Marsupial--mouse cell hybrids containing fragments of the marsupial X chromosome.

Hybrids were obtained from fusions of HPRT-deficient mouse fibroblasts and marsupial lymphocytes. These hybrids retained no identifiable marsupial chromosomes, but all expressed the marsupial form of HPRT. Half the clones also expressed marsupial PGK-A, and half of these also marsupial G6PD; no other marsupial allozyme markers were detected. Since G6PD is known to be sex linked in these species, we conclude that Hpt and Pgk-A are also located on the X chromosome and the markers lie in the order Hpt-Pgk-A-Gpd.     

The evolution of marsupial and monotreme chromosomes

Marsupial and monotreme mammals fill an important gap in vertebrate phylogeny between reptile-mammal divergence 310 million years ago (mya) and the eutherian (placental) mammal radiation 105 mya. They possess many unique features including their distinctive chromosomes, which in marsupials are typically very large and well conserved between species. In contrast, monotreme genomes are divided into several large chromosomes and many smaller chromosomes, with a complicated sex chromosome system that forms a translocation chain in male meiosis. The application of molecular cytogenetic techniques has greatly advanced our understanding of the evolution of marsupial chromosomes and allowed the reconstruction of the ancestral marsupial karyotype. Chromosome painting and gene mapping have played a vital role in piecing together the puzzle of monotreme karyotypes, particularly their complicated sex chromosome system. Here, we discuss the significant insight into karyotype evolution afforded by the combination of recently sequenced marsupial and monotreme genomes with cytogenetic analysis, which has provided a greater understanding of the events that have shaped not only marsupial and monotreme genomes, but the genomes of all mammals.     

Selected features of marsupial genetics

As a consequence of the ancient separation of the marsupial and eutherian lineages, comparative genetical studies of these two mammalian taxa can be particularly informative. The potential for marsupial genetical research has been enhanced by the development of laboratory colonies of three model species-Macropus eugenii, Monodelphis domestica andSminthopsis crassicaudata. In this paper two selected aspects of marsupial genetics are reviewed, one involving cytogenetics and the other linkage. Marsupials provide a spectacular example of karyotypic conservation. The so-called basic karyotype (2n=14) is probably ancestral in all extant marsupials. Karyotypes that do not conform to this basic arrangement are thought to have been derived from it. A notable feature of the basic karyotype is that it has been retained, possibly for as long as 150 million years, in morphologically, behaviourally and ecologically diverse species from at least five Australian and two American families; this suggests that selective forces, presently unknown, have acted to conserve the basic chromosome form and number in these species. With respect to genetic linkage, family studies inS. crassicaudata and more recentlyM. domestica have indicated extreme differences between the sexes with the recombination frequencies for linked loci being very much greater in males than in females, a situation that is strikingly different from that in eutherian mammals. These differences in linkage values are paralleled by differences in the number and distribution of chiasmata during male and female meiosis. Prospects for further research in marsupials, particularly research that builds upon the observations of karyotypic conservation and genetic linkage, are noted.     

Reversal and convergence in marsupial chromosome evolution

The karyotypes of marsupial species are characterized by their relatively low number of chromosomes, and their conservation. Most species have diploid numbers lying between the two modes, 2n = 14 and 2n = 22, but the karyotype of Aepyprymnus rufescens is exceptional in containing 2n = 32 chromosomes. Many differences in diploid number between marsupial species can be accounted for by particular fissions and fusions, which are easy to detect because of the low numbers of chromosomes in each karyotype. This should be a system in which it is possible to detect reversals and repeated chromosome rearrangements. We have used chromosome-specific paints derived from A. RUFESCENS to compare the karyotypes of eight marsupial species, representing closely and distantly related taxa, to trace chromosome change during evolution, and especially to detect reversals and convergence. From these and other painting comparisons, we conclude that there have been at least three reversals of fusions by fissions, and at least three fusions or fissions that have occurred independently in different lineages.     

X chromosome inactivation in female embryos of a marsupial mouse (Antechinus stuartii)

Blastocysts and late gestation stages of the marsupial mouse, Antechinus stuartii, were examined cytologically and electrophoretically to investigate X chromosome activity during embryogenesis. A late replicating X chromosome was identified in the protoderm cells of female unilaminar blastocysts and in the cells of embryonic and extra-embryonic regions of older blastocysts. Sex chromatin bodies were also observed in female bilaminar and trilaminar blastocysts. The X linked enzyme -galactosidase showed no evidence of paternal allele expression in the extra-embryonic region of bilaminar blastocysts or in the yolk sac and embryonic tissue of known heterozygotes. It is concluded that the late replicating X chromosome is paternal in origin and that unlike the laboratory mouse, X inactivation is not correlated with cell differentiation in Antechinus.     

