ArticlePDF Available

Abstract and Figures

Sex chromosomes are believed to be stable in endotherms, but young and evolutionary unstable in most ectothermic vertebrates. Within lacertids, the widely radiated lizard group, sex chromosomes have been reported to vary in morphology and heterochromatinization, which may suggest turnovers during the evolution of the group. We compared the partial gene content of the Z-specific part of sex chromosomes across major lineages of lacertids and discovered a strong evolutionary stability of sex chromosomes. We can conclude that the common ancestor of lacertids, living around 70 million years ago, already had the same highly differentiated sex chromosomes. Molecular data demonstrating an evolutionary conservation of sex chromosomes have also been documented for iguanas and colubroid snakes. It seems that differences in the evolutionary conservation of sex chromosomes in vertebrates do not reflect the distinction between endotherms and ectotherms, but rather between amniotes and anamniotes, or generally, the differences in the life-history of particular lineages This article is protected by copyright. All rights reserved.
Content may be subject to copyright.
Conservation of sex chromosomes in lacertid lizards
*Department of Ecology, Faculty of Science, Charles University in Prague, Vini
a 7, 128 44 Prague, Czech Republic, Institute
of Animal Physiology and Genetics, The Czech Academy of Sciences, Lib
echov, Czech Republic, Department of Zoology,
National Museum, V
am. 68, 115 79 Prague, Czech Republic
Sex chromosomes are believed to be stable in endotherms, but young and evolutionary
unstable in most ectothermic vertebrates. Within lacertids, the widely radiated lizard
group, sex chromosomes have been reported to vary in morphology and heterochroma-
tinization, which may suggest turnovers during the evolution of the group. We
compared the partial gene content of the Z-specific part of sex chromosomes across
major lineages of lacertids and discovered a strong evolutionary stability of sex
chromosomes. We can conclude that the common ancestor of lacertids, living around
70 million years ago (Mya), already had the same highly differentiated sex
chromosomes. Molecular data demonstrating an evolutionary conservation of sex chro-
mosomes have also been documented for iguanas and caenophidian snakes. It seems
that differences in the evolutionary conservation of sex chromosomes in vertebrates do
not reflect the distinction between endotherms and ectotherms, but rather between
amniotes and anamniotes, or generally, the differences in the life history of particular
Keywords: lizards, molecular sexing, reptiles, sex chromosomes
Received 29 December 2015; revision received 7 March 2016; accepted 22 March 2016
In vertebrates, the gonad is not differentiated early in
ontogeny and only later develops into testicular or ovar-
ian structures. Although the genetic framework for the
differentiation of testes or ovaries is highly conserved,
the process of sex determination which decides whether
the undifferentiated gonad will turn into a testis or an
ovary, is surprisingly variable. Also, the rate of turn-
overs of sex-determining mechanisms is notably differ-
ent among particular vertebrate lineages. Some
ectothermic lineages, such as several well-studied
groups of fish or frogs, possess a rapid turnover of sex
chromosomes (Miura 2007; Kikuchi & Hamaguchi 2013;
Dufresnes et al. 2015), while endotherms, that is mam-
mals and birds, have highly conserved sex chromosomes
(e.g. Shetty et al. 1999; Graves 2006). As ectotherms, rep-
tiles are usually considered as a group with a rapid turn-
over of sex-determining mechanisms (Sarre et al. 2004;
Organ & Janes 2008; Grossen et al. 2011), and as a whole,
they indeed exhibit a large variability in sex-determining
systems. However, this variability seems to be dis-
tributed unequally among particular reptile lineages. As
far as is known, all crocodiles share environmental sex
determination (ESD; Valenzuela & Lance 2004), where
the sex of an individual is decided by environmental
conditions during the sensitive period of embryonic
development. In comparison, turtles and lepidosaurs (tu-
ataras and squamates) possess variability in sex-deter-
mining systems, and both ESD and genotypic sex
determination (GSD, where the sex of an individual is
set by its sex-specific genotype) can be found in different
species of these lineages (Janzen & Phillips 2006;
a & Kratochv
ıl 2009; Valenzuela & Adams 2011;
Gamble et al. 2015; Johnson Pokorn
a & Kratochv
ıl 2016).
Nevertheless, based on the phylogenetic distribution of
the types of sex chromosomes given by classical cytoge-
netic data (Pokorn
a & Kratochv
ıl 2009; Gamble et al.
2015), some lineages of squamates might possess evolu-
tionary highly conserved sex chromosomes, although the
cytogenetic data might not always be reliable in
Correspondence: Luk
s Kratochv
ıl, Fax: +420 221951673;
©2016 John Wiley & Sons Ltd
Molecular Ecology (2016) doi: 10.1111/mec.13635
demonstrating the homology of sex chromosomes. Cyto-
genetically similar sex chromosomes might appear to be
nonhomologous in related lineages (Vicoso & Bachtrog
2015), and on the other hand, sex chromosomes can be
homologous in spite of observed variability in morphol-
ogy and heterochromatinization (Rovatsos et al. 2014a,b;
aet al. 2016). Despite significant progress in
recent years, molecular data on the evolutionary stability
of sex chromosomes among squamates exist only for
iguanas (Pleurodonta; Rovatsos et al. 2014a,b) and caeno-
phidian snakes (e.g. Matsubara et al. 2006; Vicoso et al.
2013; Rovatsos et al. 2015). These studies show that at
least in some cases, conservation of sex chromosomes in
ectothermic vertebrates can be comparable to
endotherms, but little is known whether snakes and
iguanas are rules or exceptions to the more general pat-
The lizards of the family Lacertidae represent a very
important part of diurnally active reptiles in Europe,
Asia, Africa and adjacent islands (for instance, they
play a very important ecological role in many Mediter-
ranean islands and in the Canary Islands). They occupy
an extensive range of environments, from rain forests
through to deserts, with the notable case of the lizard
Zootoca vivipara, having a wide distribution across the
Palearctic region, from Europe to Japan and even north
of the Polar Circle. Lacertids are mostly terrestrial, but
several species are saxicolous or even arboreal and
partly fossorial (Pough et al. 2003). Currently, 322 spe-
cies categorized into 42 genera have been recognized
(Uetz & Ho
sek 2015). The phylogenetic relationships
among lacertids are not fully resolved, and conflicting
topologies can be found among recent phylogenetic
studies based on molecular data (Arnold et al. 2007;
Mayer & Pavlicev 2007; Pyron et al. 2013). However, the
splitting of the family into two subfamilies (Gallotiinae
and Lacertinae with two tribes: Eremiadini and Lacer-
tini) has been well supported and now accepted. The
precise age of the group is not known, but the split
between lacertids and their likely sister group, limbless
fossorial amphisbaenians, has been estimated at approx-
imately 110130 Mya (Hedges et al. 2006), while the
basal divergence just within the tribe Lacertini based on
molecular clocks was estimated to be the surprisingly
young age of 1216 Mya (Arnold et al. 2007).
Studies have revealed that lacertids possess GSD
(reviewed, e.g. in Pokorn
a & Kratochv
ıl 2009, one older
report of ESD in a single species, Podarcis pityusensis,is
dubious; see, e.g. critical review in Pokorn
ıl 2009). Obligatory unisexuality exists within
the genus Darevskia (e.g. Kupriyanova 2010). Wherever
known, sex chromosomes of lacertids point to a female
heterogamety (reviewed in Olmo & Signorino 2015).
With the exception of several lineages with multiple
neo-sex chromosomes (Rojo et al. 2014; reviewed in
aet al. 2014a), the Z and W sex chromosomes
are believed to be rather homomorphic and are only
cytogenetically distinguishable by C-banding detection
of the notable heterochromatin accumulation on the W
chromosomes (Olmo et al. 1987; Pokorn
aet al. 2011a).
The heterochromatization of the W chromosome is
likely a result of its considerable degeneration. In some
species, the W chromosome contains an enormous accu-
mulation of repetitive elements (Pokorn
aet al. 2011a in
Eremias velox), while sex chromosome size differs across
lacertid species and W chromosomes are euchromatic in
certain lacertid lineages (Olmo et al. 1987). The latter
fact led to the hypothesis that differentiation of sex
chromosomes took place repeatedly and independently
in different taxa within the family (Odierna et al. 1993).
