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Cytogenet Genome Res 2014;143:251–258 Accepted: May 7, 2014 DOI: 10.1159/000366172 by M. Schmid Published online: September 6, 2014

ni semosomorhC xeS suogolomoH-noN suogolomoH-noN xeS semosomorhC ni Two Geckos (Gekkonidae: Gekkota) with Female Heterogamety

a c, d e c Kazumi Matsubara Tony Gamble Yoichi Matsuda David Zarkower a a a, b a Stephen D. Sarre Arthur Georges Jennifer A. Marshall Graves Tariq Ezaz a b Institute for Applied Ecology, University of Canberra, Canberra, A.C.T., and School of Life Science, La Trobe c d University, Melbourne, Vic. , Australia; Department of Genetics, Cell Biology, and Development, and Bell Museum e of Natural History, University of Minnesota, Minneapolis, Minn. , USA; Laboratory of Animal Genetics, Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya , Japan

sdroW yeK yeK sdroW Transitions between di erent genetic sex-determining Chromosome paint · Cytogenetics · FISH · Homology · mechanisms involving a change in male and female het- Reptilia erogamety are readily apparent and easy to identify [Chen and Reisman, 1970; Vol and Schartl, 2001; Ogata et al., 2003; Ezaz et al., 2006; Sarre et al., 2011]. On the other tcartsbA tcartsbA hand, the evolution of a new sex chromosome system that Evaluating homology between the sex chromosomes of dif- does not involve a transition between male and female ferent species is an important rst step in deducing the ori- heterogamety, i.e. a transition from one XY system to a gins and evolution of sex-determining mechanisms in a di erent XY system derived from a di erent autosomal clade. Here, we describe the preparation of Z and W chromo- pair, or a transition from one ZW system to another, can some paints via chromosome microdissection from the Aus- be identied only by assessing homology among the rel- tralian marbled gecko (Christinus marmoratus) and their sub- evant sex chromosomes [Takehana et al., 2007; Tanaka et sequent use in evaluating sex chromosome homology with al., 2007; Cnaani et al., 2008; Henning et al., 2008; Ross et the ZW chromosomes of the Kwangsi gecko (Gekko hokouen- al., 2009; Vicoso and Bachtrog, 2013]. Considered more sis) from eastern Asia. We show that the ZW sex chromo- broadly, failure to incorporate chromosome homology somes of C. marmoratus and G. hokouensis are not homolo- will probably undercount the actual number of transi- gous and represent independent origins of female hetero- tions among sex-determining mechanisms in a clade gamety within the Gekkonidae. We also show that the C. when mapping these mechanisms onto a phylogeny marmoratus Z and W chromosomes are genetically similar to [Gamble, 2010]. erefore, evaluating homology should each other as revealed by C-banding, comparative genomic be a high priority for researchers interested in studying hybridization, and the reciprocal painting of Z and W chro- mosome probes. This implies that sex chromosomes in C. marmoratus are at an early stage of dierentiation, suggest- ing a recent origin. © 2014 S. Karger AG, Basel K.M. and T.G. contributed equally to this work.

© 2014 S. Karger AG, Basel Kazumi Matsubara 1424–8581/14/1434–0251$39.50/0 Institute for Applied Ecology University of Canberra E-Mail [email protected] Canberra, ACT 2601 (Australia) www.karger.com/cgr E-Mail kazumi.matsubara @ canberra.edu.au b; Ross et al., 2009; Gamble et al., 2014]; PCR amplica- tion of sex-specic markers [Takehana et al., 2008; Gam- ble and Zarkower, 2014]; qPCR of sex-linked markers [Gamble et al., 2014; Rovatsos et al., 2014]; whole genome sequencing [Chen et al., 2014]; and chromosome paint- ing using ow-sorted or microdissected chromosomes [Phillips et al., 2001; Grützner et al., 2004; Henning et al., 2008; Pokorná et al., 2011]. Here, we describe the preparation of Z and W chromo- some paints via microdissection from the Australian marbled gecko (Christinus marmoratus) [Gray, 1845] and their subsequent use to evaluate sex chromosome homol- ogy with the ZW chromosomes of the Kwangsi gecko (Gekko hokouensis) [Pope, 1928]. Both species are in the

ing mechanisms have not been yet identied in most gek- konids, female heterogametic sex chromosomes (ZW sys-

