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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.
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Conservation of sex chromosomes in lacertid lizards
MICHAIL ROVATSOS,* JASNA VUKI
C,* MARIE ALTMANOV
A,* MARTINA JOHNSON
POKORN
A,*JI
R
I MORAVECand L UK
A
SKRATOCHV
IL*
*Department of Ecology, Faculty of Science, Charles University in Prague, Vini
cn
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
aclavsk
en
am. 68, 115 79 Prague, Czech Republic
Abstract
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
lineages.
Keywords: lizards, molecular sexing, reptiles, sex chromosomes
Received 29 December 2015; revision received 7 March 2016; accepted 22 March 2016
Introduction
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;
Pokorn
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
a
s Kratochv
ıl, Fax: +420 221951673;
E-mail: lukas.kratochvil@natur.cuni.cz
©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;
Altmanov
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-
tern.
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
a&
Kratochv
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
Pokorn
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
2M. ROVATSOS ET AL.
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
l’Am
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
(Alf
oldi et al. 2011; www.ensembl.org) 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.
Results
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).
Discussion
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
CONSERVATION OF SEX CHROMOSOMES IN LACERTIDS 3
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
4M. ROVATSOS ET AL.
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
ıl
2009; Gamble et al. 2015; Johnson Pokorn
a & Kratochv
ıl
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;
Koubov
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
a
& Kratochv
ıl 2016). Sex-determining systems, particu-
larly GSD systems and hence sex chromosomes, might
be stable in many other reptile lineages (Pokorn
a&
Kratochv
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.
Acknowledgements
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
avn
ı
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).
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M.R., L.K., J.V., M.A. and M.J.P. designed the research;
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tributed the material; M.R. analysed the data; M.R. and
L.K. wrote the first draft; all authors edited the manu-
script.
Data accessibility
All data are presented in the Table S3 (Supporting
information).
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-
gin.
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
CONSERVATION OF SEX CHROMOSOMES IN LACERTIDS 7
... 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. ...
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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]. ...
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... 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). ...
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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. ...
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... 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]). ...
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... 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]. ...
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... 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]. ...
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... 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. ...
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