Alternatively, nonhomologous sex chromosomes may
be present in different lineages in lacertids. The gene
content of the Z chromosome was reported in the
Swedish population of Lacerta agilis (Srikulnath et al.
2014), where the lacertid Z chromosome was suggested
to be homologous to a part of the third largest chromo-
some pair of Anolis carolinensis, the model species for
reptile genomics. However, the analysis of transcrip-
tome in Takydromus sexlinaeatus and the test of Z-specifi-
city based on qPCR in this species and in the Czech
population of Lacerta agilis revealed that the genes from
this region are in fact not Z-specific (Rovatsos et al.
2016). This finding suggests either a turnover of sex
chromosomes within Lacerta agilis, several turnovers of
sex chromosomes in lacertids as a whole or the
misidentification of the Z-specific region in the previous
study (Srikulnath et al. 2014). Only, further comparative
study within lacertids can resolve this issue and
uncover the degree of conservation of sex chromosomes
in this clade. In this study, we tested the competing
hypotheses on homology and differentiation of sex
chromosomes across lacertids.
Materials and methods
Material and ethics statement
Tissue or blood samples in ethanol were acquired from
32 individuals from 16 species of lacertids (one male
and one female per species), covering all major lacertid
clades (Table S1, Supporting information). Particular
attention was taken to include as many species from the
genus Lacerta and its close relatives (genera Timon and
Podarcis) as possible, as previous studies have suggested
a variation in sex chromosomes within this genus
(Srikulnath et al. 2014; cf. to Rovatsos et al. 2016).
All experimental procedures were carried out under
the supervision and with the approval of the Ethics
©2016 John Wiley & Sons Ltd
Committee of the Faculty of Science, Charles University
in Prague, followed by the Ministry of Education, Youth
and Sports (permission No. 35484/2015-14). Permissions
were granted for collecting lacertid species in Greece in
the jurisdiction area of the Management Body of Mt
Parnonas and Moustos Wetlands (Protocol No. 474, 29/
5/2013) and Management Body of Chelmos-Vouraikos
(Protocol No. 746, 11/8/2014), in Yemen (permission
No. 10/2007 issued by the Environment Protection
Agency, Sana’a, Republic of Yemen) and in France (per-
missions Nos. 29/2012 and 11/DDTM/657-SERN-NB
issued by Direction R
egionale de l’Environnement, de
enagement et du Logement).
Test of homology of sex chromosomes by qPCR
In organisms with degenerated W chromosomes, the
males (ZZ) have twice as many copies of most genes
linked to the Z-specific part of sex chromosomes than the
females (ZW), while genes in autosomal or pseudoauto-
somal regions have equal copy numbers in both sexes.
This difference in copy number between sexes can be
determined by qPCR, allowing the reliable identification
of Z- (or X-)specific genes (Rovatsos et al. 2014a,b,c, 2015,
2016; for similar application of qPCR, see also Nguyen
et al. 2013; Gamble et al. 2014; Literman et al. 2014).
Genomic DNA was extracted using a DNeasy Blood
and Tissue Kit (Qiagen). Primer pairs (see Table S2,
Supporting information for list) were designed for the
amplification of the 120200 bp exon fragment of the
single-copy gene elongation factor 1a (eef1a1), two auto-
somal ‘control’ genes (fbxw7,adarb2), five Z-specific
genes (mars2,lpar4,klhl13,angptl2,slc31a1) previously
identified in Takydromus sexlineatus and in the Czech
population of Lacerta agilis (Rovatsos et al. 2016) and
three genes (mecom,mynn,sh3pxd2a) recently identified
as Z-linked in Lacerta agilis in the study by Srikulnath
et al. (2014), using Primer-BLAST software (Ye et al.
2012). The control genes have orthologs linked to chro-
mosomes 2 and 4 in the zebrafinch (Taeniopygia guttata,
TGU), the five Z-specific genes found in the two lacer-
tids have orthologs linked to TGU 4A and TGU 17, and
the candidate genes from the study by Srikulnath et al.
(2014) are linked to TGU 6 and 9. Instead of the green
anole or the chicken genomes, the topology of the gen-
ome of the zebrafinch is used for this study. This is
because many of the genes in the green anole are still
only on scaffolds not linked to particular chromosomes
oldi et al. 2011; and chromo-
somes 4 and 4A of the zebrafinch are fused in the
chicken genome leading to a lower resolution of the
physical localization in chicken in comparison with
TGU. The qPCR with DNA template was carried out in
a LightCycler II 480 (Roche Diagnostics) with all
samples run in triplicate. The detailed qPCR protocol
and the formula for the calculation of the relative gene
dose between sexes have been presented in our previ-
ous articles (Rovatsos et al. 2014a,b). A relative female-
to-male gene dosage ratio (r) of 0.5 is expected for Z-
specific genes and 1.0 for pseudoautosomal or autoso-
mal genes.
We tested the relative gene dose (r) between sexes for 10
loci in 16 species of lacertid lizards with qPCR (see Fig. 1;
Table S3, Supporting information). Although not all of
the loci were successfully amplified, a minimum of 5 loci
(median 9 loci) were tested in each species. The two auto-
somal ‘control’ genes (fbxw7,adarb2) and the three genes
(mecom,mynn,sh3pxd2a) identified as Z-linked in Lacerta
agilis in the previous study (Srikulnath et al. 2014) pro-
vided equal gene doses between sexes in all 16 of the
tested species here (see Fig. 1; Table S3, Supporting infor-
mation) and in two species tested previously (Rovatsos
et al. 2016), indicating that these loci have autosomal or
pseudoautosomal topology in the lacertid genomes.
In contrast, the five genes (mars2,lpar4,klhl13,angptl2,
slc31a1) identified as Z-linked in Takydromus sexlineatus
and in the Czech population of Lacerta agilis (Rovatsos
et al. 2016; three pairs were tested in each of these two
species) show relative gene dose ratios of approxi-
mately 0.5, indicating their Z-specific topology (see
Fig. 1; Table S3, Supporting information). Exceptions to
this pattern were found for the gene mars2 with
(pseudo)autosomal topology in Gallotia galloti and
Podarcis tauricus, and the gene angptl2 with (pseudo)au-
tosomal topology in Podarcis muralis (tested also with
the same result in the second pair of Podarcis muralis).
Our sampling included all major lineages of lacertids
covering both subfamilies (Gallotiinae and Lacertinae)
and both tribes (Eremiadini and Lacertini) of the sub-
family Lacertinae. Based on the Z-specificity of the
tested genes, all included species demonstrated homolo-
gous sex chromosomes. As our sampling included both
lineages arisen from the basal splitting of the recent lac-
ertids (Gallotiinae and Lacertinae), we can conclude
that the common ancestor of lacertids living c. 70 Mya
(Hedges et al. 2006) possessed the same, already highly
differentiated ZZ/ZW sex chromosomes currently
found in the recent species. Female heterogamety has
been documented in a single species of amphisbaenas
(Cole & Gans 1987), the first outgroup to lacertids (e.g.
Pyron et al. 2013), while the second outgroup (families
Gymnophthalmidae and Teiidae) possesses male
©2016 John Wiley & Sons Ltd
heterogamety (reviewed in Pokorn
a & Kratochv
ıl 2009).
The homology of sex chromosomes between lacertids
and their outgroups has yet to be studied and would be
necessary to determine the age of lacertid sex chromo-
somes more precisely. It is possible that the lacertid sex
chromosomes are older than the basal splitting of the
subfamilies Gallotiinae and Lacertinae. Nevertheless,
even this age and the phylogenetic coverage of the pre-
sent study demonstrate a strong conservation of sex
chromosomes in lacertids, the highly radiated and mor-
phologically and ecologically diversified lizard clade.
Sex chromosomes in lacertids are homologous and
highly differentiated in all of the tested species regard-
less of the reported differences in the heterochromatiza-
tion of the W chromosome and in size of the Z and W
chromosome (e.g. Odierna et al. 1993). Lacertids have
largely conserved karyotypes with mostly acrocentric
chromosomes varying only in size. Such chromosomes
are difficult to distinguish morphologically even after
differential staining (see, e.g. Pokorn
aet al. 2014b).
