the sections. The
can include either a few large or numerous smaller lipid
diameters of the sexual tubules range from 140 to
globules (Fig. 3a, m).
180 mm in Lygodactylus and from 135 to 190 mm in L.
lugubris (measured on semithin sections).
Identification of cell types in the seminiferous tubules

Testes and seminiferous tubules The seminiferous tubules are lined by complex
seminiferous epithelium containing a non-proliferating
Both normal and phenotypic males have well devel- population of supporting cells – the Sertoli cells – and a
oped abdominal oval testes consisting mainly of proliferative population of germ cells.
convoluted seminiferous tubules (Fig. 2 a, b). The testes In normal and phenotypic males, nuclei of Sertoli cells
are surrounded by a tunica albuginea of connective can be recognised near the basal lamina of the
tissue. The seminiferous tubules are ensheathed by the seminiferous epithelium in more or less regular dis-
tunica propria and are interspersed with interstitial tances. The nuclei are irregularly shaped and possess

Fig. 1. Sexual characteristics in normal males of the bisexual genus Lygodactylus (left: a – L. capensis, c and e – L. picturatus) and a
phenotypic male of Lepidodactylus lugubris (right: b, d, f). (a, b) Anal region: males with preanal pores filled with wax-like secretions
(black arrowheads in a, unmarked in b), with a row of femoral pores in Lepidodactylus lugubris only (arrows in b), with hemipenis
pockets (asterisks), more conspicuous in Lepidodactylus lugubris, and with cloacal spurs in Lepidodactylus lugubris only (white
arrowheads). Scale bars: 1 mm. (c, d) Paraffin sections of preanal pores and holocrine secretion plugs of preanal epidermal glands.
Secretions are characteristic for reproductively active males. Scale bars: 100 mm. (e, f) Cross-sections of the kidneys: hypertrophied
terminal segments with tubules characterised by a central lumen and by cells full of granules. Scale bars: 50 mm.
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390 B. Röll, M.U.G. von Düring / Zoology 111 (2008) 385–400

Fig. 2. Paraffin cross-sections of testes: normal male of Lygodactylus picturatus (left: a, c, e) and phenotypic male of Lepidodactylus
lugubris (right: b, d, f). (a, b) Testes with well-developed seminiferous tubules. ST, seminiferous tubule; TA, tunica albuginea; TP,
tunica propria. Scale bars: 200 mm. Enlarged views of seminiferous tubules (c, d) and of seminiferous epithelia (e, f). In the normal
male well-arranged successive layers of spermatogonia (SG), spermatocytes (SC), developing spermatids (S) and mature spermatids
(MS) from the basement membrane towards the lumen; in the phenotypic male spermatocytes and spermatids not arranged in clear
layers. Arrow in (f) marks a large spermatid with the tail oriented towards the basal lamina. Scale bars: 50 mm (c, d) and 20 mm (e, f).
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darkly stained nucleoli (Fig. 3b, n). The Sertoli cells spermatocytes and contain pale granular chromatin
extend from the base of the seminiferous epithelium to (Fig. 3j, arrowhead).
the tubule lumen providing lateral processes around All stages of the first meiotic division are found in
which the spermatocytes and spermatids are arranged as similar frequencies in the seminiferous tubules both of
usual. Apical processes of the Sertoli cells enclose the the normal and of the phenotypic male. Differences are
late or mature spermatids. Electron microscopy reveals observed only in spermiogenesis, the differentiation
tight junctions forming the blood–testis barrier between process of spermatids. Spermatids can be divided into
adjacent Sertoli cells (Fig. 3c, o). They extend approxi- two main stages: early and late spermatids. Early
mately 0.1 mm along the cell membranes. These tight spermatids include stages with more or less spherical,
junctions are regularly found between Sertoli cells both pale nuclei with densely stained nuclear membranes and
of the normal and of the phenotypic male. stages with distinct acrosomal vesicles. Late spermatids
In the seminiferous tubules of both types of males, include stages characterised by the elongation of the
two types of spermatogonia are usually situated on the nuclei, condensation of the chromatin material and
basal membrane. They differ in nuclear staining. The formation of the flagellum and mature stages shortly
pale-stained, spherical nuclei of spermatogonia A before being released into the lumen. Both early
contain a prominent nucleolus and heterochromatin spermatids (Fig. 3j, v) and late spermatids (Fig. 3k, w)
centred close to the nuclear membrane (Fig. 3d, p). The are observed in the seminiferous tubules of both types of
spherical or elliptical nuclei of spermatogonia B contain males.