Activity map of the tammar X chromosome shows that marsupial X inactivation is incomplete and escape is stochastic

Background  

X chromosome inactivation is a spectacular example of epigenetic silencing. In order to deduce how this complex system evolved, we examined X inactivation in a model marsupial, the tammar wallaby (Macropus eugenii). In marsupials, X inactivation is known to be paternal, incomplete and tissue-specific, and occurs in the absence of an XIST orthologue.     

An autosomal gene assignment in a marsupial: The gene for LDH-A is on chromosome 5 of the red kangaroo,Macropus rufus

A number of somatic cell hybrids between red kangaroo (Macropus rufus) and mouse cells, which lose marsupial chromosomes, were found to express the kangaroo form of LDH-A. Concordance between the expression of marsupial LDH-A and the presence of chromosome 5 in the hybrid cells and selected subclones enabled the gene for LDH-A to be assigned to this chromosome. This is the first autosomal gene assignment in a marsupial and should prove important for chromosome mapping in the red kangaroo and in many other species of marsupials.     

A morphological analysis of marsupial mammal higher-level phylogenetic relationships

Phylogenetic relationships among marsupial taxa have proven to be more complex than the simple grouping of species by continent. Recent marsupials are distributed across the New World, Australia, New Guinea, and certain neighboring islands. Morphological characteristics of various groups bridge different geographical areas. We investigated the origin of these characteristics by assembling a morphological data matrix consisting of a new suite of 149 postcranial characters and incorporated a series of previously published data on the craniodental (76 characters) and soft tissue (5 characters) anatomy. Twenty‐one marsupial terminal taxa representing all the major radiations of marsupials and 10 outgroups, most of which are exceptionally well‐preserved fossils such as Vincelestes, Ukhaatherium, and a few basal metatherian taxa, were investigated. A maximum parsimony analysis was conducted, resulting in one most parsimonious tree. Relationships among outgroups are congruent with current understanding of mammalian phylogeny. All currently accepted marsupial orders were recovered by the analysis. We confirmed previous results showing the South American “monito del monte”Dromiciops nested within the Australasian radiation. Within this australidelphian clade, Dromiciops was closely allied with the Diprotodontia. The South American paucituberculates appeared more closely related to the Australidelphia than to the American Didelphimorphia. The marsupial mole Notoryctes and the Peramelia were closely allied to each other and in turn were the sister group of the Dromiciops plus Diprotodontia clade. This pattern of relationships left Dasyuromorphia as the most basal offshoot of the Australidelphia. Whereas this tree topology recovers some signal that had been detected by previous studies, morphological and/or molecular, some novel hypotheses are also supported.     

Genes on the short arm of the human X chromosome are not shared with the marsupial X.

Eight genes located on the short arm of the human X chromosome (MAOA, SYN1, OAT, OTC, CYBB, DMD, ZFX, POLA) have been mapped in several marsupial species by cell hybrid analysis and/or in situ hybridization using probes derived from human cDNA. Seven appear to be autosomal in all marsupial species examined. The eighth, CYBB, detected a site on the X, as well as major autosomal sites. Although these genes are not conserved on the X chromosome in marsupials, at least some of them are arranged together in autosomal clusters. The autosomal location of human Xp genes in marsupials could mean that this region either was lost from a large ancestral X chromosome in the marsupial lineage or was acquired by a small ancestral X (and perhaps Y) in the eutherian lineage. Either explanation demands that the region was not subject to X chromosome inactivation in a common ancestor 120-150 MyrBP.     

Fibrils in marsupial enamel tubules

Summary Serial etching of cross-sectioned prisms in undecalcified adult marsupial enamel from different species, revealed distinct cylindrical acid-resistant fibrils that were demonstrable by light microscopy and by scanning electron microscopy. No fibrils were found in the enamel of Vombatus.The fibrils and the organic matrix in the remainder of the enamel stain differently. The fibrils project from the center of prisms or the borderline between prisms and interprismatic substance.It is concluded that the fibrils are chemically different from the organic matrix in the enamel, that they constitute the compact, homogenous, and morphologically well defined organic contents of the tubules in adult marsupial enamel.Since most of the material was obtained from dry museum crania, it is concluded that the fibrils are not destroyed by prolonged drying.The scanning electron micrographs were taken at the Electron Microscopical Unit for Biological Sciences, Oslo, Norway.     