Highly degenerated W or Y chromosomes contain
dynamic repetitive sequences (Pokorn
aet al. 2011a;
Matsubara et al. 2016) and are rather variable in size
(Rutkowska et al. 2012). Assuming that sex
chromosomes are usually homomorphic in reptiles,
which used to be a common belief, one can easily
assemble the chromosomes into pairs where the W
chromosome is paired with an autosome of a similar
size, which is mistakenly assigned as the Z chromo-
some. Our present study demonstrates that Z chromo-
somes, or at least the parts of them containing the
tested genes in lacertids, are highly conserved, and we
therefore predict that lacertid Z chromosomes might in
fact also be similar in morphology. Improved cytoge-
netic characterization of the Z chromosomes across lac-
ertids would be beneficial in further studies. The
similarity of chromosomes in lacertid karyotypes might
also be responsible for a possible error in the determi-
nation of the genetic content of the Z chromosome in
the Swedish population of Lacerta agilis in the cytoge-
netic study by Srikulnath et al. (2014). It seems unlikely
that the sex chromosomes widely conserved across lac-
ertids as shown in the present study would differ only
between our tested Czech samples and the previously
studied Swedish populations of a single species. Never-
theless, the pattern consistent with the exceptional
pseudoautosomal or autosomal position from qPCR in
the gene angptl2 in Podarcis muralis and in the gene
Fig. 1 Relative gene dose ratios between
female and male genomes in 18 species
of lacertid lizards. Means +SD are
depicted. Value 1.0 is expected for auto-
somal or pseudoautosomal genes, while
the value 0.5 is consistent with Z-specifi-
city. The exceptional values consistent
with (pseudo)autosomal position in the
gene mars2 in Gallotia galloti and Podarcis
tauricus, and in the gene angptl2 in Podar-
cis muralis were excluded for simplicity
(see text for details). Phylogenetic rela-
tionships follow Pyron et al. (2013). The
legend shows the linkage of genes to
zebrafinch (TGU) chromosomes. These
data suggest that the differentiated ZZ/
ZW sex chromosomes were already pre-
sent in the common ancestor of extant
lacertids and that they have been con-
served across the evolution of the group.
©2016 John Wiley & Sons Ltd
mars2 in Gallotia galloti and Podarcis tauricus (Table S3,
Supporting information) suggests that although generally
highly conserved, sex chromosomes in lacertids might
have been subjected to certain rearrangements such as
independent translocations of these loci from the ances-
tral Z chromosome to autosomes. The further study of
the situation in Lacerta agilis is therefore warranted.
Although reptiles as a whole are often viewed as a
group with a frequent turnover of sex-determining
systems, the emerging evidence (Pokorn
a & Kratochv
2009; Gamble et al. 2015; Johnson Pokorn
a & Kratochv
2016) suggests that in actual fact the variability can only
be found in three lineages: in turtles (Valenzuela &
Adams 2011), geckos (Pokorn
a & Kratochv
ıl 2009;
Gamble 2010; Pokorn
aet al. 2010, 2011b, 2014b;
aet al. 2014; Gamble et al. 2015) and in dragon
lizards (Ezaz et al. 2009). In these three ancient lineages,
this variability might be explained by the presence of
ancestral ESD and repeated independent emergences of
sex chromosomes (Gamble et al. 2015; Johnson Pokorn
& Kratochv
ıl 2016). Sex-determining systems, particu-
larly GSD systems and hence sex chromosomes, might
be stable in many other reptile lineages (Pokorn
ıl 2009; Gamble et al. 2015), but currently there
is a lack of molecular evidence to determine this. So far
among amniotes a high evolutionary stability of sex
chromosomes has been confirmed by molecular evi-
dence in birds (ZW; Shetty et al. 1999), viviparous mam-
mals (XY; Graves 2006), iguanas (XY; Rovatsos et al.
2014b), caenophidian snakes (ZW; Matsubara et al. 2006;
Vicoso et al. 2013; Rovatsos et al. 2015) and lacertids
(ZW, this study). It is evident that evolutionary conser-
vation of sex chromosomes in amniotes is not connected
with heterogamety as lineages with both male and
female heterogamety show comparable conservation of
sex chromosomes, although XY and ZW sex chromo-
somes generally differ in many important aspects such
as in the tendency to evolve global dosage compensa-
tion (e.g. Vicoso & Bachtrog 2009; Mank 2009, 2013) or
to form multiple neo-sex chromosomes (Pokorn
aet al.
2014a; Pennell et al. 2015). It can be also concluded that
the stability of sex chromosomes has nothing to do with
endothermy versus ectothermy as was previously
suggested (e.g. Grossen et al. 2011). In contrast to
several lineages of anamniotes (cf. the situation in stick-
lebacks: Ross et al. 2009; medaka fish: Kikuchi &
Hamaguchi 2013; the frog genera Hyla: Dufresnes et al.
2015 and Rana: Miura 2007), up to now no case of fre-
quent and rapid turnovers of sex chromosomes has ever
been reported among amniotes. Therefore, we suggest
that the difference in the stability of sex chromosomes
does not follow the distinction between endotherms
and ectotherms, but more likely between amniotes ver-
sus anamniotes. Surprising recent evidence has shown
that effective population size, intensity of sexual selec-
tion and possibly also the rate of molecular evolution
reflected by intraspecific genetic polymorphism might
differ between lineages with different mortality of juve-
nile stages versus adults, respectively, different parental
investment to individual offspring reflected by propag-
ule size (Romiguier et al. 2014; Pischedda et al. 2015).
The putative link between the life history and the evo-
lutionary stability of sex chromosomes deserves further
theoretical and empirical studies.
The authors would like to express their gratitude to F. Marec
and R. Stopkov
a for sharing their knowledge of qPCR. We
thank T. Jir
asek and J. Tr
cek, Zoo Plze
n (Czech Republic)
for providing us with blood samples of Timon tangitanus,D.
Frynta for tissue samples of Gallotia galloti, T. Uller for tissue
samples of Podarcis muralis, G. Tryfonopoulos from Manage-
ment Body of Mt Parnonas and Moustos Wetlands (Greece)
and M. Kamilari from Management Body of Chelmos-Vourai-
kos (Greece) for providing us with permission to collect rep-
tiles and J.
Cervenka for taking care of our lacertids at Charles
University Animal Facilities (accreditation No. 13060/2014-
MZE-17214). The work of JM was financially supported by the
Ministry of Culture of the Czech Republic (DKRVO 2015/15,
National Museum, 00023272).
oldi J, Di Palma F, Grabherr M et al. (2011) The genome of
the green anole lizard and a comparative analysis with birds
and mammals. Nature,477, 587591.
AltmanováM, Rovatsos M, Kratochvíl L, Johnson PokornáM
(2016) Minute Y chromosomes and karyotype evolution in
Madagascan iguanas (Squamata: Iguania: Opluridae). Biologi-
cal Journal of the Linnean Society. doi: 10.1111/bij.12751.
Arnold EN, Arribas
O, Carranza S (2007) Systematics of the
Palaearctic and Oriental Lizard Tribe Lacertini (Squamata: Lacer-
tidae: Lacertinae), With Descriptions of Eight new Genera. Mag-
nolia Press, Auckland.
Cole CJ, Gans C (1987) Chromosomes of Bipes, Mesobaena, and
other amphisbaenians (Reptilia), with comments on their
evolution. American Museum Novitates,2869,19.
Dufresnes C, Borz
ee A, Horn A et al. (2015) Sex-chromosome
homomorphy in Palearctic tree frogs results from both turn-
overs and XY recombination. Molecular Biology and Evolution,
32, 23282337.
Ezaz T, Quinn AE, Sarre SD, O’meally D, Georges A, Graves
JA (2009) Molecular marker suggests rapid changes of sex-
determining mechanisms in Australian dragon lizards. Chro-
mosome Research,17,9198.
Gamble T (2010) A review of sex determining mechanisms in
geckos (Gekkota: Squamata). Sexual Development,4,88103.
Gamble T, Geneva AJ, Glor RE, Zarkower D (2014) Anolis sex
chromosomes are derived from a single ancestral pair. Evolu-
tion,68, 10271041.
Gamble T, Coryell J, Ezaz T, Lynch J, Scantlebury DP, Zar-
kower D (2015) Restriction site-associated DNA sequencing
©2016 John Wiley & Sons Ltd
(RAD-seq) reveals an extraordinary number of transitions
among gecko sex-determining systems. Molecular Biology and
Evolution,32, 12961309.
Graves JAM (2006) Sex chromosome specialization and degen-
eration in mammals. Cell,124, 901914.