homology of G. hokouensis and C. marmoratus sex chro- mosomes provides an important step in accurately esti- mating the number of transitions among sex-determin- ing mechanisms that have occurred across geckos. e sex chromosomes of geckos are also of particular interest because the G. hokouensis ZW pair is homologous with the avian ZW system [Kawai et al., 2009], supporting the hypothesis that the bird ZW system may be ancestral to reptiles and mammals [Graves, 2009]. However, non-ho- Fig. 1. Phylogenetic relationships among species in the family Gek- mology of ZW pairs in closely related species [Pokorná et konidae with known sex-determining mechanisms. Genera with al., 2011] favored the alternate hypothesis that they were multiple species that all possess the same sex-determining mecha- nism are represented by a single species. Phylogeny and sex deter- independently derived from a particularly propitious au- mination data were taken from Gamble [2010] and Gamble et al. tosome [Graves and Peichel, 2010; O’Meally et al., 2012]. [2012]. us studies of sex chromosome homology in geckos can help answer this deep question of amniotes’ sex chromo- some ancestry and evolution. Four karyomorphs (2n = 32, 2n = 34, 2n = 36, and the evolution of sex chromosomes and sex-determining 2n = 36 with heteromorphic ZZ/ZW) have been identi- mechanisms. ed in the widely distributed southern Australian C. mar- Historically, chromosome homology was established moratus [King and Rofe, 1976; King and King, 1977]. e using chromosomal morphology combined with G-, C-, distribution of these karyomorphs’ chromosomal forms or R-banding [Stock et al., 1974; Patton and Baker, 1978]. is discrete and each karyomorph is allopatrically distrib- However, these features lack su cient resolution and are uted [King and Rofe, 1976; King and King, 1977]. A sub- susceptible to convergence, so similarities in morphology set of the 2n = 36 chromosomal form that lacks hetero- and banding patterns do not imply orthology of DNA se- morphic sex chromosomes has since been described as a quences [Kluge, 1994; Stanyon et al., 1995]. Methods that separate species, Christinus alexanderi [Storr, 1987]. is either directly or indirectly compare DNA sequences be- cytogenetic diversity, coupled with molecular genetic di- tween species are now widely used, and homology be- versity across its range, suggests that C. marmoratus is a tween the sex chromosomes of dierent species can be species complex [Heinicke et al., 2014]. We examined C. properly evaluated using several techniques. ese in- marmoratus from the vicinity of Canberra (Australian clude: FISH mapping of BACs, fosmids, or cDNAs [Mat- Capital Territory), which are part of the 2n = 36 (ZZ/ZW) subara et al., 2006; Takehana et al., 2007; Ezaz et al., 2009a, group. e heteromorphic Z and W are distinguishable

252 Cytogenet Genome Res 2014;143:251–258 Matsubara /Gamble /Matsuda /Zarkower / DOI: 10.1159/000366172 Sarre /Georges /Graves /Ezaz from each other and most other chromosomes [King and a Rofe, 1976] and present an ideal opportunity to microdis- sect sex chromosomes from a ZW gecko species to gener- ate a gekkonid sex chromosome paint. e sex chromosomes of G. hokouensis represent the rst attempt to identify a sex chromosome linkage group in a gecko [Kawai et al., 2009]. is species is widely dis- tributed in southeastern China, Taiwan, some of the Ryukyu islands, and part of Kyushu, Japan. G. hokouensis , like C. marmoratus , is most likely a species complex [Shibaike et al., 2009]. All populations possess a diploid b chromosome number of 2n = 38, although heteromor- phic ZW sex chromosomes are present in only some pop- ulations [Kawai et al., 2009; Shibaike et al., 2009]. In con- trast to a dissimilarity of their karyotypes, G. hokouensis and C. marmoratus sex chromosomes have a similar mor- phology: the Z chromosomes are acrocentric with small short arms, and the W chromosomes are distinct bi- armed chromosomes [King and Rofe, 1976; Kawai et al., species is likely to be the result of a pericentric inversion in the W, although the G. hokouensis W shows evidence of at least 1 additional rearrangement [King and Rofe, Fig. 2. DAPI-stained karyotypes of females of C. marmoratus (a ) 1976; Kawai et al., 2009]. Despite their supercial similar- and G. hokouensis (b ). Scale bars = 10 µm. ity in morphology, we show that the ZW sex chromo- somes of C. marmoratus and G. hokouensis are not ho- mologous and represent independently derived ZW sex ture ask at a nal concentration of 75 ng/ml prior to harvesting. chromosomes within the Gekkonidae. Following harvesting, cultured cells were suspended in 0.075 M KCl

