Original Article

Cytogenet Genome Res

DOI: 10.1159/000447340

Accepted: March 14, 2016

by M. Schmid
Published online: July 23, 2016

First Description of the Karyotype

and Sex Chromosomes in the Komodo
Dragon (Varanus komodoensis)
Martina Johnson Pokorn a, c Marie Altmanov a Michail Rovatsos a
Petr Velensk b Roman Vodika b Ivan Rehk b Luk Kratochvl a
a

Faculty of Science, Charles University in Prague, and b Prague Zoological Garden, Prague, and c Institute of Animal
Physiology and Genetics, The Czech Academy of Sciences, Libchov, Czech Republic

Abstract
The Komodo dragon (Varanus komodoensis) is the largest lizard in the world. Surprisingly, it has not yet been cytogenetically examined. Here, we present the very first description
of its karyotype and sex chromosomes. The karyotype consists of 2n = 40 chromosomes, 16 macrochromosomes and
24 microchromosomes. Although the chromosome number
is constant for all species of monitor lizards (family Varanidae) with the currently reported karyotype, variability in the
morphology of the macrochromosomes has been previously documented within the group. We uncovered highly differentiated ZZ/ZW sex microchromosomes with a heterochromatic W chromosome in the Komodo dragon. Sex chromosomes have so far only been described in a few species of
varanids including V. varius, the sister species to Komodo
dragon, whose W chromosome is notably larger than that of
the Komodo dragon. Accumulations of several microsatellite
sequences in the W chromosome have recently been detected in 3 species of monitor lizards; however, these accumula-

2016 S. Karger AG, Basel

14248581/16/00000000$39.50/0
E-Mail karger@karger.com
www.karger.com/cgr

tions are absent from the W chromosome of the Komodo

dragon. In conclusion, although varanids are rather conservative in karyotypes, their W chromosomes exhibit substantial variability at the sequence level, adding further evidence
that degenerated sex chromosomes may represent the most
dynamic genome part.
2016 S. Karger AG, Basel

The Komodo dragon, Varanus komodoensis Ouwens,

1912, is a well-known predator and is also the largest lizard
in the world. It is a member of the monitor lizard family
(Varanidae) which is widespread in Australia, Africa,
Asia, and many islands in the Pacific, including Indonesia,
with 78 currently recognised species [Uetz and Hoek,
2016]. The Komodo dragon is endemic to the Lesser Sunda chain of islands (recently restricted to just 5 islands and
islets) between Australia and Java, where it is a dominant
terrestrial carnivore [Ciofi et al., 1999, 2011; Sastrawan
and Ciofi, 2002]. Males can grow up to 3 meters in length
and usually weigh around 70 kg (with the maximal weight
recorded at 166 kg [Ciofi, 1999]). This exceptional size

M.J.P. and M.A. contributed equally to this work.

Luk Kratochvl
Faculty of Science
Charles University in Prague
Vinin 7, CZ128 44 Prague 2 (Czech Republic)
E-Mail lukas.kratochvil@natur.cuni.cz

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Key Words
CGH Female heterogamety Heterochromatin
Microsatellite accumulation Sex chromosome evolution
Squamate reptile

Cytogenet Genome Res

DOI: 10.1159/000447340

them having female heterogamety with differentiated ZZ/

ZW sex chromosomes. The sex chromosomes are microchromosomes and, in some species, the W chromosome
is distinctively larger than the other microchromosomes
[King and King, 1975; Matsubara et al., 2014]. In V. acanthurus, V. gouldii, and V. rosenbergi, Matsubara et al.
[2014] identified accumulations of microsatellite motifs
in the W chromosome. They found an accumulation of
the (AAT)n microsatellite repeat motif in the W chromosome of V. acanthurus, whereas the (CGG)n motif was
accumulated in the W chromosome of V. gouldii and
V. rosenbergi. Sex-determining mechanisms and sex
chromosomes in other species are still unknown.
The Komodo dragon is one of the few species of squamate reptiles (and vertebrates in general) where facultative asexuality has been observed [Watts et al., 2006]. The
asexually produced offspring were homozygous for all
tested loci and were exclusively males, which strongly
points to female heterogamety with a degenerated W in
this species. Supposedly, during asexuality, connected
with the loss of heterozygosity, both ZZ and WW genotypes are produced, but the WW genotypes are not viable.
However, direct evidence of sex chromosomes and the
degree of their differentiation had not been obtained for
this species.
It is extremely difficult to determine the sex of Komodo dragon individuals by external phenotypic characteristics, especially in young animals [Auffenberg, 1981;
Halverson and Spelman, 2002; Sulandari et al., 2014; Rovatsos et al., in press]. Sulandari et al. [2014] optimised
the protocol by Halverson and Spelman [2002] for molecular sexing of this species using a sex-specific PCR
marker. However, in this procedure, the sex is not determined simply by the presence or absence of a specific
band on an electrophoretic gel as in other molecular sexing techniques based on PCR including the original protocol by Halverson and Spelman [2002], but by differences in the length, position, and intensity of bands between males and females. These results did not point to
female heterogamety, where one band specific for females
can be expected, or to male heterogamety, where the
male-specific band should be absent in females. This pattern is rather unusual, and the authors did not offer any
biological or methodological explanation for their results.
In this study, we applied methods of molecular cytogenetics to investigate the sex chromosomes of Komodo
dragons from Prague Zoo, originating from sexual as well
as asexual reproduction. Our work provides the very first
description of the karyotype and the sex chromosomes of
these enigmatic animals.
JohnsonPokorn etal.

