The multiple sex chromosomes of platypus and echidna are not completely identical and several share homology with the avian Z

Background

Sex-determining systems have evolved independently in vertebrates. Placental mammals and marsupials have an XY system, birds have a ZW system. Reptiles and amphibians have different systems, including temperature-dependent sex determination, and XY and ZW systems that differ in origin from birds and placental mammals. Monotremes diverged early in mammalian evolution, just after the mammalian clade diverged from the sauropsid clade. Our previous studies showed that male platypus has five X and five Y chromosomes, no SRY, and DMRT1 on an X chromosome. In order to investigate monotreme sex chromosome evolution, we performed a comparative study of platypus and echidna by chromosome painting and comparative gene mapping.

Results

Chromosome painting reveals a meiotic chain of nine sex chromosomes in the male echidna and establishes their order in the chain. Two of those differ from those in the platypus, three of the platypus sex chromosomes differ from those of the echidna and the order of several chromosomes is rearranged. Comparative gene mapping shows that, in addition to bird autosome regions, regions of bird Z chromosomes are homologous to regions in four platypus X chromosomes, that is, X₁, X₂, X₃, X₅, and in chromosome Y₁.

Conclusion

Monotreme sex chromosomes are easiest to explain on the hypothesis that autosomes were added sequentially to the translocation chain, with the final additions after platypus and echidna divergence. Genome sequencing and contig anchoring show no homology yet between platypus and therian Xs; thus, monotremes have a unique XY sex chromosome system that shares some homology with the avian Z.     

Gene mapping studies confirm the homology between the platypus X and echidna X1 chromosomes and identify a conserved ancestral monotreme X chromosome

The identification of the sex chromosomes in the three extant species of Prototherian mammals (the monotremes) is complicated by their involvement in a multivalent translocation chain at the first division of male meiosis. The platypus X chromosome, identified by the presence of two copies in females and one in males, has been found to possess a suite of genes that have been mapped to the X chromosomes of all eutherian and metatherian mammals. We have extended gene mapping studies to a member of the only other extant monotreme family, the echidna, which has a G-band equivalent X₁ chromosome, as well as a smaller X₂. We find that the five human X-linked genes (G6PD, GDX, F9, AR and MCF2) map to the echidna X₁ chromosome in locations equivalent to those on the platypus X. These results confirm that the echidna X₁ is the original X chromosome in this species, and identify a conserved ancestral monotreme X chromosome.     

Disruption and pseudoautosomal localization of the major histocompatibility complex in monotremes

Background

The monotremes, represented by the duck-billed platypus and the echidnas, are the most divergent species within mammals, featuring a flamboyant mix of reptilian, mammalian and specialized characteristics. To understand the evolution of the mammalian major histocompatibility complex (MHC), the analysis of the monotreme genome is vital.

Results

We characterized several MHC containing bacterial artificial chromosome clones from platypus (Ornithorhynchus anatinus) and the short-beaked echidna (Tachyglossus aculeatus) and mapped them onto chromosomes. We discovered that the MHC of monotremes is not contiguous and locates within pseudoautosomal regions of two pairs of their sex chromosomes. The analysis revealed an MHC core region with class I and class II genes on platypus and echidna X3/Y3. Echidna X4/Y4 and platypus Y4/X5 showed synteny to the human distal class III region and beyond. We discovered an intron-containing class I pseudogene on platypus Y4/X5 at a genomic location equivalent to the human HLA-B,C region, suggesting ancestral synteny of the monotreme MHC. Analysis of male meioses from platypus and echidna showed that MHC chromosomes occupy different positions in the meiotic chains of either species.

Conclusion

Molecular and cytogenetic analyses reveal new insights into the evolution of the mammalian MHC and the multiple sex chromosome system of monotremes. In addition, our data establish the first homology link between chicken microchromosomes and the smallest chromosomes in the monotreme karyotype. Our results further suggest that segments of the monotreme MHC that now reside on separate chromosomes must once have been syntenic and that the complex sex chromosome system of monotremes is dynamic and still evolving.     