dispersed heterochromatin (not shown). The diameters of the nuclei of early spermatids of the
Mitotic divisions of spermatogonia B produce pre- phenotypic male are 1.8–1.9 larger than those of the
leptotene primary spermatocytes which resemble sper- normal male (Fig. 3j, v; Table 1). Mature spermatids of
matogonia B and which are also located in the basal both types of males differ in number and often in size
compartment. The following stages of the primary and shape. Generally, mature spermatids (Fig. 3l, x and
spermatocytes, localised in the luminal compartment, spermatozoa are much more frequently observed in the
are easy to identify as they possess the largest nuclei normal than in the phenotypic male. In the seminiferous
among the germ cells. The diameters of the nuclei of epithelium of the normal male almost all mature
primary spermatocytes of the phenotypic male L. spermatids possess darkly stained, elongated nuclei
lugubris are about 1.2 times larger than those of the and tails which are oriented towards the lumen (Figs.
normal male L. capensis (Table 1). Furthermore, the 3l, and 4a). The seminiferous epithelium of the
lengthy prophase of the first meiotic division involves phenotypic male contains both spermatids with see-
extensive rearrangements of the chromatin which well mingly normal nuclei and spermatids with large non-
characterises most of its different stages. The nuclei of elongated, deformed nuclei (Fig. 3x, arrowhead). For
the leptotene spermatocytes contain filamentous chro- further characterisation of the nuclei, spermatids and
matin fibres (Fig. 3e, q) which are thickened in the nuclei both testicular and epididymal spermatozoa were
of zygotene spermatocytes (Fig. 3f, r). The nuclei of the investigated electron microscopically.
pachytene spermatocyte are slightly larger and contain
thick chromatin fibres and areas of open nucleoplasm
(Fig. 3g, s). Pachytene spermatocytes are visible in most Ultrastructure of spermatids and spermatozoa in the
cross-sections of the seminiferous tubules of the seminiferous tubules and the duct system
gekkonid males. Diplotene spermatocytes are hard to
distinguish from the pachytene spermatocytes. During The early stages of spermiogenesis in both types of
diakinesis, the final stage of the prophase, the chromo- males are characterised by the formation of the
somes are fully condensed and stain very deeply acrosomal vesicle, which grows in size and forms a
(Fig. 3h, t). The nuclear membrane is dissolved. In nuclear depression. The latter deepens with the growing
metaphase spermatocytes, the chromosomes are aligned vesicle (Fig. 3j, v). Generally, the acrosomal region
at the equatorial plate (Fig. 3i, u). It is difficult to points towards the base of the seminiferous epithelium.
distinguish between metaphase I and II based on the The nuclei migrate from a central position in the cell to a
amount of chromatin seen, as the number and area of peripheral one. Furthermore, the vesicle spreads and
chromosomes depend on the sectional plane through the ultimately covers the entire apical nucleus. Simulta-
nucleus. However, some of the metaphase cells are neously, the flagellum develops on the opposite pole of
suggested to be metaphase II cells as they are located the acrosomal vesicle. Elongation of the nucleus and
more adluminally than metaphase I cells. Usually condensation of the chromatin proceed simultaneously.
secondary spermatocytes of vertebrates are relatively During the early phase, the chromatin appears more
short-lived and thus are infrequently found in semithin granular and then filamentous. With further condensa-
sections of gekkonid testes. The nuclei of secondary tion, the chromatin filaments become thicker, and at the
spermatocytes are smaller than those of primary end of the condensation process the chromatin has lost
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Table 1. Diameters and calculated volumes of nuclei of between 0.6 and 0.8 mm (n ¼ 29). The proximal centriole
primary spermatocytes (PS) and early spermatids (ES). resides in the neck region directly beneath the nucleus
n ¼ number of measurements (Fig. 5c). The distal centriole forms the axoneme which
reveals a 9+2 arrangement of microtubules in cross-
Lygodactylus Lepidodactylus
capensis lugubris section (Fig. 5c). The axoneme extends through the
whole tail. Electron-dense fibres form a fibrous sheath
+ nuclei of primary 6.8–8.3 mm 8.4–10.2 mm around the axoneme of the midpiece and of the principal
spermatocytes (PS) (n ¼ 25) (n ¼ 24) piece. It appears as a ring in cross-section (Fig. 5c, g). In
Calculated nuclear volume 165–300 mm3 310–539 mm3 the midpiece both the axoneme and the fibrous sheath
(VolPS) are covered with mitochondria and so-called dense
+ nuclei of early spermatids 3.8–4.5 mm 6.8–8.5 mm bodies supposed to be transformed mitochondria. The
(ES) (n ¼ 24) (n ¼ 23)
endpiece contains only the axoneme. Midpiece, principal
calculated nuclear volume 29–48 mm3 165–322 mm3
piece and endpiece of the spermatozoa of the normal
(VolES)
Nuclear volume of ES 16–18% 53–60% male have diameters of 0.6–0.7 mm (n ¼ 10), 0.3–0.5 mm
relative to PSa (n ¼ 18) and 0.2–0.3 mm (n ¼ 22), respectively.