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among lizards was previously attributed to island gigantism; however, recent studies now view this species more
as a relic of the similar-sized monitor lizards that used to
live across Australia and Indonesia [Hocknull et al., 2009].
Currently, there are 3,0005,000 Komodo dragons distributed throughout the islands of Komodo, Gili Motang,
Rinca, Gili Dasami, and Flores. However, a dearth of egglaying females, poaching, human encroachment, and natural disturbances have driven the species to an endangered status, being now listed as vulnerable by the IUCN
Red List [Ciofi, 2002; Ciofi et al., 2002; Sastrawan and
Ciofi, 2002; IUCN 2016].
Monitor lizards (Varanidae) are members of the group
Anguimorpha. The closest related lineages of Anguimorpha are likely snakes and iguanians; however, the relationships between these 3 major lineages differ in phylogenetic reconstructions [Townsend at al., 2004; Vidal and
Hedges, 2005; Wiens et al., 2012]. As far as we know,
karyotypes have been described for 22 species of monitor
lizards [King and King, 1975; King et al., 1982; Olmo and
Signorino, 2016], all of them exhibiting a uniform chromosome number of 2n = 40, and all consisting of 16 macrochromosomes and 24 microchromosomes. Approximately half of the species possess 14 metacentric and 2
acrocentric macrochromosomes, while the other half exhibits variability in the morphology of their macrochromosomes. This variability was an important part of the
study by King and King [1975], where species were organised into several groups based on this characteristic. Srikulnath et al. [2013] investigated rearrangements in 2
species of monitor lizards using comparative gene mapping and came to the same conclusion as King and King
[1975] that the rearrangement of macrochromosomes is
predominantly caused by pericentric inversions and centromere repositioning rather than interchromosomal
changes. They also confirmed that the genome organisation at the chromosomal level of squamate reptiles is rather conserved as previously suggested by Pokorn et al.
[2011a, 2012] or Kasai et al. [2012].
Squamate reptiles possess variability in sex-determining systems; however, this variability is not distributed
equally among lineages [e.g., Pokorn and Kratochvl,
2009]. A sex ratio putatively consistent with temperaturedependent sex determination had been reported in a single species of varanids (V. salvator), but these records appeared dubious under closer examination [Harlow, 2004;
Pokorn and Kratochvl, 2009]. Sex chromosomes have
been detected in only 6 species of monitor lizards (one of
which is V. varius, the sister species to the Komodo dragon) [King and King, 1975; Matsubara et al., 2014], all of

Our research involved Komodo dragons captive bred in Prague

Zoo. We cytogenetically examined 2 females produced from sexual reproduction and 9 males from asexual reproduction.
Chromosome Preparation and Staining
Metaphase chromosome spreads were prepared from whole
blood cell cultures, following the protocol described in Pokorn et
al. [2014]. Briefly, a small amount (40 l) of peripheral blood was
cultured for a week at 30 C in Dulbeccos Modified Eagles Medium
(Sigma-Aldrich), enriched with 10% foetal bovine serum (Baria),
0.5% penicillin/streptomycin solution (Gibco), 1% L-glutamine
(Sigma-Aldrich), 3% phytohaemagglutinin (Gibco), and 1% lipopolysaccharide (Sigma-Aldrich). Chromosome preparations were
made following standard procedures including colcemid and hypotonic treatment and fixation in 3:1 methanol:acetic acid. Chromosome preparations from all specimens were stained with conventional Giemsa solution. C-banding was performed as described by
Sumner [1972] with slight modifications, i.e., the slides were aged at
65 C for 1 h, soaked in 0.2 N HCl for 20 min, then in 5% Ba(OH)2
solution for 4.5 min at 45 C, and rinsed in 0.2 N HCl. Finally, the
slides were incubated in 2 SSC for 1 h at 60 C, rinsed in distilled
water, and stained with 4,6-diamidino-2-phenylindole (DAPI).