Grossen C, Neuenschwander S, Perrin N (2011) Temperature-
dependent turnovers in sex-determination mechanisms: a
quantitative model. Evolution,65,6478.
Hedges SB, Dudley J, Kumar S (2006) TimeTree: a public
knowledge-base of divergence times among organisms.
Bioinformatics,22, 29712972.
Janzen FJ, Phillips PC (2006) Exploring the evolution of envi-
ronmental sex determination, especially in reptiles. Journal of
Evolutionary Biology,19, 17751784.
Johnson Pokorn
a M, Kratochv
ıl L (2016) What was the ances-
tral sex-determining mechanism in amniote vertebrates? Bio-
logical Reviews,91,112.
Kikuchi K, Hamaguchi S (2013) Novel sex-determining genes
in fish and sex chromosome evolution. Developmental Dynam-
ics,242, 339353.
a M, Johnson Pokorn
a M, Rovatsos M, Farka
a M, Kratochv
ıl L (2014) Sex determination in
Madagascar geckos of the genus Paroedura (Squamata:
Gekkonidae): are differentiated sex chromosomes indeed so
evolutionary stable? Chromosome Research,22, 441452.
Kupriyanova L (2010) Cytogenetic and genetic trends in the
evolution of unisexual lizards. Cytogenetic and Genome
Research,127, 273279.
Literman R, Badenhorst D, Valenzuela N (2014) qPCR-based
molecular sexing by copy number variation in rRNA genes
and its utility for sex identification in soft-shell turtles. Meth-
ods in Ecology and Evolution,5, 872880.
Mank JE (2009) The W, X, Y and Z of sex-chromosome dosage
compensation. Trends in Genetics,25, 226233.
Mank JE (2013) Sex chromosome dosage compensation: defi-
nitely not for everyone. Trends in Genetics,29, 677683.
Matsubara K, Tarui H, Toriba M et al. (2006) Evidence for dif-
ferent origin of sex chromosomes in snakes, birds, and mam-
mals and step-wise differentiation of snake sex
chromosomes. Proceedings of the National Academy of Sciences
USA,103, 1819018195.
Matsubara K, O’Meally D, Azad B et al. (2016) Amplification of
microsatellite repeat motifs is associated with the evolution-
ary differentiation and heterochromatinization of sex chro-
mosomes in Sauropsida. Chromosoma,125, 111123.
Mayer W, Pavlicev M (2007) The phylogeny of the family
Lacertidae (Reptilia) based on nuclear DNA sequences: conver-
gent adaptations to arid habitats within the subfamily Eremi-
ainae. Molecular Phylogenetics and Evolution,44, 11551163.
Miura I (2007) An evolutionary witness: the frog Rana rugosa
underwent change of heterogametic sex from XY male to
ZW female. Sexual Development,1, 323331.
Nguyen P, S
aJet al. (2013) Neo-sex chromo-
somes and adaptive potential in tortricid pests. Proceedings of
the National Academy of Sciences USA,110, 69316936.
Odierna G, Kupriyanova LA, Caprigilone T, Olmo E (1993) Fur-
ther data on sex chromosomes of Lacertidae and a hypothesis
on their evolutionary trend. Amphibia-Reptilia,14,111.
Olmo E, Signorino GG (2015) Chromorep: a reptile chromosomes
database. Accessed 21 March
Olmo E, Odierna G, Capriglione T (1987) Evolution of sex-
chromosomes in lacertid lizards. Chromosoma,96,3338.
Organ CL, Janes DE (2008) Evolution of sex chromosomes
in Sauropsida. Integrative and Comparative Biology,48,
Pennell MW, Kirkpatrick M, Otto SP et al. (2015) Y fuse? Sex
chromosome fusions in fishes and reptiles. PLoS Genetics,11,
Pischedda A, Friberg U, Stewart AD, Miller PM, Rice WR
(2015) Sexual selection has minimal impact on effective pop-
ulation sizes in species with high rates of random offspring
mortality: an empirical demonstration using fitness distribu-
tions. Evolution,69, 26382647.
a M, Kratochv
ıl L (2009) Phylogeny of sex-determining
mechanisms in squamate reptiles: are sex chromosomes an
evolutionary trap? Zoological Journal of the Linnean Society,
156, 168183.
ab P, Ferguson-Smith MA, Rens W,
ıl L (2010) Differentiation of sex chromosomes and
karyotypic evolution in the eye-lid geckos (Squamata:
Gekkota: Eublepharidae), a group with different modes of
sex determination. Chromosome Research,18, 809820.
a M, Kratochv
ıl L, Kejnovsk
y E (2011a) Microsatellite
distribution on sex chromosomes at different stages of
heteromorphism and heterochromatinization in two lizard
species (Squamata: Eublepharidae: Coleonyx elegans and
Lacertidae: Eremias velox). BMC Genetics,12, 90.
a M, Giovannotti M, Kratochv
ıl L et al. (2011b) Strong
conservation of the bird Z chromosome in reptilian genomes
is revealed by comparative painting despite 275 million
years divergence. Chromosoma,120, 455468.
a M, Altmanov
a M, Kratochv
ıl L (2014a) Multiple sex
chromosomes in the light of female meiotic drive in amniote
vertebrates. Chromosome Research,22,3544.
a M, Rens W, Rovatsos M, Kratochv
ıl L (2014b) A ZZ/
ZW sex chromosome system in the thick-tailed gecko (Under-
woodisaurus milii; Squamata: Gekkota: Carphodactylidae), a
member of the ancient gecko lineage. Cytogenetic and Genome
Research,142, 190196.
Pough FH, Andrews RM, Cadle JE, Crump ML, Savitsky AH,
Wells KD (2003) Herpetology, 3rd edn. Benjamin Cummings,
San Francisco, California.
Pyron RA, Burbrink FT, Wiens JJ (2013) A phylogeny and
revised classification of Squamata, including 4161 species of
lizards and snakes. BMC Evolutionary Biology,13, 93.
Rojo V, Giovannotti M, Naveira H et al. (2014) Karyological char-
acterization of the endemic iberian rock lizard, Iberolacerta
monticola (Squamata, Lacertidae): insights into sex chromo-
some evolution. Cytogenetic and Genome Research,142,2839.
Romiguier J, Gayral P, Ballenghien M et al. (2014) Comparative
population genomics in animals uncovers the determinants
of genetic diversity. Nature,515, 261263.
Ross JA, Urton JR, Boland JE, Shapiro MD, Peichel CL (2009)
Turnover of sex chromosomes in the stickleback fishes (Gas-
terosteidae). PLoS Genetics,5,112.
Rovatsos M, Altmanov
a M, Pokorn
a M, Kratochv
ıl L (2014a)
Conserved sex chromosomes across adaptively radiated Ano-
lis lizards. Evolution,68, 20792085.
Rovatsos M, Pokorn
a M, Altmanov
a M, Kratochv
ıl L (2014b)
Cretaceous park of sex determination: sex chromosomes are
conserved across iguanas. Biology Letters,10, 20131093.
©2016 John Wiley & Sons Ltd
Rovatsos M, Altmanova M, Pokorna MJ, Kratochvil L (2014c)
Novel X-linked genes revealed by quantitative polymerase
chain reaction in the green anole, Anolis carolinensis.G3,
Genes|Genomes|Genetics,4, 21072113.
Rovatsos M, Vuki
c J, Lymberakis P, Kratochv
ıl L (2015) Evolu-
tionary stability of sex chromosomes in snakes. Proceedings of
the Royal Society B,282, 20151992.
Rovatsos M, Vukic J, Kratochv
ıl L (2016) Mammalian X homo-
log acts as sex chromosome in lacertid lizards. Heredity,
Rutkowska J, Lagisz M, Nakagawa S (2012) The long and the
short of avian W chromosomes: no evidence for gradual W
shortening. Biology Letters,8, 636638.
Sarre SD, Georges A, Quinn A (2004) The ends of a continuum:
genetic and temperature-dependent sex determination in
reptiles. BioEssays,26, 639645.
Shetty S, Griffin DK, Graves JAM (1999) Comparative painting
reveals strong chromosome homology over 80 million years
of bird evolution. Chromosome Research,7, 289295.
Srikulnath K, Matsubara K, Uno Y, Nishida C, Olsson M, Mat-
suda Y (2014) Identification of the linkage group of the Z sex
chromosomes of the sand lizard (Lacerta agilis, Lacertidae)
and elucidation of karyotype evolution in lacertid lizards.