and xed in 3: 1 methanol:acetic acid, and the cell suspension was dropped onto glass slides, air-dried and stored at –80 ° C. sdohteM dna slairetaM slairetaM dna sdohteM C-Banding Animals e C-banded chromosomes were obtained with the CBG Tail tips were cut from 4 male and 5 female C. marmoratus and method [Sumner, 1972]. Chromosome slides were treated in 0.2 N used for cell culture. Animal collection, handling, sampling, and HCl for 40 min and rinsed by distilled water. en the slides were all other relevant procedures were performed following the guide- denatured in 5% Ba(OH) 2 for 5 min at 50 ° C. Denaturation was lines of the Australian Capital Territory Animal Welfare Act 1992 stopped by rinsing the slides in 0.2 N HCl and distilled water. e (Section 40), and conducted under CEAE 11/07 (the Committee chromosome slides were renatured by incubation in 2× SSC for 60 for Ethics in Animal Experimentation at the University of Can- min at 60 ° C. en the slides were rinsed with distilled water and berra). Fibroblasts of a male and a female G. hokouensis originally stained with 4% Giemsa for 30 min. collected at Nakagusuku, the southern part of Okinawajima Island, the Ryukyu Archipelago, Japan, were used for cell culture [Kawai Microdissection of Sex Chromosomes and Preparation of et al., 2009]. Chromosome Paints We performed microdissection using an inverted phase-con- Chromosome Preparation trast microscope Zeiss Axiovert.A1 (Zeiss, Oberkochen, Germa- Metaphase chromosome spreads were prepared from broblast ny) equipped with Eppendorf TransferMan NK 2 micromanipula- cell lines of tail tissues following the protocol described in Ezaz et tor (Eppendorf, Hamburg, Germany). Glass needles were made al. [2008]. Briey, minced tail tissues were implanted in a T25 cul- from 1.0 mm diameter capillary glass using a glass capillary puller, ture ask containing AmnioMax medium (Life Technologies, Sutter P-30 Micropipette Puller (Sutter Instrument, Novato, Calif., Carlsbad, Calif., USA) and were allowed to propagate under the USA) and sterilized by ultraviolet irradiation. Z and W chromo- condition of 28 ° C and 5% CO2 . Once the broblast cells had grown somes were scratched from freshly prepared slides of a female C. to about 80% conuency, they were split into T75 asks and subse- marmoratus with a glass needle using the micromanipulation sys- quently split up to 4 passages before the chromosomes were har- tem and transferred into 0.2-ml PCR tubes. Because the Z chromo- vested. Colcemid (Roche, Basel, Switzerland) was added to the cul- some could be confused with several autosomes of similar size and