Comparative Genomic Hybridization

Male and female genomic DNA of V. komodoensis was labelled
with biotin-dUTP and digoxigenin-dUTP, respectively, using a
Nick Translation Kit (Abbott Molecular). From each sample, 1 g
of male and 1 g of female labelled genomic DNA was co-precipitated overnight with 5 l salmon sperm DNA (10 mg/ml, Sigma)
and 10 l sodium acetate. After precipitation, the dry pellets were
resuspended in 11 l hybridization buffer per slide (50% formamide, 2 SSC, 10% SDS, 10% dextran sulphate, 1 Denhardts
buffer, pH 7), denatured at 75 C for 10 min, and then chilled on
ice for 10 min prior to hybridization. At the same time, the metaphase slides were treated with RNase and pepsin, fixed with 4%
formaldehyde, dehydrated through a 70, 85, and 100% ethanol series, denatured in 70% formamide/2 SSC at 75 C for 3 min, and
dehydrated again. For the next step, 11 l of the probe was applied
to each slide and incubated at 37 C for 48 h. Post-hybridization
washes were performed in 50% formamide/2 SSC at 42 C and in
2 SSC. Each slide was incubated with 100 l of 4 SSC/5% blocking reagent (Roche) at 37 C for 30 min and then with 100 l detection solution 4 SSC/5% blocking reagent including 2 l of avidinFITC (Vector Laboratories) and 10 l of anti-digoxigenin-rhodamine (Roche) at 37 C for 30 min. The slides were subsequently
washed in 4 SSC/0.05% Tween 20, dehydrated through an ethanol series, and air dried. Finally, the slides were mounted with
Fluoroshield antifade medium containing DAPI (Sigma-Aldrich).

CMA3/DAPI Staining
Chromomycin A3 (CMA3, a DNA dye specific for GC-rich regions) and DAPI (AT-specific) fluorescence staining was performed as described by Sola et al. [1992]. Briefly, the slides were
incubated in McIlvaine buffer containing MgCl2 for 10 min and
stained with 150 l of CMA3 solution in a wet chamber for 15 min.
The slides were then incubated in McIlvaine buffer containing
Methyl Green for 15 min, rinsed in McIlvaine buffer, and finally
stained with DAPI.

Cytogenetics of the Komodo Dragon

FISH with Telomeric Probe

We used the commercial telomeric probe Telomere PNA
Probe/Cy3 (DAKO) directly labelled with Cy3 fluorochrome and
followed the protocol enclosed with only the slight modification of
longer hybridization time (1.5 h).
FISH with rRNA Genes and Microsatellite Motifs
The rDNA was localised by performing FISH with the rRNA
gene probe on both sexes. The probe carrying rRNA genes was
prepared from a plasmid (pDm r.a51#1) with an 11.5-kb insert,
encoding the 18S and 28S rRNA units of Drosophila melanogaster
Meigen, 1830 [Endow, 1982] and labelled with biotin-dUTP using
a Nick Translation Kit (Abbott Molecular). The probe was ethanol-precipitated with sonicated salmon sperm DNA and resuspended in hybridization buffer (50% formamide/2 SSC).
In order to detect particular repetitive sequences in the Komodo dragon genome and to compare the content of sex chromosomes with other species [Matsubara et al., 2014], the microsatellites (AAT)10, (CGG)10, and (GATA)8 were hybridised to metaphases of both sexes. For each slide, 500 ng of commercially
prepared, biotin-labelled oligonucleotides (Macrogen) were resuspended in 10 l hybridization buffer (50% formamide, 2 SSC,
10% SDS, 10% dextran sulphate, 1 Denhardts buffer, pH 7). All
probes (rRNA genes and microsatellites) were denatured at 75 C
for 6 min and chilled on ice for 10 min before use. Prior to hybridization, the chromosomes were treated with RNase A and pepsin,
fixed in 1% formaldehyde, dehydrated through a 70, 85 and 96%
ethanol series, denatured in 70% formamide/2 SSC at 75 C for
3 min, and dehydrated again (for a detailed FISH protocol, see Pokorn et al. [2014]). Hybridization was performed at 37 C overnight.
For the rRNA gene probe, 3 post-hybridization washes in 50%
formamide/2 SSC at 37 C for 5 min each were followed by 2
washes in 2 SSC for 5 min. For the microsatellites, the washes
were carried out in 0.4 SSC/0.3% Nonidet P-40 (Sigma-Aldrich)
at 40 C for 2 min and in 2 SSC/0.1% Nonidet P-40 at room temperature for 30 s.
Each slide was incubated in 100 l of 4 SSC/5% blocking reagent (Roche) at 37 C for 30 min in a wet chamber. The probe
signal was enhanced and detected using the avidin-FITC/biotinylated anti-avidin system (Vector Laboratories). Finally, the slides
were mounted with Fluoroshield containing DAPI.