Chromosome chains and platypus sex: kinky connections

Mammal sex determination depends on an XY chromosome system, a gene for testis development and a means of activating the X chromosome. The duckbill platypus challenges these dogmas.(1,2) Gutzner et al.(1) find no recognizable SRY sequence and question whether the mammalian X was even the original sex chromosome in the platypus. Instead they suggest that the original platypus sex chromosomes were derived from the ZW chromosome system of birds and reptiles. Unraveling the puzzles of sex determination and dosage compensation in the platypus has been complicated by the fact that it has a surplus of sex chromosomes. Rather than a single X and Y chromosome, the male platypus has five Xs and five Ys.     

Karyotypic conservation in the mammalian order monotremata (subclass Prototheria)

The order Monotremata, comprising the platypus and two species of echidna (Australian and Nuigini) is the only extant representative of the mammalian subclass Prototheria, which diverged from subclass Theria (marsupials and placental mammals) 150–200 million years ago. The 2n=63, 64 karyotype (newly described here) of the Nuigini echidna is almost identical in morphology and G-band pattern to that of the Australian echidna, from which it diverged about a million years ago. The karyotype of the platypus (2n=52) has several features in common with those of the echidna species; six pairs of large autosomes, many pairs of small (but not micro-) chromosomes, and a series of small unpaired chromosomes which form a multivalent at meiosis. Comparison of the G-band patterns of platypus and echidna autosomes reveals considerable homology. Chromomycin banding demonstrates GC-rich heterochromatin at the centromeres of many platypus and echidna chromosomes, and at the nucleolar organizing regions; some of this heterochromatin C-bands weakly in platypus (but not echidna) spreads. Late replication banding patterns resemble G-banding patterns and confirm the homologies between the species. Striking heteromorphism between chromosomes of some of the large autosomal pairs can be accounted for in the echidna by differences in amount of chromomycin-bright, late replicating heterochromatin. The sex chromosomes in all three species also bear striking homology, despite the difference in sex determination mechanism between platypus (XX/XY) and the echidna species (X₁X₁X₂X₂/X₁X₂Y). The platypus X and echidna X₁ each represent about 5.8% of haploid chromosome length, and are G-band identical. Y chromosomes are similar between species, and are largely homologous to the X (or X₁).     

The origin of multiple sex chromosomes in the gerbil Gerbillus gerbillus (Rodentia: Gerbillinae)

The sex chromosomes of the partly sympatric species of gerbils Gerbillus pyramidum and G. gerbillus (Mammalia: Gerbillinae) were investigated by a variety of light- and electron-microscope methods, including DNA replication banding and synaptonemal complex (SC) techniques. The sex-chromosome mechanism of G. pyramidum is of the maleXY:femaleXX type, whereas that of G. gerbillus is of the less common maleXY1Y2:femaleXX system. The results include the demonstration that the X chromosomes of both species are compound. One segment is added to the X chromosome of G. pyramidum, leading to an increase in length from the standard 5% to approximately 7.3%, whereas two different extra segments increase the length of the X chromosome of G. gerbillus to approximately 11% of the length of the haploid genome. In both cases the extra material is autosomal and is also represented in the respective Y chromosomes. Classifying heterochromatin by the variation in staining quality was helpful in elucidating the possible origin of the different chromosome segments, including the pericentromeric regions. Observations on meiotic chromosome pairing and chiasma formation have confirmed the homologies established by band comparisons. The occurrence of chiasmata between the sex chromosomes supports the autosomal origin of the pairing segments. These and other findings have been interpreted in the framework of a multistep evolutionary model. This sequence starts from a hypothetical pair of sex chromosomes, the X element of which amounts to 5% of the haploid genome, and leads through three translocations involving two pairs of autosomes and one pericentric inversion to the most complex situation of this series, manifested in G. gerbillus. The adaptive value, if any, of autosome incorporation into the sex chromosomes repeatedly occurring here is unknown. It is, however, a remarkable fact that in one species, G. gerbillus, the complex sex-chromosome constitution is conserved over vast geographic distances, and in the other, G. pyramidum, the compound X and Y chromosomes withstand change in the face of extreme autosome restructuring.     