In the phenotypic male L. lugubris apparently
a
R ¼ (VolES/VolPS) * 100. ‘normal’ spermatozoa as well as spermatozoa with
deformed nuclei and irregular tails are observed.
Spermatozoa with ‘normal’ nuclei and deformed nuclei
its filamentous structure and is devoid of any substruc- are found in testis and epididymidis in a ratio of 1:0.8
tures. Mitochondria congregate next to the centrioles to (n ¼ 97). Compared with L. capensis, the ductus
constitute the midpieces of the tails, which are oriented epididymidis and ductus deferens contain relatively
towards the lumen (Fig. 4a). Up to this stage few spermatozoa (Fig. 5d, e).
spermiogenesis proceeds similarly in the normal and in The ultrastructure of the ‘normal’ spermatozoa of the
the phenotypic male; after this stage conspicuous phenotypic male is similar to that of the spermatozoa of
differences can be found. the normal male described above (Fig. 5h and inset).
Chromatoid bodies are observed within the cytoplasm Some spermatozoa with an apparently normally elon-
of late spermatids. The chromatoid bodies are cloud-like gated nucleus reveal an abnormal acrosome with a
accumulations of dense fibrous material called nuage. vacuole-like structure (Fig. 4b). The nuclei of the
They are irregularly shaped and are conspicuously larger ‘normal’ spermatozoa have diameters between 0.7 and
and appear more frequently in spermatids of the 0.9 mm (n ¼ 17). The midpieces, principal pieces and
phenotypic male than in those of the normal male endpieces have diameters of 0.8–1.1 mm (n ¼ 12),
(Fig. 4f). 0.3–0.5 mm (n ¼ 14) and 0.2–0.3 mm (n ¼ 11), respec-
Mature spermatids and spermatozoa of the normal tively. The most conspicuous features of many sperma-
male L. capensis have elongated, entirely black stained tozoa of the phenotypic male are the sizes and shapes of
nuclei with an acrosomal complex at its apical end and a their nuclei. The maximal diameters of the nuclei vary
flagellar tail consisting of midpiece, principal piece and between 0.6 and 6.9 mm (n ¼ 21); however, most are
endpiece. As in the main part of the tubular lumen of the between 3 and 5 mm. Their nuclei can be more or less
testis, the lumina of the ductus epididymidis and ductus round, cup-shaped or bell-shaped (Fig. 4b–d, and
deferens are full of spermatozoa (Fig. 5a, b). Almost all Fig. 5f). Furthermore, some midpieces contain 2 or 3
spermatozoa have elongated nuclei and only sporadic axonemes, each of which has its own fibrous sheath;
spermatozoa with larger nuclei are found (Fig. 5a, b). however, they share the mitochondria and part of the
The acrosomal piece consists of the acrosome vesicle dense body lying between them (Fig. 4c, e, and Fig. 5g).
and the subacrosomal cone which surround the nuclear Only one comparable spermatozoon with two
attenuation, also called nuclear point (Fig. 5c, inset). axonemes has been observed in the normal male.
The tip of the acrosome contains the perforatorium. However, here the two axonemes were surrounded by
Proximally, the spermatozoal nuclei have diameters a single fibrous sheath.

Fig. 3. Examples of cell types of the seminiferous epithelia. Lygodactylus capensis (left: a–l), Lepidodactylus lugubris (right: m–x).