Microscopy and Image Analyses

Images were captured using a Provis AX70 (Olympus) fluorescence microscope equipped with a DP30BW digital camera (Olympus). The karyotype was arranged using Ikaros karyotyping software (Metasystems). DP manager imaging software (Olympus)
was used to capture grayscale images and to superimpose the
source images with colours to visualise the results of the FISH.

Results

The karyotype of the Komodo dragon is composed of

2n = 40 chromosomes including 8 pairs of macrochromosomes and 12 pairs of microchromosomes (fig.1A, C).
Within the macrochromosomes, there are 4 metacentric,
Cytogenet Genome Res
DOI: 10.1159/000447340

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Materials and Methods

Fig. 1. Giemsa-stained karyotype (A, C) and C-banded metaphase (B, D) of a male (A, B) and female (C, D) of
V. komodoensis. The W chromosome is depicted. In the female karyotype it was identified by C-banding of the
metaphase used for karyotyping. Bars = 10 m.

Cytogenet Genome Res

DOI: 10.1159/000447340

CMA3/DAPI fluorescence staining revealed GC-rich

regions inside the heterochromatic blocks (fig. 2D, E),
with no differences being seen in the pattern between sexes. The probe for rDNA bound to the pericentromeric
region of the largest chromosome pair in both sexes (for
demonstration in female see fig.2F). Probes with all tested microsatellite motifs (CGG, AAT, and GATA) did not
reveal any significant accumulation on any of the chromosomes in either sex (not shown).

Discussion

The Komodo dragon shares the same basic cytogenetic characteristics with other members of the family Varanidae. It has the diploid chromosome number 2n = 40
and possesses ZZ/ZW sex chromosomes. In fact, the
whole family appears to be very conservative in chromosome number, with also the number of macrochromosomes and microchromosomes being very stable in the
whole group [King and King, 1975; Chaiprasertsri et al.,
2013; Srikulnath et al., 2013; Matsubara et al., 2014].
Based on the modern phylogeny, the monospecific famJohnsonPokorn etal.

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3 submetacentric, and 1 acrocentric chromosome pairs.

The morphology of the microchromosomes is not identifiable due to their small size.
C-banding revealed bands of heterochromatin in the
pericentromeric positions of the 2 largest pairs of macrochromosomes as well as in the only acrocentric pair of
macrochromosomes (fig. 1B, D). Heterochromatin in
centromeric or pericentromeric regions was also detectable in 1 pair of microchromosomes in both sexes. A
strongly heterochromatic microchromosome was detected via C-banding in all female metaphases examined, and
as it was absent in all male metaphases, we can therefore
conclude that it is the W chromosome. It is also clearly
visualised by CGH, where only 1 chromosome hybridised
predominantly with female-specific sequences (fig.2B).
The Z chromosome could not be distinguished by Cbanding or by CGH. Integrating the C-banded chromosomes into the karyotype, the W falls within the range of
chromosomes 912 (fig.1C).
The telomeric probe provided typical signals restricted
to the terminal regions of all chromosomes in both sexes
(for illustration see female metaphase, fig.2C). We did not
detect any accumulation of interstitial telomeric sequences.

Fig. 2. A, B Results of CGH on male (A) and female (B) metaphases of V. komodoensis. The male genome is stained with FITC (green
colour), the female genome with rhodamine (red colour). Regions
common for genomes of both sexes are yellow (combination of
green and red). The W chromosome is indicated in the female

metaphase. C FISH with telomeric probe on a female metaphase.