Chromosoma,123, 563575.
Uetz P, Ho
sek J, eds. (2015) The Reptile Database. http:// Accessed 21 March 2015.
Valenzuela N, Adams DC (2011) Chromosome number and sex
determination coevolve in turtles. Evolution,65, 18081813.
Valenzuela N, Lance V (2004) Temperature-Dependent sex Deter-
mination in Vertebrates. Smithsonian Books, Washington, Dis-
trict of Columbia.
Vicoso B, Bachtrog D (2009) Progress and prospects toward
our understanding of the evolution of dosage compensation.
Chromosome Research,17, 585602.
Vicoso B, Bachtrog D (2015) Numerous transitions of sex chro-
mosomes in diptera. PLoS Biology,13, e1002078.
Vicoso B, Emerson JJ, Zektser Y, Mahajan S, Bachtrog D (2013)
Comparative sex chromosome genomics in snakes: differenti-
ation, evolutionary strata, and lack of global dosage compen-
sation. PLoS Biology,11, e1001643.
Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden T
(2012) Primer-BLAST: a tool to design target-specific primers
for polymerase chain reaction. BMC Bioinformatics,13, 134.
M.R., L.K., J.V., M.A. and M.J.P. designed the research;
M.R., J.V. and L.K. performed the research; J.M. con-
tributed the material; M.R. analysed the data; M.R. and
L.K. wrote the first draft; all authors edited the manu-
Data accessibility
All data are presented in the Table S3 (Supporting
Supporting information
Additional supporting information may be found in the online ver-
sion of this article.
Table S1 Lacertid specimens used in the study and their ori-
Table S2 Primers used for the measurement of relative gene
dosage by qPCR.
Table S3 Relative gene dose ratios (r) between female and
male genomes in lacertids.
©2016 John Wiley & Sons Ltd
... The hypothesis on the multiple independent transitions from the ancestral ESD to GSD in geckos predicts that ESD in this genus is either not supported, or that GSD species of carphodactylids phylogenetically separated by ESD should have nonhomologous sex chromosomes. In order to expand our knowledge on sex determination in geckos and to test this specific prediction, here, we sequenced the whole genome from a male and a female individual of U. milii and a male and a female individual of Saltuarius cornutus, with the aim to uncover gene content of their sex chromosomes by comparative genome coverage analysis (e.g., Vicoso and Bachtrog 2011;Vicoso et al. 2013;Picard et al. 2018), and to validate sex linkage of a subset of genes revealed from the bioinformatic analysis by quantitative real-time PCR (qPCR) (e.g., Rovatsos, Altmanov a, Johnson Pokorn a, et al. 2014;Rovatsos, Vuki c, et al. 2015;Rovatsos, Vuki c, et al. 2016). The sex linkage of genes was further tested by qPCR in other species from the family Carphodactylidae to explore the homology of sex chromosomes across the phylogenetic spectrum of this gecko lineage and to test homology and stability of GSD across gekkotan lizards. ...
... The knowledge of sex-linked genes can be used in the qPCR-based method of molecular sexing in members of the genera Underwoodisaurus, Nephrurus, and Saltuarius, as was previously developed for anguimorphan reptiles, caenophidian snakes, iguanas, lacertids, skinks, and trionychid turtles Rovatsos, Vuki c, et al. 2015;Rovatsos, Vuki c, et al. 2016;Rovatsos et al. 2017;Rovatsos, Reh ak, et al. 2019;Rovatsos, Vuki c, et al. 2019;Kostmann et al. 2021). Such molecular sexing method can be important for instance in breeding projects and developmental studies requiring knowledge of the sex of embryos. ...
... These genes have homologs to the genomic regions GGA10, GGA17, GGA22, and GGA24, which are involved on the sex chromosomes of either U. milii or S. cornutus ( fig. 1 and supplementary tables S2 and S3, Supplementary Material online). In addition, we also tested by qPCR five genes homologous to GGA4 (bmf, maml3, mbnl3, zdhhc9) and to GGA15 (derl3) which were Z-linked in several species of geckos from the genus Paroedura (Rovatsos, Farka cov a, et al. 2019) and a single gene homologous to GGA5 (noct), which is X-specific in pygopodid geckos We used a qPCR method to calculate the relative gene copy number variation between the male and female genome and to test the Z-specificity of the candidate Z-specific genes Rovatsos, Vuki c, et al. 2015;Rovatsos, Vuki c, et al. 2016;Rovatsos et al. 2017;Nielsen, Guzm an-M endez, et al. 2019;Rovatsos, Farka cov a, et al. 2019;Rovatsos, Reh ak, et al. 2019;Rovatsos, Vuki c, et al. 2019). With the same reasoning as for the comparative genome coverage, males (ZZ) have double copies of Z-specific genes compared with females (ZW) in species with degenerated nonrecombining W chromosomes. ...
Full-text available
Amniotes possess astonishing variability in sex determination ranging from environmental sex determination (ESD) to genotypic sex determination (GSD) with highly differentiated sex chromosomes. Geckos are one of the few amniote groups with substantial variability in sex determination. What makes them special in this respect? We hypothesized that the extraordinary variability of sex determination in geckos can be explained by two alternatives: 1) unusual lability of sex determination, predicting that the current GSD systems were recently formed and are prone to turnovers; 2) independent transitions from the ancestral ESD to later stable GSD, which assumes that geckos possessed ancestrally ESD, but once sex chromosomes emerged, they remain stable in the long-term. Here, based on genomic data, we document that the differentiated ZZ/ZW sex chromosomes evolved within carphodactylid geckos independently from other gekkotan lineages and remained stable in the genera Nephrurus, Underwoodisaurus and Saltuarius for at least 15 million of years (MY) and potentially up to 45 MY. These results together with evidence for the stability of sex chromosomes in other gekkotan lineages support more our second hypothesis suggesting that geckos do not dramatically differ from the evolutionary transitions in sex determination observed in the majority of the amniote lineages.
... those on the X chromosome but absent in the degenerated part of the Y chromosome). The differences in gene copy numbers between sexes triggered by the degeneration of the Y chromosome can also be directly measured by qPCR applied to genomic DNA [16,28,48,49]. In L. burtonis, we used this approach for the validation of X-specificity in a subset of loci from the candidate putative syntenic blocks. ...
... A relative male-to-female gene dose ratio (r) of 0.5 is expected for X-specific genes and of 1.0 for autosomal and pseudoautosomal genes, and genes with poorly differentiated gametologs. We recently used similar methodology to discover sex-linked genes in lacertid and anguimorphan lizards and in the gecko genus Paroedura [16,28,49]. ...
Full-text available
Differentiation of sex chromosomes is thought to have evolved with cessation of recombination and subsequent loss of genes from the degenerated partner (Y and W) of sex chromosomes, which in turn leads to imbalance of gene dosage between sexes. Based on work with traditional model species, theory suggests that unequal gene copy numbers lead to the evolution of mechanisms to counter this imbalance. Dosage compensation, or at least achieving dosage balance in expression of sex-linked genes between sexes, has largely been documented in lineages with male heterogamety (XX/XY sex determination), while ZZ/ZW systems are assumed to be usually associated with the lack of chromosome-wide gene dose regulatory mechanisms. Here, we document that although the pygopodid geckos evolved male heterogamety with a degenerated Y chromosome 32–72 Ma, one species in particular, Burton's legless lizard ( Lialis burtonis ), does not possess dosage balance in the expression of genes in its X-specific region. We summarize studies on gene dose regulatory mechanisms in animals and conclude that there is in them no significant dichotomy between male and female heterogamety. We speculate that gene dose regulatory mechanisms are likely to be related to the general mechanisms of sex determination instead of type of heterogamety. This article is part of the theme issue ‘Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part II)’.
