Fixed Allele Differences Associated With the Centromere Reveal Chromosome Morphology and Rearrangements in a Reptile (Varanus acanthurus BOULENGER).

Chromosome rearrangements are often implicated with genomic divergence and are proposed to be associated with species evolution. Rearrangements alter the genomic structure and interfere with homologous recombination by isolating a portion of the genome. Integration of multiplatform next-generation DNA sequencing technologies has enabled putative identification of chromosome rearrangements in many taxa; however, integrating these data sets with cytogenetics is still uncommon beyond model genetic organisms. Therefore, to achieve the ultimate goal for the genomic classification of eukaryotic organisms, physical chromosome mapping remains critical. The ridge-tailed goannas (Varanus acanthurus BOULENGER) are a group of dwarf monitor lizards comprised of several species found throughout northern Australia. These lizards exhibit extreme divergence at both the genic and chromosomal levels. The chromosome polymorphisms are widespread extending across much of their distribution, raising the question if these polymorphisms are homologous within the V. acanthurus complex. We used a combined genomic and cytogenetic approach to test for homology across divergent populations with morphologically similar chromosome rearrangements. We showed that more than one chromosome pair was involved with the widespread rearrangements. This finding provides evidence to support de novo chromosome rearrangements have occurred within populations. These chromosome rearrangements are characterized by fixed allele differences originating in the vicinity of the centromeric region. We then compared this region with several other assembled genomes of reptiles, chicken, and the platypus. We demonstrated that the synteny of genes in Reptilia remains conserved despite centromere repositioning across these taxa.

Widespread chromosomal rearrangements preceded genetic divergence in a monitor lizard, Varanus acanthurus (Varanidae).

Chromosomal rearrangements are often associated with local adaptation and speciation because they suppress recombination, and as a result, rearrangements have been implicated in disrupting gene flow. Although there is strong evidence to suggest that chromosome rearrangements are a factor in genetic isolation of divergent populations, the underlying mechanism remains elusive. Here, we applied an integrative cytogenetics and genomics approach testing whether chromosomal rearrangements are the initial process, or a consequence, of population divergence in the dwarf goanna, Varanus acanthurus. Specifically, we tested whether chromosome rearrangements are indicators of genetic barriers that can be used to identify divergent populations by looking at gene flow within and between populations with rearrangements. We found that gene flow was present between individuals with chromosome rearrangements within populations, but there was no gene flow between populations that had similar chromosome rearrangements. Moreover, we identified a correlation between reduced genetic variation in populations with a higher frequency of homozygous submetacentric individuals. These findings suggest that chromosomal rearrangements were widespread prior to divergence, and because we found populations with higher frequencies of submetacentric chromosomes were associated with lower genetic diversity, this could indicate that polymorphisms within populations are early indicators of genetic drift.

Sex-specific splicing of Z- and W-borne nr5a1 alleles suggests sex determination is controlled by chromosome conformation.

Pogona vitticeps has female heterogamety (ZZ/ZW), but the master sex-determining gene is unknown, as it is for all reptiles. We show that nr5a1 (Nuclear Receptor Subfamily 5 Group A Member 1), a gene that is essential in mammalian sex determination, has alleles on the Z and W chromosomes (Z-nr5a1 and W-nr5a1), which are both expressed and can recombine. Three transcript isoforms of Z-nr5a1 were detected in gonads of adult ZZ males, two of which encode a functional protein. However, ZW females produced 16 isoforms, most of which contained premature stop codons. The array of transcripts produced by the W-borne allele (W-nr5a1) is likely to produce truncated polypeptides that contain a structurally normal DNA-binding domain and could act as a competitive inhibitor to the full-length intact protein. We hypothesize that an altered configuration of the W chromosome affects the conformation of the primary transcript generating inhibitory W-borne isoforms that suppress testis determination. Under this hypothesis, the genetic sex determination (GSD) system of P. vitticeps is a W-borne dominant female-determining gene that may be controlled epigenetically.

High elevation increases the risk of Y chromosome loss in Alpine skink populations with sex reversal.