D, E CMA3/DAPI staining of male (D) and female (E) metaphases.
The W chromosome is identified by sequential C-banding and does
not contain any AT- or GC-rich regions. F FISH with the rDNA
probe on a female metaphase. Arrowheads indicate the signals.

ily Lanthanotidae is likely to be a sister group to Varanidae [Pyron et al., 2013], but as the karyotype has not yet
been determined, we cannot reconstruct how far in the
phylogeny this uniformity extends. The next closest and
cytogenetically studied group is the family Helodermatidae [Pyron et al., 2013], where the karyotype of 1 out of 2
species (Gila monster, Heloderma suspectum) was re-described recently [Johnson Pokorn et al., 2014]. In comparison with the varanids, the karyotype of the Gila monster is composed of 2n = 36, with 14 macrochromosomes
and 22 microchromosomes, similar to that of the other
varanid outgroups (Iguania, snakes) [Singh, 1972; Altmanov et al., 2016; Olmo and Signorino, 2016]. It is
therefore clear that some rearrangements (probably concerning microchromosomes [Srikulnath et al., 2013])
must have happened during the evolutionary pathway
from the last common ancestor of the varanids and the
Gila monster, most likely in the lineage leading to the varanids.

The uniformity in the number of chromosomes inside

the family Varanidae does not, however, correspond with
uniformity in macrochromosome morphology. Species
differ in the number of metacentric, submetacentric, and
acrocentric chromosomes. King and King [1975] classified varanid species into 6 groups based on the morphology of chromosomes. Interestingly these karyotype
groups correspond to the species geographical distribution and with the phylogenetic positions of all the lineages [Pianka et al., 2004]. The only exception is V. indicus
belonging to the same karyological group as V. varius, but
being phylogenetically more distant. Although the cytogenetic groups mostly hold together with the major lineages of monitor lizards, the evolutionary scenario placing the cytogenetic group around V. salvator as ancestral
[King and King, 1975] does not correspond to the current
phylogeny [Pianka et al., 2004; Srikulnath et al., 2013].
The Australian sister species of the Komodo dragon,
V. varius, and V. komodoensis share similar karyotypes

Cytogenetics of the Komodo Dragon

Cytogenet Genome Res

DOI: 10.1159/000447340

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Cytogenet Genome Res

DOI: 10.1159/000447340

rectly determine the sex of Komodo dragons, which otherwise can be rather difficult. We tested molecular sexing
based on the protocol of Sulandari et al. [2014] on all
available Komodo dragon individuals in Prague Zoo and
found it concordant with our cytogenetic examination of
the species [Rovatsos et al., in press].
The W chromosome of the Komodo dragon is not
identifiable by Giemsa staining since it does not differ
noticeably in morphology from the other similar-sized
microchromosomes. It is, however, highly heterochromatic and thus visible by C-banding. It therefore appears
that whereas the sex chromosomes in Komodo dragons
may not differ extensively in morphology (however, we
stress that we did not unequivocally identify the Z chromosome), they are highly differentiated in sequences, as
also proved by CGH showing that the W chromosome is
enriched with female-specific sequences.
Sex chromosomes have been described in V. varius,
the sister species of the Komodo dragon. King and King
[1975] noticed a difference in the size and morphology
within the largest pair of microchromosomes in the female, while in the male this pair of chromosomes is homomorphic. The W chromosome in V. varius is acrocentric and larger than the metacentric Z and all other microchromosomal pairs, whereas in V. komodoensis the W
chromosome is not larger than several other microchromosomes (fig.1C). It is interesting that the karyotypes of
these 2 species differ notably only in the morphology of
the W chromosomes. The W chromosomes are significantly larger compared to the unidentifiable Z chromosomes (which can be any other microchromosome) in
V. exanthematicus and V. niloticus [King and King 1975]
as well as in V. acanthurus and V. rosenbergi [Matsubara
et al., 2014]. Interestingly, in V. acanthurus the situation
is slightly blurred by the presence of 1 large microchromosome pair carrying a heterochromatic block in the
karyotype of both sexes. In this species, the well-identified W sex chromosome is however clearly larger than the
other microchromosomes. King and King [1975] did not
examine the male karyotype in V. exanthematicus and
V. niloticus. Therefore, although they observed a distinctly heteromorphic pair of microchromosomes in the females, due to the lack of comparison with male karyotypes, we cannot rule out the possibility of it being the
same situation as in V. acanthurus. When Matsubara et
al. [2014] identified the W chromosome in V. gouldii,
they found that it did not differ in size from some of the
other microchromosomes, i.e., a similar case to that of the
Komodo dragon.