... Squamates (lizards and snakes) as a whole have more diverse sex-determination systems than other amniotes, but there is a high degree of sex chromosome conservation within most suborders or families (Pokorna & Kratochvíl, 2009). These include the XX/XY sex chromosomes of skinks (Kostmann et al., 2021), the ZZ/ZW sex chromosomes of lacertids (Rovatsos, Vukić, et al., 2016), the XX/ XY of most pleurodonts (Nielsen, Guzmán-Méndez, et al., 2019;Rovatsos et al., 2014) and the ZZ/ZW of caenophidian snakes (Matsubara et al., 2006;Vicoso, Emerson, et al., 2013). Gecko lizards, on the other hand, have evolved multiple sex chromosome systems (Gamble, Coryell, et al., 2015), likely owing to a common ancestor that possessed temperature-dependent sex determination (TSD) (Pokorna & Kratochvíl, 2009;Gamble, Coryell, et al., 2015) with different daughter lineages subsequently evolving sex-determining loci on different syntenic blocks (Augstenová et al., 2021). ...
Full-text available
Sex‐determination systems are highly variable amongst vertebrate groups, and the prevalence of genomic data has greatly expanded our knowledge of how diverse some groups truly are. Gecko lizards are known to possess a variety of sex‐determination systems, and each new study increases our knowledge of this diversity. Here, we used RADseq to identify male‐specific markers in the banded gecko Coleonyx brevis, indicating this species has a XX/XY sex‐determination system. Furthermore, we show that these sex‐linked regions are not homologous to the XX/XY sex chromosomes of two related Coleonyx species, C. elegans and C. mitratus, suggesting that a cis‐sex chromosome turnover—a change in sex chromosomes without a concomitant change in heterogamety—has occurred within the genus. These findings demonstrate the utility of genome‐scale data to uncover novel sex chromosomes and further highlight the diversity of gecko sex chromosomes. Male‐specific genetics markers in the banded gecko, Coleonyx brevis, indicate an XX/XY sex chromosome system. These results support a sex chromosome turnover within the genus Coleonyx.
... In particular, reports of TSD in C. chamaeleon is anecdotical [145], and recent studies on this species and on the congeneric C. calyptratus evidenced the presence of homomorphic XY sex chromosomes [13,99]. Concerning p. pityusensis and V. salvator, qPCR studies by Rovatsos et al. [113,[146][147][148] evidenced a ZW sex chromosome system, while TDS in Elgaria multicarinata was not supported by incubation experiments by Reference [147]. Furthermore, the probable lack of TSD in Lacertidae has been discussed by Rovatsos et al. [149], who found evidence of a conserved ZW system in the family. ...
Full-text available
Lizards represent unique model organisms in the study of sex determination and sex chromosome evolution. Among tetrapods, they are characterized by an unparalleled diversity of sex determination systems, including temperature-dependent sex determination (TSD) and genetic sex determination (GSD) under either male or female heterogamety. Sex chromosome systems are also extremely variable in lizards. They include simple (XY and ZW) and multiple (X1X2Y and Z1Z2W) sex chromosome systems and encompass all the different hypothesized stages of diversification of heterogametic chromosomes, from homomorphic to heteromorphic and completely heterochro-matic sex chromosomes. The co-occurrence of TSD, GSD and different sex chromosome systems also characterizes different lizard taxa, which represent ideal models to study the emergence and the evolutionary drivers of sex reversal and sex chromosome turnover. In this review, we present a synthesis of general genome and karyotype features of non-snakes squamates and discuss the main theories and evidences on the evolution and diversification of their different sex determination and sex chromosome systems. We here provide a systematic assessment of the available data on lizard sex chromosome systems and an overview of the main cytogenetic and molecular methods used for their identification, using a qualitative and quantitative approach.
... The ancestral clades show deep divergences (discussed by Avise [46]) and possibly can all be traced back to a few initial hybridization events [39], seemingly supporting the 'rare formation hypothesis' (see §3(a)). Murphy et al. [289] proposed sex chromosomes to play key roles in the formation of unisexual Darevskia, which like most lacertid lizards [303] feature female heterogamety (ZW). Murphy et al. [289] stated that unisexual D. dahli and D. armeniaca express the micro-heteromorphic W chromosome from their maternal ancestry, D. mixta [304,305], while D. unisexualis expresses the derived micro-heteromorphic chromosome from its maternal lineage, D. raddei [295,304]). ...
Full-text available
We review knowledge about the roles of sex chromosomes in vertebrate hybridization and speciation, exploring a gradient of divergences with increasing reproductive isolation (speciation continuum). Under early divergence, well-differentiated sex chromosomes in meiotic hybrids may cause Haldane-effects and introgress less easily than autosomes. Undifferentiated sex chromosomes are more susceptible to introgression and form multiple (or new) sex chromosome systems with hardly predictable dominance hierarchies. Under increased divergence, most vertebrates reach complete intrinsic reproductive isolation. Slightly earlier, some hybrids (linked in ‘the extended speciation continuum’) exhibit aberrant gametogenesis, leading towards female clonality. This facilitates the evolution of various allodiploid and allo- polyploid clonal (‘asexual’) hybrid vertebrates, where ‘asexuality’ might be a form of intrinsic reproductive isolation. A comprehensive list of ‘asexual’ hybrid vertebrates shows that they all evolved from parents with divergences that were greater than at the intraspecific level (K2P-distances of greater than 5–22% based on mtDNA). These ‘asexual’ taxa inherited genetic sex determi- nation by mostly undifferentiated sex chromosomes. Among the few known sex-determining systems in hybrid ‘asexuals’, female heterogamety (ZW) occurred about twice as often as male heterogamety (XY). We hypothesize that pre-/meiotic aberrations in all-female ZW-hybrids present Haldane- effects promoting their evolution. Understanding the preconditions to produce various clonal or meiotic allopolyploids appears crucial for insights into the evolution of sex, ‘asexuality’ and polyploidy. This article is part of the theme issue ‘Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part II)’.
... Facultative parthenogenesis yielding genetically variable offspring of both sexes was discovered in a xantusiid lizard [211]. Five squamate clades (iguanas, lacertid lizards, varanids, skinks and caenophidian snakes) covering approximately 60% of extant squamates show evolutionary conserved sex chromosomes [206,[212][213][214][215][216], while other lineages, particularly Acrodonta (agamid lizards and chameleons), boas and pythons, and geckos exhibit more variable SD [18,205,[217][218][219]. In two snake families and the Komodo dragon (Varanus komodoensis) with female heterogamety, substantial W-chromosome degeneration and the absence of global Z-chromosome dosage compensation has been shown, dosage balance is largely lacking in Z-specific genes in these species [215,220,221]. ...
Full-text available
Triggers and biological processes controlling male or female gonadal differentiation vary in vertebrates, with sex determination (SD) governed by environmental factors or simple to complex genetic mechanisms that evolved repeatedly and independently in various groups. Here, we review sex evolution across major clades of vertebrates with information on SD, sexual development and reproductive modes. We offer an up-to- date review of divergence times, species diversity, genomic resources, genome size, occurrence and nature of polyploids, SD systems, sex chromosomes, SD genes, dosage compensation and sex-biased gene expression. Advances in sequencing technologies now enable us to study the evolution of SD at broader evolutionary scales, and we now hope to pursue a sexomics integrative research initiative across vertebrates. The vertebrate sexome comprises interdisciplinary and integrated information on sexual differentiation, development and reproduction at all biological levels, from genomes, transcriptomes and proteomes, to the organs involved in sexual and sex-specific processes, including gonads, secondary sex organs and those with transcriptional sex-bias. The sexome also includes ontogenetic and behavioural aspects of sexual differentiation, including malfunction and impairment of SD, sexual differentiation and fertility. Starting from data generated by high-through- put approaches, we encourage others to contribute expertise to building understanding of the sexomes of many key vertebrate species. This article is part of the theme issue ‘Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part I)’.
... Beyond mammals and birds, conserved sex chromosomes have recently been discovered in several amniote (specifically reptile) clades [22][23][24][25], all of which feature differentiated sex chromosomes. Evolutionarily very old, conserved and homomorphic ZZ/ZW sex chromosomes are known in some ratite birds (Ratidae), dating back more than 130 Myr [26,27]. ...