Cell types centred within the figures. (c, o) Ultrathin sections (scale bars: 0.5 mm), all others semithin sections to same scale (scale bar
in n: 10 mm). (a, m) Leydig cells in the interstitial tissue between the seminiferous tubules. (b, n) Nuclei of Sertoli cells on the base of
the seminiferous epithelium. (c, o) Tight junctions (arrowheads) of the blood–testis barrier between adjacent Sertoli cells. (d, p)
Spermatogonia of type A. Primary spermatocytes: (e, q) leptotene spermatocyte; (f, r) zygotene spermatocyte; (g, s) pachytene
spermatocyte; (h, t) diakinesis; (i, u) metaphase (presumably I). Spermatids: (j, v) early stages of spermatids, partly with acrosomal
vesicle and a secondary spermatocyte (arrowhead in j); (k, w) late stages of spermatids; (l, x) spermatids shortly before being released
into the lumen, and in (x) a conspicuously deformed spermatid (arrowhead).
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Discussion are less conspicuous and do not show secreted material.

The secretions may be involved in the production of
Macroscopic morphology of testes pheromones for socio-sexual communication, as after
castration the femoral pores of male lacertid lizards
Normal and the phenotypic males have similar, regress (Bellairs, 1970).
normally developed testes with seminiferous tubules The renal sexual segment in the kidneys of male
and associated organs like epididymes and ducts for the lizards and snakes consists of hypertrophied regions
storage and transport of spermatozoa. Thus, the usually situated terminally in the nephrons (Fox, 1977).
phenotypic males obviously do not suffer from gonadic It can include the preterminal and terminal portions of
sterility, one of the various sterility patterns of hybrids the collecting ducts, also called preterminal and terminal
characterised by an abnormal development and reduced segments, and parts of the ureter. Both the normal males
size of gonads. Gonadic sterility occurs in phenotypic of Lygodactylus and the phenotypic males of L. lugubris
triploid males originated through backcrossing of a had well-developed sexual segments with hypertrophied
parthenogenetic female with a male of a related bisexual regions in at least the terminal segments of the collecting
species (Saint-Girons and Ineich, 1992). Apparently, the ducts. In the Madagascan gecko Phelsuma dubia the
haploid genome from the male interferes with the sexual segment also involves the terminal portion of the
diploid genome of the parthenogenetic female if the collecting ducts and additionally parts of the ureter
spermatozoon contains genes for the determination of a (Osadnik, 1987). In lizards, the hypertrophy of these
male sex of the offspring, as triploid females originated urinary ducts follows a seasonal cycle which is related to
through backcrossing have normal female sexual char- the cyclic activity of the testes (Fox, 1977; Sever and
acters. Hopkins, 2005). In P. dubia, a species with a seasonal
spermatogenic cycle, spermiation coincides with the
maximal development of the sexual segment (Osadnik,
Secondary sexual characteristics and reproductive 1987). After the mating season, the sexual segment
activity undergoes recrudescence and can hardly be distin-
guished from the adjacent tubular regions. After
External secondary sexual characteristics of gekkonid castration, the male sexual segment regresses in male
males are usually specific for each genus and comprise lizards and snakes either before or during reproduction
characters like hemipenis pockets, preanal and/or (Fox, 1977).
femoral pores, escutcheons and cloacal spurs. The As both types of males investigated here possess both
phenotypic males of L. lugubris exhibit all of these preanal pores completely filled with secretions and a
characters with the exception of an escutcheon. As in sexual segment with distinctly hypertrophied regions, it
most gekkonid species, the males of L. lugubris are can be concluded that they are not in a sexually
larger than the females. Furthermore, the phenotypic quiescent phase, but are reproductively active.