The influence of the Robertsonian translocation Rb(X.2)2Ad on anaphase I non-disjunction in male laboratory mice

A Robertsonian translocation in the mouse between the X chromosome and chromosome 2 is described. The male and female carriers of the Rb(X.2)2Ad were fertile. A homozygous/hemizygous line was maintained. The influence of the X-autosomal Robertsonian translocation on anaphase I non-disjunction in male mice was studied by chromosome counts in cells at metaphase II of meiosis and by assessment of aneuploid progeny. The results conclusively show that the inclusion of Rb2Ad in the male genome induces non-disjunction at the first meoitic division. In second metaphase cells the frequency of sex-chromosomal aneuploidy was 10.8%, and secondary spermatocytes containing two or no sex chromosome were equally frequent. The Rb2Ad males sired 3.9% sex-chromosome aneuploid progeny. The difference in aneuploidy frequencies in the germ cells and among the progeny suggests that the viability of XO and XXY individuals is reduced. The pairing configurations of chromosomes 2, Rb2Ad and Y were studied during meiotic prophase by light and electron microscopy. Trivalent pairing was seen in all well spread nuclei. Complete pairing of the acrocentric autosome 2 with the corresponding segment of the Rb2Ad chromosome was only seen in 3.2% of the cells analysed in the electron microscope. The pairing between the X and Y chromosome in the Rb2Ad males corresponded to that in males with normal karyotype. Reasons for sex-chromosomal non-disjunction despite the normal pairing pattern between the sex chromosomes may be seen in the terminal chiasma location coupled with the asynchronous separation of the sex chromosomes and the autosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Verification of the structure of the complex sex chromosome system in Lagorchestes conspicillatus Gould (Marsupialia: Mammalia)

The complex sex chromosome system of Lagorchestes conspicillatus has been reinvestigated using G-banding, Hoechst 33258 sensitivity, and Ag staining. These investigations demonstrate that, as proposed, three exchanges have been involved in the evolution of this system. An autosome was translocated to the original X and the homologue of that autosome was translocated to the original Y. An additional autosome has been translocated to the Y. There is no sex vesicle at meiosis in the male, and no association between the original X and Y elements of the compound chromosomes. The inadequacies of the present terminology for complex sex chromosomes are considered and an alternative system suggested.     

Weird mammals provide insights into the evolution of mammalian sex chromosomes and dosage compensation

The deep divergence of mammalian groups 166 and 190 million years ago (MYA) provide genetic variation to explore the evolution of DNA sequence, gene arrangement and regulation of gene expression in mammals. With encouragement from the founder of the field, Mary Lyon, techniques in cytogenetics and molecular biology were progressively adapted to characterize the sex chromosomes of kangaroos and other marsupials, platypus and echidna—and weird rodent species. Comparative gene mapping reveals the process of sex chromosome evolution from their inception 190 MYA (they are autosomal in platypus) to their inevitable end (the Y has disappeared in two rodent lineages). Our X and Y are relatively young, getting their start with the evolution of the sex-determining SRY gene, which triggered progressive degradation of the Y chromosome. Even more recently, sex chromosomes of placental mammals fused with an autosomal region which now makes up most of the Y. Exploration of gene activity patterns over four decades showed that dosage compensation via X-chromosome inactivation is unique to therian mammals, and that this whole chromosome control process is different in marsupials and absent in monotremes and reptiles, and birds. These differences can be exploited to deduce how mammalian sex chromosomes and epigenetic silencing evolved.     

Aberrant chromosomal sex-determining mechanisms in mammals, with special reference to species with XY females