The view that has genotypic sex determination and environmental sex determination as mutually exclusive states in fishes and reptiles has been contradicted by the discovery that chromosomal sex and environmental influences can co-exist within the same species, hinting at a continuum of intermediate states. Systems where genes and the environment interact to determine sex present the opportunity for sex reversal to occur, where the phenotypic sex is the opposite of that predicted by their sex chromosome complement. The skink Bassiana duperreyi has XX/XY sex chromosomes with sex reversal of the XX genotype to a male phenotype, in laboratory experiments, and in field nests, in response to exposure to cold incubation temperatures. Here we studied the frequency of sex reversal in adult populations of B. duperreyi in response to climatic variation, using elevation as a surrogate for environmental temperatures. We demonstrate sex reversal in the wild for the first time in adults of a reptile species with XX/XY sex determination. The highest frequency of sex reversal occurred at the highest coolest elevation location, Mt Ginini (18.46%) and decreased in frequency to zero with decreasing elevation. We model the impact of this under Fisher's frequency-dependent selection to show that, at the highest elevations, populations risk the loss of the Y chromosome and a transition to temperature-dependent sex determination. This study contributes to our understanding of the risks of extinction from climate change in species subject to sex reversal by temperature, and will provide focus for future research to test on-the-ground management strategies to mitigate the effects of climate in local populations.

Comparative Cytogenetic Mapping and Telomere Analysis Provide Evolutionary Predictions for Devil Facial Tumour 2.

The emergence of a second transmissible tumour in the Tasmanian devil population, devil facial tumour 2 (DFT2), has prompted questions on the origin and evolution of these transmissible tumours. We used a combination of cytogenetic mapping and telomere length measurements to predict the evolutionary trajectory of chromosome rearrangements in DFT2. Gene mapping by fluorescence in situ hybridization (FISH) provided insight into the chromosome rearrangements in DFT2 and identified the evolution of two distinct DFT2 lineages. A comparison of devil facial tumour 1 (DFT1) and DFT2 chromosome rearrangements indicated that both started with the fusion of a chromosome, with potentially critically short telomeres, to chromosome 1 to form dicentric chromosomes. In DFT1, the dicentric chromosome resulted in breakage-fusion-bridge cycles leading to highly rearranged chromosomes. In contrast, the silencing of a centromere on the dicentric chromosome in DFT2 stabilized the chromosome, resulting in a less rearranged karyotype than DFT1. DFT2 retains a bimodal distribution of telomere length dimorphism observed on Tasmanian devil chromosomes, a feature lost in DFT1. Using long term cell culture, we observed homogenization of telomere length over time. We predict a similar homogenization of telomere lengths occurred in DFT1, and that DFT2 is unlikely to undergo further substantial rearrangements due to maintained telomere length.

Evolution of Marsupial Genomes.

Marsupial genomes, which are packaged into large chromosomes, provide a powerful resource for studying the mechanisms of genome evolution. The extensive and valuable body of work on marsupial cytogenetics, combined more recently with genome sequence data, has enabled prediction of the 2n = 14 karyotype ancestral to all marsupial families. The application of both chromosome biology and genome sequencing, or chromosomics, has been a necessary approach for various aspects of mammalian genome evolution, such as understanding sex chromosome evolution and the origin and evolution of transmissible tumors in Tasmanian devils. The next phase of marsupial genome evolution research will employ chromosomics approaches to begin addressing fundamental questions in marsupial genome evolution and chromosome evolution more generally. The answers to these complex questions will impact our understanding across a broad range of fields, including the genetics of speciation, genome adaptation to environmental stressors, and species management.

Chromosomics: Bridging the Gap between Genomes and Chromosomes.

The recent advances in DNA sequencing technology are enabling a rapid increase in the number of genomes being sequenced. However, many fundamental questions in genome biology remain unanswered, because sequence data alone is unable to provide insight into how the genome is organised into chromosomes, the position and interaction of those chromosomes in the cell, and how chromosomes and their interactions with each other change in response to environmental stimuli or over time. The intimate relationship between DNA sequence and chromosome structure and function highlights the need to integrate genomic and cytogenetic data to more comprehensively understand the role genome architecture plays in genome plasticity. We propose adoption of the term 'chromosomics' as an approach encompassing genome sequencing, cytogenetics and cell biology, and present examples of where chromosomics has already led to novel discoveries, such as the sex-determining gene in eutherian mammals. More importantly, we look to the future and the questions that could be answered as we enter into the chromosomics revolution, such as the role of chromosome rearrangements in speciation and the role more rapidly evolving regions of the genome, like centromeres, play in genome plasticity. However, for chromosomics to reach its full potential, we need to address several challenges, particularly the training of a new generation of cytogeneticists, and the commitment to a closer union among the research areas of genomics, cytogenetics, cell biology and bioinformatics. Overcoming these challenges will lead to ground-breaking discoveries in understanding genome evolution and function.