Non-Homology between Sex Cytogenet Genome Res 2014;143:251–258 253 Chromosomes of Two Geckos DOI: 10.1159/000366172 of the W chromosome. is C-band pattern indicates metaphase were transferred into separate PCR tubes. In contrast that most of the W chromosome is not heterochromatic. to the Z, the W chromosome was easily distinguishable from other We performed CGH on chromosome spreads from 3 somes individually, others were collected from multiple metaphas- female and 2 male C. marmoratus to examine whether the es and placed into 1 PCR tube. Chromosome DNAs were ampli ed W chromosome contains any female-speci c DNA. CGH using GenomePlex® Single Cell Whole Genome Ampli cation Kit images showed equal intensity of red signal (female ge- (Sigma-Aldrich, St. Louis, Mo., USA) according to the manufac- nomic DNA) and green signal (male genomic DNA) on turer’s protocol with slight modi cation. e volume for all reac- tion steps was scaled down to half, and PCR ampli cation was whole chromosomes and detected no accumulation of increased to 30 cycles. somes of this species are morphologically dierentiated FISH with Chromosome Paints but the DNA sequence of the W chromosome is not FISH and CGH were conducted according to our previous prominently divergent from that of the Z chromosome. study [Ezaz et al., 2005] with slight modi cation as follows. Chro- mosome paints were labeled by nick translation incorporating SpectrumGreen-dUTP (Abbott, North Chicago, Ill., USA) or Chromosome Painting with Z and W Paints SpectrumOrange-dUTP (Abbott). e labeled paint was precipi- We prepared 4 W chromosome probes and 10 Z chro- tated with 20 µg glycogen as carrier, and dissolved in 15 µl hybrid- mosome probes. While 2 of the 4 W chromosome probes ization buer (50% formamide, 10% dextran sulfate, 2× SSC, 40 were ampli ed from single W chromosomes, the other 2 mM sodium phosphate pH 7.0 and 1× Denhardt’s solution). e hybridization mixture was placed on a chromosome slide and were ampli ed from 5 and 4 W chromosomes, respec- sealed with a coverslip and rubber cement. e probe DNA and tively. All 10 Z probes were ampli ed from single Z chro- chromosome DNA were denatured by heating the slide on a heat mosomes. plate at 68.5 ° C for 5 min. e slides were hybridized overnight in We tested the quality of each probe and the homology a humid chamber at 37 ° C. Hybridization was carried out for 2 days between the Z and W by painting each back onto meta- in cross-species chromosome painting. e slides were then washed by the following series: 0.4× SSC, 0.3% IGEPAL (Sigma- phase spreads of female C. marmoratus . All 4 W probes Aldrich) at 55 ° C for 2 min followed by 2× SSC, 0.1% IGEPAL at successfully produced bright hybridization signals on room temperature for 1 min. e slides were dehydrated by etha- e 2 probes from multiple W chromosomes showed DAPI, 2× SSC and mounted with anti-fade medium, Vectashield more intense signals than the 2 probes from single chro- (Vector Laboratories, Burlingame, Calif., USA). FISH images were captured using a Zeiss Axioplan epiuorescence microscope mosomes (data not shown). ree of the 10 Z probes equipped with a CCD camera (Zeiss). ISIS soware or AxioVision failed to hybridize, probably because of loss of the single (Zeiss) was used for microphotography and analyzing images. chromosome at the microdissection step. e other 7 Z probes produced bright hybridization signals on whole CGH Total genomic DNA was extracted from cultured broblasts using the DNeasy kit (Qiagen, Venlo, e Netherlands) and fol- lowing the manufacturer’s protocol. Genomic DNA was labeled by probes of both chromosomes produced intense signals nick translation incorporating SpectrumOrange-dUTP (Abbott) around the C-band-positive centromeric region of the W for females and SpectrumGreen-dUTP (Abbott) for males. e chromosome, suggesting accumulation of some repeti- labeled male and female DNA was coprecipitated with 20 µg gly- tive sequences in this region. e hybridization of Z and cogen as carrier, and dissolved in 15 µl hybridization buer. e hybridization and washes were carried out as above. W probes to both Z and W chromosomes provides fur- ther evidence that DNA sequences of the 2 chromosomes are not highly dierentiated from each other. Both Z and W probes produced hybridization signals on telomeric stluseR stluseR regions of several autosomes and faint hybridization sig- nals on interstitial regions of a few autosomes. is sug- C-Banding and CGH gests that some repetitive sequences are shared between All C. marmoratus examined for this study had kar- sex chromosomes and autosomes. yotypes with 2n = 36 chromosomes, and all females had morphologically dierentiated ZW chromosomes Cross-Species Chromosome Painting to G. hokouensis Chromosomes We carried out cross-species chromosome painting A faint C-band was also detected on the proximal region using C. marmoratus sex chromosome probes to meta-

254 Cytogenet Genome Res 2014;143:251–258 Matsubara /Gamble /Matsuda /Zarkower / DOI: 10.1159/000366172 Sarre /Georges /Graves /Ezaz a b c

d e f

Fig. 3. C-banding and chromosome painting of female C. marmoratus . a C-banding distinguishes Z and W chro- mosomes. b CGH with female (red) and male (green) genomic DNA shows no obvious sex-speci c sequences. c DAPI staining of the same metaphase. d , e Chromosome painting with W ( d ) and Z chromosome probes ( e ) to the same metaphase spread and the DAPI-stained image ( f ). Larger images of Z and W chromosomes are shown in insets ( a , b , d , e ). Scale bars = 10 µm.

a b

Fig. 4. Cross-species chromosome painting with C. marmoratus W (a ) and Z ( b ) chromosome probes to meta- phase spreads of a female G. hokouensis . Z and W chromosomes are indicated. Arrowheads point to hybridiza- .mµ 01 = srab elacS .slangis noit .slangis elacS srab = 01 .mµ