JohnsonPokorn etal.

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with the exception of W chromosome size as discussed

below. The other species of the family with known karyotypes differ in the morphology of their macrochromosomes, which range from metacentric to submetacentric
up to acrocentric, although the first 4 pairs always seem
to be metacentric [King and King, 1975; King et al., 1982;
Srikulnath et al., 2013; Matsubara et al., 2014]. As far as
known, rearrangements in macrochromosome morphology within the family are restricted to centromere shifts
most likely caused by pericentric inversions [King and
King, 1975; Srikulnath et al., 2013]. Such rearrangements
could have occurred in the common ancestor of V. varius
and V. komodoensis, and the shape of their macrochromosomes could be their synapomorphy.
The telomeric probe showed signals only in the terminal positions of all chromosomes, and we did not detect
any accumulations of interstitial telomeric or telomerelike sequences in the Komodo dragon (fig.2C). This corresponds to the findings in other species of varanids, although telomeric sequences have been mapped in metaphases of only 3 additional species [Srikulnath et al., 2013;
Rovatsos et al., 2015]. The probe for rDNA hybridised in
the pericentromeric region of the largest chromosome
pair in the Komodo dragon (fig. 2F). The same results
have been reported in V. salvator and V. exanthematicus
[Srikulnath et al., 2013]. AgNOR staining was performed
in more than 18 species of the family to detect the nucleolar organiser regions [Olmo and Signorino, 2016]. In all
species studied, the AgNOR sites were present on the
largest chromosome pair along with the rDNA signal in
the Komodo dragon [King and King, 1975], which further supports the stability of the sequential content of the
macrochromosomes. In Komodo dragons, the rDNA region is part of the heterochromatin localised in the pericentromeric region of the largest pairs. The heterochromatin on several other macrochromosomes is also localised in pericentromeric positions. Our CMA3/DAPI
staining shows that within this heterochromatin there are
clear GC-rich regions. Contrastingly, the microchromosomes do not possess any specific GC or AT accumulations. This technique has not yet been applied in any other species of the family, and so we are currently unable to
further compare the GC/AT genome composition in varanids.
As expected from data in other varanid species examined and from the pattern of facultative asexuality, we
showed that the Komodo dragon possesses female heterogamety with a differentiated W chromosome. The
identification of ZZ/ZW sex chromosomes proves that
the cytogenetic inspection of offspring can be used to cor-

The differences in size and morphology of W chromosomes among monitor lizards can be partially explained
by the differences in the accumulation of microsatellite
repeats. Matsubara et al. [2014] identified an accumulation of AAT microsatellite motifs in the W chromosome
of V. acanthurus and of the CGG motif in the W chromosomes of V. rosenbergi and V. gouldii. However, neither
the probes with these microsatellite motifs, nor the probe
with the GATA microsatellite motif, which was found to
accumulate in sex chromosomes of other reptiles
[OMeally et al., 2010], revealed accumulations in the W
chromosome of V. komodoensis. Also, our CMA3/DAPI
staining shows that there is no GC-rich region present on
the W chromosome of this species (fig.2D, E). The relatively small size of the W in the Komodo dragon in comparison to other microchromosomes cannot be fully explained by the absence of accumulated repetitive motives,
because, in the case of V. gouldii, the W is also the same
size as some of the other microchromosomes and yet contains microsatellite accumulation [Matsubara et al., 2014].
Comparison among varanids further supports the observation that the repetitive content and morphology of unpaired sex chromosomes are evolutionarily highly plastic and dynamic [Matsubara et al., 2006; Pokorn et al.,

2011b; Rutkowska et al., 2012; Altmanov et al., 2016].

Degenerated sex chromosomes may therefore represent
the most dynamic part of the genome.

Acknowledgements
The authors would like to express their gratitude to Petr Rb
for his ongoing support. We are also grateful to Prague Zoo
namely Zdenk Kymla and Nataa Velensk for their cooperation
on this project. Alexandr Sember kindly provided the telomeric
probe, and Chris Johnson performed the language revision of the
manuscript and provided valuable comments.

Statement of Ethics
The processing of the biological material was carried out under
the supervision and with the approval of the Ethics Committee of
the Faculty of Science, Charles University in Prague, followed by
the Committee for Animal Welfare of the Ministry of Agriculture
of the Czech Republic.

Disclosure Statement
The authors have no conflicts of interest to declare.

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