Sexual conflict in action: An antagonistic relationship between maternal and paternal sex allocation in the tammar wallaby, Notamacropus eugenii.

Sex ratio biases are often inconsistent, both among and within species and populations. While some of these inconsistencies may be due to experimental design, much of the variation remains inexplicable. Recent research suggests that an exclusive focus on mothers may account for some of the inconsistency, with an increasing number of studies showing variation in sperm sex ratios and seminal fluids. Using fluorescent in-situ hybridization, we show a significant population-level Y-chromosome bias in the spermatozoa of wild tammar wallabies, but with significant intraindividual variation between males. We also show a population-level birth sex ratio trend in the same direction toward male offspring, but a weaning sex ratio that is significantly female-biased, indicating that males are disproportionately lost during lactation. We hypothesize that sexual conflict between parents may cause mothers to adjust offspring sex ratios after birth, through abandonment of male pouch young and reactivation of diapaused embryos. Further research is required in a captive, controlled setting to understand what is driving and mechanistically controlling sperm sex ratio and offspring sex ratio biases and to understand the sexually antagonistic relationship between mothers and fathers over offspring sex. These results extend beyond sex allocation, as they question studies of population processes that assume equal input of sex chromosomes from fathers, and will also assist with future reproduction studies for management and conservation of marsupials.

Understanding the Evolution of Reptile Chromosomes through Applications of Combined Cytogenetics and Genomics Approaches.

Studies of reptile (nonavian reptiles) chromosomes began well over a century ago (1897) with the initial report on the description of sand lizard (Lacerta agilis) chromosomes. Since then, chromosome analysis in reptiles has contributed significantly to understanding chromosome evolution in vertebrates. Reptile karyotypes are also unique, as being the only vertebrate group where the majority of the species possess variable numbers of macro- and microchromosomes, which was first reported for iguanids and teiids in 1921. In addition, many reptiles have microchromosomes as sex chromosomes, highlighting their evolutionary significance, yet very little is known about their evolutionary origin and significance in shaping amniote genomes. Advances in genomic technologies in recent years have accelerated our capacity to understand how sequences are arranged within a genome. However, genomic and cytogenetic analyses have been combined for only 3 species to provide a deeper understanding of reptile chromosome evolution and sequence organization. In this review, we highlight how a combined approach of cytogenetic analysis and sequence analysis in reptiles can help us answer fundamental questions of chromosome evolution in reptiles, including evolution of microchromosomes and sex chromosomes.

Marsupial chromosomics: bridging the gap between genomes and chromosomes.

Marsupials have unique features that make them particularly interesting to study, and sequencing of marsupial genomes is helping to understand their evolution. A decade ago, it was a huge feat to sequence the first marsupial genome. Now, the advances in sequencing technology have made the sequencing of many more marsupial genomes possible. However, the DNA sequence is only one component of the structures it is packaged into: chromosomes. Knowing the arrangement of the DNA sequence on each chromosome is essential for a genome assembly to be used to its full potential. The importance of combining sequence information with cytogenetics has previously been demonstrated for rapidly evolving regions of the genome, such as the sex chromosomes, as well as for reconstructing the ancestral marsupial karyotype and understanding the chromosome rearrangements involved in the Tasmanian devil facial tumour disease. Despite the recent advances in sequencing technology assisting in genome assembly, physical anchoring of the sequence to chromosomes is required to achieve a chromosome-level assembly. Once chromosome-level assemblies are achieved for more marsupials, we will be able to investigate changes in the packaging and interactions between chromosomes to gain an understanding of the role genome architecture has played during marsupial evolution.

The methylation and telomere landscape in two families of marsupials with different rates of chromosome evolution.