males of L. lugubris vocalise much louder than the
females with multiple chirp calls (Röll, 2002). Hawaiian
phenotypic males of L. lugubris displayed behaviours Testicular non-spermatogenic cell types
which are usually associated with courtship, e.g. neck
biting and crawling on top of the female (Brown and Generally, reproduction among amniotes is under
Murphy-Walker, 1996). However, intromission of a hormonal control, with testosterone playing an impor-
hemipenis could not be observed. tant role in male reproduction. In the testes, this
Males of the bisexual Lygodactylus and the pheno- hormone is produced in Leydig cells and partly
typic male of L. lugubris possess preanal pores which are transported via Sertoli cells to the germ cells. The
conspicuously filled with wax-like secretions projecting interstitial tissue between the seminiferous tubules of the
like small pins. The pores are ducts of epidermal normal and the phenotypic males investigated contains
holocrine glands which become hyperactive during the relatively few Leydig cells. Apparently, Leydig cells of
breeding period. Outside the breeding period, the pores different lizard species vary seasonally in number, size

Fig. 4. Late spermatids in the seminiferous tubules of Lygodactylus capensis (a) and Lepidodactylus lugubris (b–e). Ultrathin
sections. (a) Well-arranged spermatids with elongated nuclei and developing midpieces and flagella. The tails are aligned towards the
lumen of the tubule (asterisk). (b) Spermatids: cell on the right with large, deformed nucleus and tail pointing towards the lumen, cell
on the left with an almost normally elongated nucleus, but an atypical acrosome (arrowhead). (c) Longitudinal section of a
spermatid with deformed nucleus and at least two, presumably three flagella (arrowheads). (d) Spermatid with a deformed, bell-
shaped nucleus in longitudinal section. Proximal centriole in the centre of the figure. (e) Cross-section of a midpiece with two
flagella. (f) Late spermatid of the phenotypic male with a large chromatoid body (black arrowhead) and annulate lamellae (black
arrow). Scale bars: 2 mm (a), 1 mm (b, c, f), 0.2 mm (d, e).
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B. Röll, M.U.G. von Düring / Zoology 111 (2008) 385–400 397

and cytological content (Fox, 1977). In many lizards, males, and therefore meiosis takes place without
including the geckos Hemidactylus flaviviridis and P. obvious impairment in the phenotypic male. However,
dubia, Leydig cells are most numerous in quiescent testes spermiogenesis, the process following the meiotic divi-
and sparsest in active testes (Dutta, 1944; Fox, 1977; sions, seems to be disturbed.
Osadnik, 1987). However, the low number in active In the phenotypic male, approximately half of the
testes could be due to dispersion of the interstitial cells nuclei of the spermatids are not elongated, but more or
as a result of the expansion of the seminiferous tubules. less deformed. For a further characterisation of this
In the Leydig cells of P. dubia lipid inclusions are mainly apparent dysfunction, the ultrastructure of spermatids
observed in reproductively active testes, not in quiescent and spermatozoa has been investigated using electron
testes (Osadnik, 1987). Most of the Leydig cells of the microscopy. This technique revealed – apart from the
males investigated here also include lipid droplets. This difference in the nuclear diameters of early spermatids –
observation firstly supports the conclusion that both the that the early stages of spermiogenesis including the
normal and the phenotypic males are in the reproduc- beginning of the nuclear elongation and condensation
tively active phase. Secondly, it indicates a normal and the formation of the tail are similar in the normal
production of testicular testosterone in both males. and the phenotypic males. However, during the last
Sertoli cells have various functions essential for stages of the chromatin condensation the spermatids of
spermatogenesis, e.g. support and nutrition of the germ both male types differ in three main points: size of the
cells, release of mature spermatids into the lumen of the chromatoid body, shape of the nucleus and number of
seminiferous tubule, phagocytosis both of residuals of flagella.
released and of degenerated spermatids, secretion of the The chromatoid body is a special form of nuage that
androgen-binding protein, and formation of the blood– occurs in the cytoplasm of all germ cells throughout the
testis barrier. Between adjacent Sertoli cells of both the animal kingdom (Parvinen, 2005). The function of the
normal and the phenotypic male, tight junctions are chromatoid body is currently not well understood, but it
regularly found. Tight junctions are the morphological is thought to be associated with germ cell determination
sites of the blood–testis barrier which isolates sperma- and post-transcriptional processing of RNA. The
tocytes and spermatids of the luminal part and protects chromatoid body is obviously larger and much more
them from harmful substances or from plasma proteins, frequently found in late spermatids of the phenotypic
e.g. antibodies which are directed against antigens of male than in those of the normal male. It is suggested
spermatozoa. The density and morphology of Sertoli that the genetic material that cannot be stored in the
cells are identical in both the normal and the phenotypic small nucleus of a spermatozoon is discarded within the
males indicating a normal function of the blood–testis chromatoid body.
