Highly Differentiated ZW Sex Microchromosomes in the Australian Varanus Species Evolved through Rapid Amplification of Repetitive Sequences

Abstract

Transitions between sex determination systems have occurred in many lineages of squamates and it follows that novel sex chromosomes will also have arisen multiple times. The formation of sex chromosomes may be reinforced by inhibition of recombination and the accumulation of repetitive DNA sequences. The karyotypes of monitor lizards are known to be highly conserved yet the sex chromosomes in this family have not been fully investigated. Here, we compare male and female karyotypes of three Australian monitor lizards, Varanus acanthurus, V. gouldii and V. rosenbergi, from two different clades. V. acanthurus belongs to the acanthurus clade and the other two belong to the gouldii clade. We applied C-banding and comparative genomic hybridization to reveal that these species have ZZ/ZW sex micro-chromosomes in which the W chromosome is highly differentiated from the Z chromosome. In combination with previous reports, all six Varanus species in which sex chromosomes have been identified have ZZ/ZW sex chromosomes, spanning several clades on the varanid phylogeny, making it likely that the ZZ/ZW sex chromosome is ancestral for this family. However, repetitive sequences of these ZW chromosome pairs differed among species. In particular, an (AAT)n microsatellite repeat motif mapped by fluorescence in situ hybridization on part of W chromosome in V. acanthurus only, whereas a (CGG)n motif mapped onto the W chromosomes of V. gouldii and V. rosenbergi. Furthermore, the W chromosome probe for V. acanthurus produced hybridization signals only on the centromeric regions of W chromosomes of the other two species. These results suggest that the W chromosome sequences were not conserved between gouldii and acanthurus clades and that these repetitive sequences have been amplified rapidly and independently on the W chromosome of the two clades after their divergence.

First Description of the Karyotype and Sex Chromosomes in the Komodo Dragon <b><i>(Varanus komodoensis)</i></b>

“…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 fam- 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.…”

“…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 μl hybridization buffer (50% formamide, 2× SSC, 10% SDS, 10% dextran sulphate, 1× Denhardt's buffer, pH 7).…”

“…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;Pokorná and Kratochvíl, 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 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].…”

“…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 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.…”

“…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 .…”