Two marsupial families exemplify divergent rates of karyotypic change. The Dasyurid family has an extremely conserved karyotype. In contrast, there is significant chromosomal variation within the Macropodidae family, best exemplified by members of the genus Petrogale (rock-wallabies). Both families are also distinguished by their telomere landscape (length and epigenetics), with the dasyurids having a unique telomere length dimorphism not observed in other marsupials and hypothesised to be regulated in a parent-of-origin fashion. Previous work has shown that proximal ends of chromosomes are enriched in cytosine methylation in dasyurids, but that the chromosomes of a macropod, the tammar wallaby, have DNA methylation enrichment of pericentric regions. Using a combination of telomere and 5-methylcytosine immunofluorescence staining, we investigated the telomere landscape of four dasyurid and three Petrogale species. As part of this study, we also further examined the parent-of-origin hypothesis for the regulation of telomere length dimorphism in dasyurids, using epigenetic modifications known to differentiate the active maternal X chromosome, including 5-methylcytosine methylation and histone modifications H3K4me2, H3K9ac and H4Kac. Our results give further support to the parent-of-origin hypothesis for the regulation of telomere length dimorphism in dasyurids, where the paternally derived X chromosome in females was associated with long telomeres and the maternally derived with short telomeres. In contrast to the tammar wallaby, rock-wallabies demonstrated a similar 5-methylcytosine staining pattern across all chromosomes to that of dasyurids, suggesting that DNA methylation of telomeric regions is not responsible for differences in the rates of chromosome evolution between these two families.

Adaptation and conservation insights from the koala genome.

The koala, the only extant species of the marsupial family Phascolarctidae, is classified as 'vulnerable' due to habitat loss and widespread disease. We sequenced the koala genome, producing a complete and contiguous marsupial reference genome, including centromeres. We reveal that the koala's ability to detoxify eucalypt foliage may be due to expansions within a cytochrome P450 gene family, and its ability to smell, taste and moderate ingestion of plant secondary metabolites may be due to expansions in the vomeronasal and taste receptors. We characterized novel lactation proteins that protect young in the pouch and annotated immune genes important for response to chlamydial disease. Historical demography showed a substantial population crash coincident with the decline of Australian megafauna, while contemporary populations had biogeographic boundaries and increased inbreeding in populations affected by historic translocations. We identified genetically diverse populations that require habitat corridors and instituting of translocation programs to aid the koala's survival in the wild.

Chromosome Evolution in Marsupials.

Marsupials typically possess very large, distinctive chromosomes that make them excellent subjects for cytogenetic analysis, and the high level of conservation makes it relatively easy to track chromosome evolution. There are two speciose marsupial families with contrasting rates of karyotypic evolution that could provide insight into the mechanisms driving genome reshuffling and speciation. The family Dasyuridae displays exceptional karyotype conservation with all karyotyped species possessing a 2n = 14 karyotype similar to that predicted for the ancestral marsupial. In contrast, the family Macropodidae has experienced a higher rate of genomic rearrangement and one genus of macropods, the rock-wallabies (Petrogale), has experienced extensive reshuffling. For at least some recently diverged Petrogale species, there is still gene flow despite hybrid fertility issues, making this species group an exceptional model for studying speciation. This review highlights the unique chromosome features of marsupial chromosomes, particularly for these two contrasting families, and the value that a combined cytogenetics, genomics, and epigenomics approach will have for testing models of genome evolution and speciation.

Identification of candidate genes for devil facial tumour disease tumourigenesis.

Devil facial tumour (DFT) disease, a transmissible cancer where the infectious agent is the tumour itself, has caused a dramatic decrease in Tasmanian devil numbers in the wild. The purpose of this study was to take a candidate gene/pathway approach to identify potentially perturbed genes or pathways in DFT. A fusion of chromosome 1 and X is posited as the initial event leading to the development of DFT, with the rearranged chromosome 1 material now stably maintained as the tumour spreads through the population. This hypothesis makes chromosome 1 a prime chromosome on which to search for mutations involved in tumourigenesis. As DFT1 has a Schwann cell origin, we selected genes commonly implicated in tumour pathways in human nerve cancers, or cancers more generally, to determine whether they were rearranged in DFT1, and mapped them using molecular cytogenetics. Many cancer-related genes were rearranged, such as the region containing the tumour suppressor NF2 and a copy gain for ERBB3, a member of the epidermal growth factor receptor family of receptor tyrosine kinases implicated in proliferation and invasion of tumours in humans. Our mapping results have provided strong candidates not previously detected by sequencing DFT1 genomes.

Chromosomal Speciation in the Genomics Era: Disentangling Phylogenetic Evolution of Rock-wallabies.