barrier in the seminiferous tubules. The ultrastructure of the spermatozoa of the normal
male and of the ‘normal’ spermatozoa of the phenotypic
male corresponds well to that of other gekkonid species
Identification of spermatogenic stages and and in main parts to that of other squamate lizards
ultrastructure of late spermatids and spermatozoa (Furieri, 1970; Jamieson et al., 1996; Giugliano et al.,
2002; Teixeira et al., 2002; Vieira et al., 2004; Gribbins et
As observed in paraffin and semithin sections, the al., 2007). The nuclear diameters of the seemingly
normal arrangement of the spermatogenic stages in ‘normal’ spermatozoa of L. lugubris and those of L.
successive layers is disturbed in the phenotypic male of capensis are similar and correspond well to the diameters
L. lugubris. However, in both types of males all stages of of spermatozoa of a male of a bisexual population of the
spermatogenesis can be recognised in the seminiferous gecko Heteronotia binoei (Jamieson et al., 1996).
epithelia. Spermatogonia and all stages of the meiotic Here the nucleus has a width of 0.4 mm below the end
phase of spermatogenesis are identical in both types of of the acrosomal cone and of 0.7 mm shortly before its

Fig. 5. Cross-sections of ductus epididymidis and ductus deferens in Lygodactylus capensis (a–c) and Lepidodactylus lugubris (d–h).
Paraffin section (e), semithin sections (a, b, d) and ultrathin sections (c, f–h). (a) Ductus epididymidis and (b) ductus deferens filled
with normally developed, small spermatozoa. (c) Longitudinal section of a normal spermatozoon in the ductus epididymidis with
elongated, thin nucleus, neck region containing the proximal centriole (arrowhead) and a midpiece housing the flagellum,
mitochondria and electron-dense intermitochondrial material. The arrow marks a cross-section of a principal piece. Inset: Acrosome
with acrosomal vesicle, subacrosomal cone, nuclear point. The perforatorium is cut basally. (d) Ductus epididymidis and (e) ductus
deferens of the phenotypic male containing relatively few spermatozoa, the majority of them with large, densely stained heads. (f)
Longitudinal section of a deformed spermatozoon in the ductus epididymidis with large, cup-shaped nucleus and two flagella. (g)
Cross-section of a midpiece with two flagella (arrowhead) and several cross-sections of principal pieces (arrows). (h) Longitudinal
section of a seemingly normal spermatozoon of Lepidodactylus lugubris with elongated head. Inset: Seemingly normal acrosome with
acrosomal vesicle, subacrosomal cone, nuclear point and perforatorium. Scale bars: 50 mm (a, b, d, e), 1 mm (c, f, g, h, insets).
 ARTICLE IN PRESS
398 B. Röll, M.U.G. von Düring / Zoology 111 (2008) 385–400

posterior end. Apparently, no further data about the cytes of the normal male are reduced to about 16–18%
sizes of spermatids or spermatozoa of other gekkonid in the early spermatids, whereas those of the phenotypic
species are available in the literature. male are only reduced to about 53–60% (Table 1). This
However, almost half of the mature spermatids and limited reduction may be at least partly due to the
spermatozoa of the phenotypic male have very large, increased content of genetic material. During spermio-
deformed nuclei. Furthermore, a number of spermato- genesis the genetic material of the early spermatid is
zoa possess two or three axonemes in their tails. These further strongly condensed in order to produce small
spermatozoa are presumably not capable of fertilisation. spermatozoa for the transportation of this material,
It seems that the release of these deformed spermatids normally only a haploid set of chromosomes. Appar-
into the tubular lumen is impaired. As a consequence, ently, the nuclei of the developing spermatids of the
the spermatids remain within the epithelium for a phenotypic males of L. lugubris are unable to contain a
prolonged time, and this disturbs the normal arrange- complete diploid set of chromosomes in normally
ment in the seminiferous epithelium. condensed nuclei. Thus, many nuclei of late spermatids
and spermatozoa are larger and deformed compared to
nuclei of normal gekkonid spermatids. The abnormal
Possible reason for the dysfunction of spermiogenesis spermatids and spermatozoa of L. lugubris resemble the
spermatozoon-like cells of the medaka hybrid male
As a hybrid, L. lugubris possesses high heterozygosity, (Shimizu et al., 1997). The doubled content of chromo-
which renders correct homologous chromosome pairing somes may also cause regulatory problems leading to
impossible. In the parthenogenetic females, a premeiotic the occurrence of more than one flagellum.