Both mouse and man have the common XX/XY sex chromosome mechanism. The X chromosome is of original size (5-6% of female haploid set) and the Y is one of the smallest chromosomes of the complement. But there are species, belonging to a variety of orders, with composite sex chromosomes and multiple sex chromosome systems: XX/XY1Y2 and X1X1X2X2/X1X2Y. The original X or the Y, respectively, have been translocated on to an autosome. The sex chromosomes of these species segregate regularly at meiosis; two kinds of sperm and one kind of egg are produced and the sex ratio is the normal 1:1. Individuals with deviating sex chromosome constitutions (XXY, XYY, XO or XXX) have been found in at least 16 mammalian species other than man. The phenotypic manifestations of these deviating constitutions are briefly discussed. In the dog, pig, goat and mouse exceptional XX males and in the horse XY females attract attention. Certain rodents have complicated mechanisms for sex determination: Ellobius lutescens and Tokudaia osimensis have XO males and females. Both sexes of Microtus oregoni are gonosomic mosaics (male OY/XY, female XX/XO). The wood lemming, Myopus schisticolor, the collared lemming, Dirostonyx torquatus, and perhaps also one or two species of the genus Akodon have XX and XY females and XY males. The XX, X*X and X*Y females of Myopus and Dicrostonyx are discussed in some detail. The wood lemming has proved to be a favourable natural model for studies in sex determination, because a large variety of sex chromosome aneuploids are born relatively frequently. The dosage model for sex determination is not supported by the wood lemming data. For male development, genes on both the X and the Y chromosomes are necessary.     

Genetic evidence that nonhomologous disjunction and meiotic drive are properties of wild-type Drosophila melanogaster male meiosis

We have followed sex and second chromosome disjunction, and the effects of these chromosomes on sperm function, in four genotypes: wild-type males, males deficient for the Y-linked crystal locus, males with an X chromosome heterochromatic deficiency that deletes all X-Y pairing sites, and males with both deficiencies. Both mutant situations provoke chromosome misbehavior, but the disjunctional defects are quite different. Deficiency of the X heterochromatin, consonant with the lack of pairing sites, mostly disrupts X-Y disjunction with a decidedly second-level effect on major autosome behavior. Deleting crystal, consonant with the cytological picture of postpairing chromatin-condensation problems, disrupts sex and autosome disjunction equally. Even when the mutant-induced nondisjunction has very different mechanics, however, and even more importantly, even in the wild type, there is strong, and similar, meiotic drive. The presence of meiotic drive when disjunction is disrupted by distinctly different mechanisms supports the notion that drive is a normal cellular response to meiotic problems rather than a direct effect of particular mutants. Most surprisingly, in both wild-type and crystal-deficient males the Y chromosome moves to the opposite pole from a pair of nondisjoined second chromosomes nearly 100% of the time. This nonhomologous interaction is, however, absent when the X heterochromatin is deleted. The nonhomologous disjunction of the sex and second chromosomes may be the genetic consequence of the chromosomal compartmentalization seen by deconvolution microscopy, and the absence of Y-2 disjunction when the X heterochromatin is deleted suggests that XY pairing itself, or a previously unrecognized heterochromatic function, is prerequisite to this macrostructural organization of the chromosomes.     

Multicopy genes uniquely amplified in the Y chromosome-specific repeats of the liverwort Marchantia polymorpha

Sex of the liverwort Marchantia polymorpha is determined by the sex chromosomes Y and X, in male and female plant, respectively. Approximately half of the Y chromosome is made up of unique repeat sequences. Here, we report that part of the Y chromosome, represented by a 90-kb insert of a genomic clone pMM2D3, contains five putative genes in addition to the ORF162 gene, which is present also within the Y chromosome-specific repeat region. One of the five putative genes shows similarity to a male gamete-specific protein of lily and is expressed predominantly in male sex organs, suggesting that this gene has a male reproductive function. Furthermore, Southern blot analysis revealed that these five putative genes are amplified on the Y chromosome, but they also probably have homologs on the X chromosome and/or autosomes. These observations suggest that the Y chromosome evolved by co-amplifying protein-coding genes with unique repeat sequences.     