The association of chromosome rearrangements (CRs) with speciation is well established, and there is a long history of theory and evidence relating to "chromosomal speciation." Genomic sequencing has the potential to provide new insights into how reorganization of genome structure promotes divergence, and in model systems has demonstrated reduced gene flow in rearranged segments. However, there are limits to what we can understand from a small number of model systems, which each only tell us about one episode of chromosomal speciation. Progressing from patterns of association between chromosome (and genic) change, to understanding processes of speciation requires both comparative studies across diverse systems and integration of genome-scale sequence comparisons with other lines of evidence. Here, we showcase a promising example of chromosomal speciation in a non-model organism, the endemic Australian marsupial genus Petrogale. We present initial phylogenetic results from exon-capture that resolve a history of divergence associated with extensive and repeated CRs. Yet it remains challenging to disentangle gene tree heterogeneity caused by recent divergence and gene flow in this and other such recent radiations. We outline a way forward for better integration of comparative genomic sequence data with evidence from molecular cytogenetics, and analyses of shifts in the recombination landscape and potential disruption of meiotic segregation and epigenetic programming. In all likelihood, CRs impact multiple cellular processes and these effects need to be considered together, along with effects of genic divergence. Understanding the effects of CRs together with genic divergence will require development of more integrative theory and inference methods. Together, new data and analysis tools will combine to shed light on long standing questions of how chromosome and genic divergence promote speciation.

Implications of monotreme and marsupial chromosome evolution on sex determination and differentiation.

Studies of chromosomes from monotremes and marsupials endemic to Australasia have provided important insight into the evolution of their genomes as well as uncovering fundamental differences in their sex determination/differentiation pathways. Great advances have been made this century into solving the mystery of the complicated sex chromosome system in monotremes. Monotremes possess multiple different X and Y chromosomes and a candidate sex determining gene has been identified. Even greater advancements have been made for marsupials, with reconstruction of the ancestral karyotype enabling the evolutionary history of marsupial chromosomes to be determined. Furthermore, the study of sex chromosomes in intersex marsupials has afforded insight into differences in the sexual differentiation pathway between marsupials and eutherians, together with experiments showing the insensitivity of the mammary glands, pouch and scrotum to exogenous hormones, led to the hypothesis that there is a gene (or genes) on the X chromosome responsible for the development of either pouch or scrotum. This review highlights the major advancements made towards understanding chromosome evolution and how this has impacted on our understanding of sex determination and differentiation in these interesting mammals.

Marsupials as models for understanding the role of chromosome rearrangements in evolution and disease.

Chromosome rearrangements have been implicated in diseases, such as cancer, and speciation, but it remains unclear whether rearrangements are causal or merely a consequence of these processes. Two marsupial families with very different rates of karyotype evolution provide excellent models in which to study the role of chromosome rearrangements in a disease and evolutionary context. The speciose family Dasyuridae displays remarkable karyotypic conservation, with all species examined to date possessing nearly identical karyotypes. Despite the seemingly high degree of chromosome stability within this family, they appear prone to developing tumours, including transmissible devil facial tumours. In contrast, chromosome rearrangements have been frequent in the evolution of the species-rich family Macropodidae, which displays a high level of karyotypic diversity. In particular, the genus Petrogale (rock-wallabies) displays an extraordinary level of chromosome rearrangement among species. For six parapatric Petrogale species, it appears that speciation has essentially been caught in the act, providing an opportunity to determine whether chromosomal rearrangements are a cause or consequence of speciation in this system. This review highlights the reasons that these two marsupial families are excellent models for testing hypotheses for hotspots of chromosome rearrangement and deciphering the role of chromosome rearrangements in disease and speciation.

Identification of interleukin genes in Pogona vitticeps using a de novo transcriptome assembly from RNA-seq data.

Interleukins are a group of cytokines with complex immunomodulatory functions that are important for regulating immunity in vertebrate species. Reptiles and mammals last shared a common ancestor more than 350 million years ago, so it is not surprising that low sequence identity has prevented divergent interleukin genes from being identified in the central bearded dragon lizard, Pogona vitticeps, in its genome assembly. To determine the complete nucleotide sequences of key interleukin genes, we constructed full-length transcripts, using the Trinity platform, from short paired-end read RNA sequences from stimulated spleen cells. De novo transcript reconstruction and analysis allowed us to identify interleukin genes that are missing from the published P. vitticeps assembly. Identification of key cytokines in P. vitticeps will provide insight into the essential molecular mechanisms and evolution of interleukin gene families and allow for characterization of the immune response in a lizard for comparison with mammals.