Full-text available
Several hypotheses explain the prevalence of undifferentiated sex chromo- somes in poikilothermic vertebrates. Turnovers change the master sex determination gene, the sex chromosome or the sex determination system (e.g. XY to WZ). Jumping master genes stay main triggers but translocate to other chromosomes. Occasional recombination (e.g. in sex-reversed females) prevents sex chromosome degeneration. Recent research has uncovered con- served heteromorphic or even homomorphic sex chromosomes in several clades of non-avian and non-mammalian vertebrates. Sex determination in sturgeons (Acipenseridae) has been a long-standing basic biological question, linked to economical demands by the caviar-producing aquaculture. Here, we report the discovery of a sex-specific sequence from sterlet (Acipenser ruthenus). Using chromosome-scale assemblies and pool-sequencing, we first identified an approximately 16 kb female-specific region. We developed a PCR-genotyping test, yielding female-specific products in six species, spanning the entire phylogeny with the most divergent extant lineages (A. sturio, A. oxyrinchus versus A. ruthenus, Huso huso), stemming from an ancient tetraploidization. Similar results were obtained in two octoploid species (A. gueldenstaedtii, A. baerii). Conservation of a female-specific sequence for a long period, representing 180 Myr of sturgeon evolution, and across at least one polyploidization event, raises many interesting biological questions. We discuss a conserved undifferen- tiated sex chromosome system with a ZZ/ZW-mode of sex determination and potential alternatives. This article is part of the theme issue ‘Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part I)’.
... Usage and distribution for commercial purposes requires written permission. Ehl Rovatsos et al., 2016Rovatsos et al., , 2019a. Some species -among amniotes forming a minority -rely on environmental sex determination (ESD), where sexes do not differ in their genotypes and the sex of an individual is set by environmental conditions during a sensitive developmental period. ...
Full-text available
Transitions from environmental sex determination (ESD) to genotypic sex determination (GSD) require an intermediate step of sex reversal, i.e., the production of individuals with a mismatch between the ancestral genotypic and the phenotypic sex. Among amniotes, the sole well-documented transition in this direction was shown in the laboratory in the central bearded dragon, Pogona vitticeps, where very high incubation temperatures led to the production of females with the male-typical (ZZ) genotype. These sex-reversed females then produced offspring whose sex depended on the incubation temperature. Sex-reversed animals identified by molecular and cytogenetic markers were also reported in the field, and their increasing incidence was speculated as a climate warming-driven transition in sex determination. We show that the molecular and cytogenetic markers normally sex-linked in P. vitticeps are also sex-linked in P. henrylawsoni and P. minor, which points to quite ancient sex chromosomes in this lineage. Nevertheless, we demonstrate, based on a crossing experiment with a male bearded dragon who possesses a mismatch between phenotypic sex and genotype, that the used cytogenetic and molecular markers might not be reliable for the identification of sex reversal. Sex reversal should not be considered as the only mechanism causing a mismatch between genetic sex-linked markers and phenotypic sex, which can emerge also by other processes, here most likely by a rare recombination between regions of sex chromosomes which are normally sex-linked. We warn that sex-linked, even apparently for a long evolutionary time, and sex-specific molecular and cytogenetic markers are not a reliable tool for the identification of sex-reversed individuals in a population and that sex reversal has to be verified by other approaches, particularly by observation of the sex ratio of the progeny.
Full-text available
Embryos, juveniles, and even adults of many bird species lack pronounce external sexually dimorphic characteristics. Accurate identification of sex is crucial for research (e.g. developmental, population, and evolutionary studies), management of wildlife species, and captive breeding programs for both conservation and poultry. An accurate molecular sexing method applicable across the entire bird radiation is theoretically possible thanks to the long‐term stability of their ZZ/ZW sex chromosomes, but current methods are not applicable in a wide range of bird lineages. Here, we developed a novel molecular sexing method based on the comparison of gene copy number variation by quantitative real‐time PCR (qPCR) in conserved Z‐specific genes (CHRNA6, DDX4, LPAR1, TMEM161B, VPS13A), i.e. genes linked to Z but absent from W chromosomes. We tested the method across three paleognath and 70 neognath species covering the avian phylogeny. In addition, we designed primers for four Z‐specific genes (DOCK8, FUT10, PIGG and PSD3) for qPCR‐based molecular sexing in three paleognath species. We have demonstrated that the genes DOCK8, FUT10, PIGG and PSD3 can identify sex in paleognath birds and the genes CHRNA6, DDX4, TMEM161B, and VPS13A can reveal sex in neognath birds. The gene LPAR1 can be used to accurately identify sex in both paleognath and neognath species. Along with outlining a novel method of practical importance for molecular sexing in birds, our study also documents in detail the conservation of sex chromosomes across the avian phylogeny.
Full-text available
Squamate reptiles show high diversity in sex determination ranging from environmental sex determination to genotypic sex determination with varying degrees of differentiation of sex chromosomes. Unfortunately, we lack even basic information on sex determination mode in several lineages of squamates, which prevents full understanding of their diversity and evolution of sex determination. One of the reptilian lineages with missing information on sex determination is the family Gerrhosauridae, commonly known as the plated lizards. Several species of gerrhosaurids have been studied in the past by conventional cytogenetic methods, but sex-specific differences were not identified. In this study, we applied both conventional and molecular cytogenetic methods to metaphases from both sexes of the Peters' keeled plated lizard (Tracheloptychus petersi). We identified accumulations of rDNA loci in a pair of microchromosomes in metaphases from males, but only in a single microchromosome in females. The restriction of the observed heterozygosity to females suggests a putative ZZ/ZW system of sex chromosomes, which represents the first report of sex chromosomes in a gerrhosaurid lizard. The lack of sex-specific signals in all other cytogenetic methods implies that the sex chromosomes of T. petersi are poorly differentiated in sequence content.
Full-text available
Among amniotes, squamate reptiles are especially variable in their mechanisms of sex determination; however, based largely on cytogenetic data, some lineages possess highly evolutionary stable sex chromosomes. The still very limited knowledge of the genetic content of squamate sex chromosomes precludes a reliable reconstruction of the evolutionary history of sex determination in this group and consequently in all amniotes. Female heterogamety with a degenerated W chromosome typifies the lizards of the family Lacertidae, the widely distributed Old World clade including several hundreds of species. From the liver transcriptome of the lacertid Takydromus sexlineatus female, we selected candidates for Z-specific genes as the loci lacking single-nucleotide polymorphisms. We validated the candidate genes through the comparison of the copy numbers in the female and male genomes of T. sexlineatus and another lacertid species, Lacerta agilis, by quantitative PCR that also proved to be a reliable technique for the molecular sexing of the studied species. We suggest that this novel approach is effective for the detection of Z-specific and X-specific genes in lineages with degenerated W, respectively Y chromosomes. The analyzed gene content of the Z chromosome revealed that lacertid sex chromosomes are not homologous with those of other reptiles including birds, but instead the genes have orthologs in the X-conserved region shared by viviparous mammals. It is possible that this part of the vertebrate genome was independently co-opted for the function of sex chromosomes in viviparous mammals and lacertids because of its content of genes involved in gonad differentiation.
Full-text available
Iguanas (Pleurodonta) are predominantly distributed in the New World, but one previously cytogenetically understudied family, Opluridae, is endemic to Madagascar and the adjacent Grand Comoro archipelago. The aim of our contribution is to fill a gap in the cytogenetic understanding of this biogeographically puzzling lineage. Based on examination of six species, we found that oplurids are rather conservative in karyotype, which is composed of 36 chromosomes as in most iguanas. However, the species differ in the position of the nucleolar organizer region and heterochromatic blocks and in the accumulation and distribution of interstitial telomeric sequences (ITSs), which suggests cryptic intra- and interchromosomal rearrangements. All tested species share the XY sex-determining system homologous to most other iguana families. The oplurid Y chromosome is degenerated, very small in size but mostly euchromatic. Fluorescence in situ hybridization with probes composed of microsatellite motifs revealed variability among species in the accumulation of particular repeats on the Y chromosome. This variability accounts for the differences in the detection of sex chromosomes across the species of the family using comparative genome hybridization (CGH) technique. Our study demonstrates the limits of the commonly used CGH technique to uncover sex chromosomes even in organisms with heteromorphic and sequentially largely differentiated sex chromosomes.