Shared DNA sequences between the X and Y chromosomes in the tammar wallaby – evidence for independent additions to eutherian and marsupial sex chromosomes

Marsupial sex chromosomes are smaller than their eutherian counterparts and are thought to reflect an ancestral mammalian X and Y. The gene content of this original X is represented largely by the long arm of the human X chromosome. Genes on the short arm of the human X are autosomal in marsupials and monotremes, and represent a recent addition to the eutherian X and Y. The marsupial X and Y apparently lack a pseudoautosomal region and show only end-to-end pairing at meiosis. However, the sex chromosomes of macropodid marsupials (kangaroos and wallabies) are larger than the sex chromosomes of other groups, and a nucleolus organizer is present on the X and occasionally the Y. Chromosome painting using DNA from sorted and microdissected wallaby X and Y chromosomes reveals homologous sequences on the tammar X and Y chromosomes, concentrated on the long arm of the Y chromosome and short arm of the X. Ribosomal DNA sequences were detected by fluorescence in situ hybridization on the wallaby Xp but not the Y. Since no chiasmata have been observed in marsupial sex chromosomes, it is unlikely that these shared sequences act as a pseudoautosomal region within which crossing over may occur, but they may be required for end-to-end associations. The shared region of wallaby X and Y chromosomes bears no homology with the recently added region of the eutherian sex chromosomes, so we conclude that independent additions occurred to both sex chromosomes in a eutherian and macropodid ancestor, as predicted by the addition-attrition hypothesis of sex chromosome evolution. Received: 18 October 1996 / Accepted: 21 February 1997     

The synaptic behaviour of the X and Y chromosomes in the marsupial Monodelphis dimidiata

The pairing behaviour of the X and Y chromosomes of Monodelphis dimidiata was studied with light and electron microscopy. Pairing of the sex chromosomes is delayed with respect to autosome synapsis. Both the X and the minute Y chromosome show an axis attached by its two ends to the nuclear envelope. Synapsis of the sex chromosomes occurs by the joining of the chromatin sheaths that surround the axes and by a small, three-layered structure close to the nuclear envelope. The X and Y chromosomes remain joined to each other during the diffuse stage and diplotene-diakinesis but they do not show a synaptonemal complex. During the diffuse stage a dense plate is formed at the boundary between the X-Y body and the nuclear envelope. During early metaphase a folded sheet is attached to the periphery of the X-Y body. This sheet is formed by a piece of the nuclear envelope carrying the dense plate and it shows transverse fibrils and a central element similar to synaptonemal-complex remains. No evidence of a non-chiasmate segregation mechanism was observed. Polarization of the axial ends of the sex chromosomes is observed after X-Y synapsis. These important departures from the X-Y pairing pattern of eutherian mammals are discussed and assumed to present a special mechanism for holding the minute Y joined to the X chromosome in this marsupial.     

Sex chromosomes and associated rDNA form a heterochromatic network in the polytene nuclei of Bactrocera oleae (Diptera: Tephritidae)

The olive fruit fly, Bactrocera oleae, has a diploid set of 2n?=?12 chromosomes including a pair of sex chromosomes, XX in females and XY in males, but polytene nuclei show only five polytene chromosomes, obviously formed by five autosome pairs. Here we examined the fate of the sex chromosomes in the polytene complements of this species using fluorescence in situ hybridization (FISH) with the X and Y chromosome-derived probes, prepared by laser microdissection of the respective chromosomes from mitotic metaphases. Specificity of the probes was verified by FISH in preparations of mitotic chromosomes. In polytene nuclei, both probes hybridized strongly to a granular heterochromatic network, indicating thus underreplication of the sex chromosomes. The X chromosome probe (in both female and male nuclei) highlighted most of the granular mass, whereas the Y chromosome probe (in male nuclei) identified a small compact body of this heterochromatic network. Additional hybridization signals of the X probe were observed in the centromeric region of polytene chromosome II and in the telomeres of six polytene arms. We also examined distribution of the major ribosomal DNA (rDNA) using FISH with an 18S rDNA probe in both mitotic and polytene chromosome complements of B. oleae. In mitotic metaphases, the probe hybridized exclusively to the sex chromosomes. The probe signals localized a discrete rDNA site at the end of the short arm of the X chromosome, whereas they appeared dispersed over the entire dot-like Y chromosome. In polytene nuclei, the rDNA was found associated with the heterochromatic network representing the sex chromosomes. Only in nuclei with preserved nucleolar structure, the probe signals were scattered in the restricted area of the nucleolus. Thus, our study clearly shows that the granular heterochromatic network of polytene nuclei in B. oleae is formed by the underreplicated sex chromosomes and associated rDNA.     