Anchoring genome sequence to chromosomes of the central bearded dragon (Pogona vitticeps) enables reconstruction of ancestral squamate macrochromosomes and identifies sequence content of the Z chromosome.

BACKGROUND: Squamates (lizards and snakes) are a speciose lineage of reptiles displaying considerable karyotypic diversity, particularly among lizards. Understanding the evolution of this diversity requires comparison of genome organisation between species. Although the genomes of several squamate species have now been sequenced, only the green anole lizard has any sequence anchored to chromosomes. There is only limited gene mapping data available for five other squamates. This makes it difficult to reconstruct the events that have led to extant squamate karyotypic diversity. The purpose of this study was to anchor the recently sequenced central bearded dragon (Pogona vitticeps) genome to chromosomes to trace the evolution of squamate chromosomes. Assigning sequence to sex chromosomes was of particular interest for identifying candidate sex determining genes. RESULTS: By using two different approaches to map conserved blocks of genes, we were able to anchor approximately 42 % of the dragon genome sequence to chromosomes. We constructed detailed comparative maps between dragon, anole and chicken genomes, and where possible, made broader comparisons across Squamata using cytogenetic mapping information for five other species. We show that squamate macrochromosomes are relatively well conserved between species, supporting findings from previous molecular cytogenetic studies. Macrochromosome diversity between members of the Toxicofera clade has been generated by intrachromosomal, and a small number of interchromosomal, rearrangements. We reconstructed the ancestral squamate macrochromosomes by drawing upon comparative cytogenetic mapping data from seven squamate species and propose the events leading to the arrangements observed in representative species. In addition, we assigned over 8 Mbp of sequence containing 219 genes to the Z chromosome, providing a list of genes to begin testing as candidate sex determining genes. CONCLUSIONS: Anchoring of the dragon genome has provided substantial insight into the evolution of squamate genomes, enabling us to reconstruct ancestral macrochromosome arrangements at key positions in the squamate phylogeny, demonstrating that fusions between macrochromosomes or fusions of macrochromosomes and microchromosomes, have played an important role during the evolution of squamate genomes. Assigning sequence to the sex chromosomes has identified NR5A1 as a promising candidate sex determining gene in the dragon.

Global DNA Methylation patterns on marsupial and devil facial tumour chromosomes.

BACKGROUND: Despite DNA methylation being one of the most widely studied epigenetic modifications in eukaryotes, only a few studies have examined the global methylation status of marsupial chromosomes. The emergence of devil facial tumour disease (DFTD), a clonally transmissible cancer spreading through the Tasmanian devil population, makes it a particularly pertinent time to determine the methylation status of marsupial and devil facial tumour chromosomes. DNA methylation perturbations are known to play a role in genome instability in human tumours. One of the interesting features of the devil facial tumour is its remarkable karyotypic stability over time as only four strains with minor karyotypic differences having been reported. The cytogenetic monitoring of devil facial tumour (DFT) samples collected over an eight year period and detailed molecular cytogenetic analysis performed on the different DFT strains enables chromosome rearrangements to be correlated with methylation status as the tumour evolves. RESULTS: We used immunofluorescent staining with an antibody to 5-methylcytosine on metaphase chromosomes prepared from fibroblast cells of three distantly related marsupials, including the Tasmanian devil, as well as DFTD chromosomes prepared from samples collected from different years and representing different karyotypic strains. Staining of chromosomes from male and female marsupial cell lines indicate species-specific differences in global methylation patterns but with the most intense staining regions corresponding to telomeric and/or centromeric regions of autosomes. In males, the X chromosome was hypermethylated as was one X in females. Similarly, telomeric regions on DFTD chromosomes and regions corresponding to material from one of the two X chromosomes were hypermethylated. No difference in global methylation in samples of the same strain taken in different years was observed. CONCLUSIONS: The methylation patterns on DFTD chromosomes suggests that the hypermethylated active X was shattered in the formation of the tumour chromosomes, with atypical areas of methylation on DFTD chromosomes corresponding to locations of X chromosome material from the shattered X. The incredibly stable broad methylation patterns observed between strains and over time may reflect the overall genomic stability of the devil facial tumour.