endomitosis enables both a quasi-normal meiosis and The seemingly ‘normal’ spermatozoa of the pheno-
the restoration of diploidy of oocytes. typic male L. lugubris are thought to have lost genetic
Endomitosis may be a phenomenon specific to material during condensation of their nuclei and thus
oogenesis in hybrid females, as it occurs in female are obviously aneuploid. This lost material may be
hybrids of fishes, amphibians and reptiles (Cuellar, 1971; phagozytised by Sertoli cells or it may be found in the
Kobel and DuPasquier, 1975). E.g., female hybrids of markedly increased amounts of nuage of spermatids of
the medaka fish (genus Oryzias) double their chromo- L. lugubris compared with the amounts of nuage in
somes by premeiotic endomitosis and produce fertile normal spermatids of male Lygodactylus.
diploid eggs (Shimizu et al., 2000); in contrast, male F1 One phenotypic male L. lugubris from the Ryukyu
hybrids of the medaka are not able to perform normal Archipelago also arose within an all-female strain
meiosis after a preceding endomitosis. The meiosis of (Yamashiro and Ota, 1998). This male possessed
their spermatogenic cells is arrested before reaching preanal and femoral pores, hemipenes and normally
metaphase I. Still, spermatogenesis continues and some developed testes, epididymes and ductus epididymidis.
spermatozoon-like cells with abnormally large nuclei However, mature spermatids or spermatozoa were
and abnormal flagella develop (Shimizu et al., 1997). nearly absent both in the seminiferous tubules and in
Here, and probably in other comparable situations, the ductus epididymidis as revealed in paraffin sections
male F1 hybrids are not capable of premeiotic endomi- (Yamashiro and Ota, 1998). Perhaps there was an
tosis. This could well be one of the primary mechanisms almost complete breakdown during spermiogenesis as
for sterility in F1 hybrid males (Haldane’s rule), even in the Japanese phenotypic male belonged to a triploid
species with homomorphic sex chromosomes. clone and therefore the triplicate content of genetic
However, the phenoytpic males of L. lugubris material had greatly enlarged the problem of storing this
investigated here are not F1 hybrids, but males which mass in a tiny nucleus.
arose spontaneously within an all-female strain. Thus, In conclusion, the ‘‘phenotypic’’ males of L. lugubris
these males have genomes identical to those of the are normal males, as far as the presence of male
females; they inherited on one side the high hetero- secondary sexual characters, gross morphology of the
zygosity that prevents a normal meiosis and on the other testes, and even microscopical appearance of Leydig and
side the ability for a premeiotic endomitosis and thus for Sertoli cells and the existence of normal meiotic stages
the realisation of a quasi-normal meiosis. We assume during spermatogenesis are concerned. There is just one
that – similar to the situation during oogenesis – a defect occurring during spermiogenesis which is never-
premeiotic endomitosis precedes spermatogenesis which theless decisive for these males: the inability to produce
should result in diploid spermatids and spermatozoa. functional diploid spermatozoa. Because of the high
The nuclei of early spermatids are much larger in number of deformed spermatozoa and possibly uncon-
diameter in the phenotypic males than those of the trolled loss of genetic material in structurally ‘normal’,
normal males of Lygodactylus. In contrast, the nuclei of but aneuploid spermatozoa, we conclude that these
primary spermatocytes are only slightly larger. The otherwise perfect males are infertile. Thus, they con-
calculated nuclear volumes of the primary spermato- stitute another example of gametic sterility.
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B. Röll, M.U.G. von Düring / Zoology 111 (2008) 385–400 399

Acknowledgements Ineich, I., 1988. Mise en évidence d’un complex unisexué-

bisexué chez le gecko Lepidodactylus lugubris (Sauria,
Special thanks are due to Mr. Erwin Schröder, Kiel, for Lacertilia) en Polynésie française. C.R. Acad. Sci. Paris
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Lepidodactylus lugubris. B.R. would like to thank the Jamieson, B.G.M., Oliver, S.C., Scheltinga, D.M., 1996. The
authorities of South African provinces Mpumalanga and ultrastructure of the spermatozoa of Squamata. I. Scinci-
dae, Gekkonidae and Pygopodidae (Reptilia). Acta Zool.
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Kobel, H.R., DuPasquier, L., 1975. Strains and species of
prepared the semithin and ultrathin sections and to Mrs. Xenopus for immunological research. In: Solomon, J.B.,
Helga Pollmann for preparing the paraffin sections. Horton, J.D. (Eds.), Developmental Immunology. Elsevier/
North-Holland Biomedical Press, Amsterdam, pp.
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