Evolution of karyotype,sex chromosomes,and meiosis in mygalomorph spiders (Araneae: Mygalomorphae)

Spider diversity is partitioned into three primary clades, namely Mesothelae, Mygalomorphae, and Araneomorphae. Mygalomorph cytogenetics is largely unknown. Our study revealed a remarkable karyotype diversity of mygalomorphs. Unlike araneomorphs, they show no general trend towards a decrease of 2n, as the chromosome number was reduced in some lineages and increased in others. A biarmed karyotype is a symplesiomorphy of mygalomorphs and araneomorphs. Male meiosis of some mygalomorphs is achiasmatic, or includes the diffuse stage. The sex chromosome system X₁X₂0, which is supposedly ancestral in spiders, is uncommon in mygalomorphs. Many mygalomorphs exhibit more than two (and up to 13) X chromosomes in males. The evolution of X chromosomes proceeded via the duplication of chromosomes, fissions, X–X, and X‐autosome fusions. Spiders also exhibit a homomorphic sex chromosome pair. In the germline of mygalomorph males these chromosomes are often deactivated; their deactivation and pairing is initiated already at spermatogonia. Remarkably, pairing of sex chromosomes in mygalomorph females is also initiated at gonial cells. Some mygalomorphs have two sex chromosome pairs. The second pair presumably arose in early‐diverging mygalomorphs, probably via genome duplication. The unique behaviour of spider sex chromosomes in the germline may promote meiotic pairing of homologous sex chromosomes and structural differentiation of their duplicates, as well as the establishment of polyploid genomes. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 377–408.     

Independent Evolution of Transcriptional Inactivation on Sex Chromosomes in Birds and Mammals

X chromosome inactivation in eutherian mammals has been thought to be tightly controlled, as expected from a mechanism that compensates for the different dosage of X-borne genes in XX females and XY males. However, many X genes escape inactivation in humans, inactivation of the X in marsupials is partial, and the unrelated sex chromosomes of monotreme mammals have incomplete and gene-specific inactivation of X-linked genes. The bird ZW sex chromosome system represents a third independently evolved amniote sex chromosome system with dosage compensation, albeit partial and gene-specific, via an unknown mechanism (i.e. upregulation of the single Z in females, down regulation of one or both Zs in males, or a combination). We used RNA-fluorescent in situ hybridization (RNA-FISH) to demonstrate, on individual fibroblast cells, inactivation of 11 genes on the chicken Z and 28 genes on the X chromosomes of platypus. Each gene displayed a reproducible frequency of 1Z/1X-active and 2Z/2X-active cells in the homogametic sex. Our results indicate that the probability of inactivation is controlled on a gene-by-gene basis (or small domains) on the chicken Z and platypus X chromosomes. This regulatory mechanism must have been exapted independently to the non-homologous sex chromosomes in birds and mammals in response to an over-expressed Z or X in the homogametic sex, highlighting the universal importance that (at least partial) silencing plays in the evolution on amniote dosage compensation and, therefore, the differentiation of sex chromosomes.     

Heterochromatin (C-bands) in somatic and male germ cells in three species ofMicrotinae

The C-banding patterns in the chromosomes ofMicrotus oeconomus, M. arvalis andM. ochrogaster demonstrate differences in the amount and distribution of heterochromatin. Autosomal centromeric heterochromatin appears as conspicuous blocks or as small dots, and in several chromosomes no heterochromatin was detected; interstitial heterochromatin was observed in one autosome pair ofM. ochrogaster. The sex chromosomes also demonstrate differences in the C-banding pattern. InM. oeconomus, the X chromosome exhibits a block of centromeric heterochromatin which is larger than that of the autosomes; this characteristic helps to recognize the X chromosomes in the karyotype. InM. arvalis no heterochromatin was appreciated in the sex chromosomes. The Y chromosomes ofM. ochrogaster andM. oeconomus are entirely heterochromatic. During male meiosis heterochromatin shows condensation, association and chiasma prevention; the sex chromosomes pair end to end in the three species. At pairing, the Y chromosome ofM. arvalis is despiralized, but it appears condensed again shortly before separation of the bivalent.