Full-text available
Amniote vertebrates possess various mechanisms of sex determination, but their variability is not equally distributed. The large evolutionary stability of sex chromosomes in viviparous mammals and birds was believed to be connected with their endothermy. However, some ectotherm lineages seem to be comparably conserved in sex determination, but previously there was a lack of molecular evidence to confirm this. Here, we document a stability of sex chromosomes in advanced snakes based on the testing of Z-specificity of genes using quantitative PCR (qPCR) across 37 snake species (our qPCR technique is suitable for molecular sexing in potentially all advanced snakes). We discovered that at least part of sex chromosomes is homologous across all families of caenophidian snakes (Acrochordidae, Xenodermatidae, Pareatidae, Viperidae, Homalopsidae, Colubridae, Elapidae and Lamprophiidae). The emergence of differentiated sex chromosomes can be dated back to about 60 Ma and preceded the extensive diversification of advanced snakes, the group with more than 3000 species. The Z-specific genes of caenophidian snakes are (pseudo)autosomal in the members of the snake families Pythonidae, Xenopeltidae, Boidae, Erycidae and Sanziniidae, as well as in outgroups with differentiated sex chromosomes such as monitor lizards, iguanas and chameleons. Along with iguanas, advanced snakes are therefore another example of ectothermic amniotes with a long-term stability of sex chromosomes comparable with endotherms.
Full-text available
The effective population size (Ne) is a fundamental parameter in population genetics that influences the rate of loss of genetic diversity. Sexual selection has the potential to reduce Ne by causing the sex-specific distributions of individuals that successfully reproduce to diverge. To empirically estimate the effect of sexual selection on Ne, we obtained fitness distributions for males and females from an outbred, laboratory-adapted population of Drosophila melanogaster. We observed strong sexual selection in this population (the variance in male reproductive success was ∼14 times higher than that for females), but found that sexual selection had only a modest effect on Ne, which was 75% of the census size. This occurs because the substantial random offspring mortality in this population diminishes the effects of sexual selection on Ne, a result that necessarily applies to other high fecundity species. The inclusion of this random offspring mortality creates a scaling effect that reduces the variance/mean ratios for male and female reproductive success and causes them to converge. Our results demonstrate that measuring reproductive success without considering offspring mortality can underestimate Ne and overestimate the genetic consequences of sexual selection. Similarly, comparing genetic diversity among different genomic components may fail to detect strong sexual selection.
Full-text available
The sex chromosomes in Sauropsida (reptiles and birds) have evolved independently many times. They show astonishing diversity in morphology ranging from cryptic to highly differentiated sex chromosomes with male (XX/XY) and female heterogamety (ZZ/ZW). Comparing such diverse sex chromosome systems thus provides unparalleled opportunities to capture evolution of morphologically differentiated sex chromosomes in action. Here, we describe chromosomal mapping of 18 microsatellite repeat motifs in eight species of Sauropsida. More than two microsatellite repeat motifs were amplified on the sex-specific chromosome, W or Y, in five species (Bassiana duperreyi, Aprasia parapulchella, Notechis scutatus, Chelodina longicollis, and Gallus gallus) of which the sex-specific chromosomes were heteromorphic and heterochromatic. Motifs (AAGG)n and (ATCC)n were amplified on the W chromosome of Pogona vitticeps and the Y chromosome of Emydura macquarii, respectively. By contrast, no motifs were amplified on the W chromosome of Christinus marmoratus, which is not much differentiated from the Z chromosome. Taken together with previously published studies, our results suggest that the amplification of microsatellite repeats is tightly associated with the differentiation and heterochromatinization of sex-specific chromosomes in sauropsids as well as in other taxa. Although some motifs were common between the sex-specific chromosomes of multiple species, no correlation was observed between this commonality and the species phylogeny. Furthermore, comparative analysis of sex chromosome homology and chromosomal distribution of microsatellite repeats between two closely related chelid turtles, C. longicollis and E. macquarii, identified different ancestry and differentiation history. These suggest multiple evolutions of sex chromosomes in the Sauropsida.
Full-text available
Chromosomal fusion plays a recurring role in the evolution of adaptations and reproductive isolation among species, yet little is known of the evolutionary drivers of chromosomal fusions. Because sex chromosomes (X and Y in male heterogametic systems, Z and W in female heterogametic systems) differ in their selective, mutational, and demographic environments, those differences provide a unique opportunity to dissect the evolutionary forces that drive chromosomal fusions. We estimate the rate at which fusions between sex chromosomes and autosomes become established across the phylogenies of both fishes and squamate reptiles. Both the incidence among extant species and the establishment rate of Y-autosome fusions is much higher than for X-autosome, Z-autosome, or W-autosome fusions. Using population genetic models, we show that this pattern cannot be reconciled with many standard explanations for the spread of fusions. In particular, direct selection acting on fusions or sexually antagonistic selection cannot, on their own, account for the predominance of Y-autosome fusions. The most plausible explanation for the observed data seems to be (a) that fusions are slightly deleterious, and (b) that the mutation rate is male-biased or the reproductive sex ratio is female-biased. We identify other combinations of evolutionary forces that might in principle account for the data although they appear less likely. Our results shed light on the processes that drive structural changes throughout the genome.
Full-text available
Contrasting with birds and mammals, poikilothermic vertebrates often have homomorphic sex chromosomes, possibly resulting from high rates of sex-chromosome turnovers and/or occasional X-Y recombination. Strong support for the latter mechanism was provided by four species of European tree frogs, which inherited from a common ancestor (~ 5 Mya) the same pair of homomorphic sex chromosomes (linkage group 1, LG1), harboring the candidate sex-determining gene Dmrt1. Here we test sex linkage of LG1 across six additional species of the Eurasian Hyla radiation with divergence times ranging from 6 to 40 Mya. LG1 turns out to be sex linked in six out of nine resolved cases. Mapping the patterns of sex linkage to the Hyla phylogeny reveals several transitions in sex-determination systems within the last 10 My, including one switch in heterogamety. Phylogenetic trees of DNA sequences along LG1 are consistent with occasional X-Y recombination in all species where LG1 is sex linked. These patterns argue against one of the main potential causes for turnovers, namely the accumulation of deleterious mutations on non-recombining chromosomes. Sibship analyses show that LG1 recombination is strongly reduced in males from most species investigated, including some in which it is autosomal. Intrinsically low male recombination might facilitate the evolution of male heterogamety, and the presence of important genes from the sex-determination cascade might predispose LG1 to become a sex chromosome. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail:
Full-text available
Many species groups, including mammals and many insects, determine sex using heteromorphic sex chromosomes. Diptera flies, which include the model Drosophila melanogaster, generally have XY sex chromosomes and a conserved karyotype consisting of six chromosomal arms (five large rods and a small dot), but superficially similar karyotypes may conceal the true extent of sex chromosome variation. Here, we use whole-genome analysis in 37 fly species belonging to 22 different families of Diptera and uncover tremendous hidden diversity in sex chromosome karyotypes among flies. We identify over a dozen different sex chromosome configurations, and the small dot chromosome is repeatedly used as the sex chromosome, which presumably reflects the ancestral karyotype of higher Diptera. However, we identify species with undifferentiated sex chromosomes, others in which a different chromosome replaced the dot as a sex chromosome or in which up to three chromosomal elements became incorporated into the sex chromosomes, and others yet with female heterogamety (ZW sex chromosomes). Transcriptome analysis shows that dosage compensation has evolved multiple times in flies, consistently through up-regulation of the single X in males. However, X chromosomes generally show a deficiency of genes with male-biased expression, possibly reflecting sex-specific selective pressures. These species thus provide a rich resource to study sex chromosome biology in a comparative manner and show that similar selective forces have shaped the unique evolution of sex chromosomes in diverse fly taxa.
Full-text available
Gekkotan lizards are a highly specious (∼1600 described species) clade of squamate lizards with nearly cosmopolitan distribution in warmer areas. The clade is primarily nocturnal and forms an ecologically dominantpart of the world nocturnal herpetofauna. However, molecular cytogenetic methods to study the evolution of karyotypes have not been widely applied in geckos. Our aim here was to uncover the extent of chromosomal rearrangements across the whole group Gekkota and to search for putative synapomorphies supporting the newly proposed phylogenetic relationships within this clade. We applied cross-species chromosome painting with the recently derived whole-chromosomal probes from the gekkonid species Gekko japonicus to members of the major gekkotan lineages. We included members of the families Diplodactylidae, Carphodactylidae, Pygopodidae, Eublepharidae, Phyllodactylidae and Gekkonidae. Our study demonstrates relatively high chromosome conservatism across the ancient group of gekkotan lizards. We documented that many changes in chromosomal shape across geckos can be attributed to intrachromosomal rearrangements. The documented rearrangements are not totally in agreement with the recently newly erected family Phyllodactylidae.The results also pointed to homoplasy, particularly in the reuse of chromosome breakpoints, in the evolution of gecko karyotypes.