Non-Homology between Sex Cytogenet Genome Res 2014;143:251–258 255 Chromosomes of Two Geckos DOI: 10.1159/000366172 phase spreads of female G. hokouensis in order to test for sex-determining mechanisms within Gekkonidae. is homology of the sex chromosomes between the 2 species. means sex chromosome evolution within Gekkonidae, e C. marmoratus W probe hybridized to a large region and Gekkota more generally, is more complex than ini- of the h-largest autosomal pair of G. hokouensis tially thought [Janzen and Krenz, 2004; Janzen and Phil- lips, 2006; Pokorná and Kratochvíl, 2009; Gamble, 2010], pair, although the hybridization signal was not as strong and may indicate substantial cryptic diversity in the ZW mechanisms of sex determination in this group. Evaluat- C. marmoratus sex chromosomes share homology with ing sex chromosome homology among more gecko spe- G. hokouensis autosome number 5, and are not homolo- cies should be a high priority for future research. e de- gous to the sex chromosomes. Hybridization signals were velopment of the C. marmoratus ZW paints, along with also detected on interstitial and telomeric regions of ad- chromosome paints from ow-sorted chromosomes in ditional chromosomes. Such signals were also observed additional gecko species [Trifonov et al., 2011], will be in the painting within C. marmoratus useful in this regard. signals were probably caused by hybridization of the re- Homology between the ZW sex chromosomes of birds petitive sequences shared among the chromosomes of the and G. hokouensis , along with homology to monotreme 2 species. sex chromosomes, led to the hypothesis that an avian-like ZW may be the ancestral sex chromosome for all amni- otes [Graves, 2009]. Our results are the rst to explore sex noissucsiD noissucsiD chromosome homology between gecko species with fe- male heterogamety and suggest that all instances of ZW Dierences between C. marmoratus Z and W appear sex determination in the Gekkonidae cannot be automat- to be small. Evidence for similarity between the Z and W ically viewed as homologous. Further testing of this hy- includes limited heterochromatin accumulation on the pothesis through sampling additional gecko species is W based on C-banding, absence of female-specic signal particularly important because geckos are the sister clade using CGH, and the reciprocal hybridization of Z and W to the remaining squamates, exclusive of the limbless di- paints along nearly the entire length of both chromo- bamids [Townsend et al., 2004; Vidal and Hedges, 2009]; somes. is implies that the sex chromosomes are at an and, of course, one of the species central to generating the early stage of dierentiation, and suggests that they hypothesis is a gecko. Examining sex chromosome ho- evolved recently. Both CGH and chromosome painting mology between the avian ZW and additional squamate are not able to detect tiny sex-specic chromosomal re- species, including additional geckos, yielded little support gions so that ner-scale analyses such as gene mapping for this hypothesis. Pokorná et al. [2011] examined 2 and whole chromosome sequencing are necessary to gecko species with male heterogamety and heteromor- identify dierentiated chromosomal regions between the phic sex chromosomes, Lialis burtonis (Pygopodidae) Z and W chromosomes. Since the C. marmoratus species and Coleonyx elegans (Eublepharidae). In neither species complex is relatively young, at most 10 million years old were sex chromosomes homologous with the avian Z [Heinicke et al., 2014], this might mean that the dieren- (and the G. hokouensis ZW). e discovery here that sex tiation of new sex chromosomes occurred aer diver- chromosomes of C. marmoratus, an additional gecko gence with C. alexanderi . However, the sex determination species in the same family (Gekkonidae), do not share system of C. alexanderi is unknown so far and sex chro- homology with the G. hokouensis and avian ZW further mosomes can remain morphologically undierentiated weakens support for the ancestral avian-like ZW hypoth- over long periods of time [Ogawa et al., 1998; Matsubara esis. Rather, this region of the genome must have been et al., 2006; Gamble et al., 2014]; so these results should chosen independently at least 3 times, suggesting that it be interpreted with caution. Further work that includes contains genes (perhaps such as DMRT1 ) that are par- other members of the species complex will be needed to ticularly suitable for the job of determining sex [Graves better understand the origin and evolution of Christinus and Peichel, 2010; Matson and Zarkower, 2012; O’Meally sex chromosomes. et al., 2012]. Hybridization of the C. marmoratus Z and W paints with G. hokouensis chromosomes revealed no homology between the ZW sex chromosomes of the 2 species. is lack of homology suggests independently derived ZW

256 Cytogenet Genome Res 2014;143:251–258 Matsubara /Gamble /Matsuda /Zarkower / DOI: 10.1159/000366172 Sarre /Georges /Graves /Ezaz stnemegdelwonkcA stnemegdelwonkcA ery Grant (ARC DP110102262 – to T.E., S.D.S., A.G., J.A.M.G. and Y.M.); Australian Research Council future fellowship We thank M. Pepper and M. Young for help with the eldwork. (FT110100733 – to T.E.); and the U.S. National Science Founda- is work was supported by: Australian Research Council Discov- tion (IOS1146820 – to D.Z.).

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