Gene mapping in marsupials and monotremes

Somatic cell genetic mapping of marsupial and monotreme species will greatly extend the power of comparative gene mapping to detect ancient mammalian gene arrangements. The use of eutherian-marsupial cell hybrids for such mapping is complicated by the frequent retention of deleted and rearranged marsupial chromosomes. We used staining techniques, involving the fluorochromes Hoechst 33258 and chromomycin A₃, to facilitate rapid and unequivocal identification of marsupial chromosomes and chromosome segments and to make chromosome assignment and regional localization of marsupial genes possible. Chromosome segregation in rodent-macropod hybrids was consistent with preferential loss of the marsupial complement. The extent of loss was very variable. Some hybrids retained 30% of the marsupial complement; some retained small centric fragments; and some, no cytologically identifiable marsupial material. We examined the chromosomes and gene products of a number of rodent-grey kangaroo Macropus giganteus hybrids, and have assigned the genes Pgk-A (phosphoglycerate kinase-A), Hpt (Hypoxanthine phosphoribosyl transferase), and Gpd (Glucose-6-phosphate dehydrogenase) to the long arm of the kangaroo X chromosome, and provisionally established the gene order Pgk-A -Hpt -Gpd.     

Mapping human X-linked genes in the phalangerid marsupial Trichosurus vulpecula.

We mapped 15 human X-chromosome markers in the common brush-tailed possum, Trichosurus vulpecula (Kerr), which represents the Australian marsupial family Phalangeridae. In situ hybridization was used to localize highly conserved human X-linked genes to chromosomes of T. vulpecula diploid lines. Ten genes located on the long arm of the human X (human Xq genes) all mapped to the possum X chromosome. However, all five genes located on the short arm of the human X (human Xp genes) mapped to autosomes. These findings confirm our previous work, which showed that the X chromosome in macropodid and dasyurid marsupials bears all the human Xq genes but none of the human Xp genes studied. This suggests that the marsupial X is highly conserved, but its gene content reflects that of only part of the eutherian X, a result consistent with our hypothesis that an autosomal region was added to the X early in eutherian divergence.     

The minimal mammalian Y chromosome - the marsupial Y as a model system

The mammalian X and Y chromosomes are very different in size and gene content. The Y chromosome is much smaller than the X and consists largely of highly repeated non-coding DNA, containing few active genes. The 65-Mb human Y is homologous to the X over two small pseudoautosomal regions which together contain 13 active genes. The heterochromatic distal half of the human Yq is entirely composed of highly repeated non-coding DNA, and even the euchromatic portion of the differential region is largely composed of non-coding repeated sequences, amongst which about 30 active genes are located. The basic marsupial Y chromosome (about 10 Mb) is much smaller than that of humans or other eutherian mammals. It appears to include no PAR, since it does not undergo homologous pairing, synaptonemal complex formation or recombination with the X. We show here that the tiny dunnart Y chromosome does not share cytogenetically detectable sequences with any other chromosome, suggesting that it contains many fewer repetitive DNA sequences than the human or mouse Y chromosomes. However, it shares several genes with the human and/or mouse Y chromosome, including the sex determining gene SRY and the candidate spermatogenesis gene RBMY, implying that the marsupial and eutherian Y are monophyletic. This minimal mammalian Y chromosome might provide a good model Y in which to hunt for new mammalian Y specific genes.     

Fluorescent localization of thiols and disulfides in marsupial spermatozoa by bromobimane labelling

The acrosome of marsupial spermatozoa is a robust structure which, unlike its placental counterpart, resists disruption by detergent or freeze/thawing and does not undergo a calcium ionophore induced acrosome reaction. In this study specific fluorescent thiol labels, bromobimanes, were used to detect reactive thiols in the intact marsupial spermatozoon and examine whether disulfides play a role in the stability of the acrosome. Ejaculated brushtail possum (Trichosurus vulpecula) and tammar wallaby (Macropus eugenii) spermatozoa were washed by swim up and incubated with or without dithiothreitol (DTT) in order to reduce disulfides to reactive thiols. Spermatozoa were then washed by centrifugation and treated with monobromobimane (mBBr), a membranepermeable bromobimane, or with monobromotrimethylammoniobimane (qBBr), a membrane-impermeable bromobimane. Labelled spermatozoa were examined by fluorescence microscopy and sperm proteins (whole sperm proteins and basic nuclear proteins) were analysed by gel electrophoresis. The membrane-permeable agent mBBr lightly labelled the perimeter of the acrosome of non-DTT-treated possum and wallaby spermatozoa, indicating the presence of peri-acrosomal thiol groups. After reduction of sperm disulfides by DTT, mBBr labelled the entire acrosome of both species. The membrane-impermeable agent qBBr did not label any part of the acrosome in non-DTT or DTT-treated wallaby or possum spermatozoa. Thiols and disulfides are thus associated with the marsupial acrosome. They are not found on the overlying plasma membrane but are either in the acrosomal membranes and/or matrix. The sperm midpiece and tail were labelled by mBBr, with increased fluorescence observed in DTT-treated spermatozoa. The nucleus was not labelled in non-DTT or DTT-treated spermatozoa. Electrophoretic analysis confirmed the microscopic observations: Basic nuclear protein (protamines) lacked thiols or disulfide groups. Based on these findings, the stability of the marsupial acrosome may be due in part to disulfide stabilization of the acrosomal membranes and/or acrosomal matrix. In common with placental mammals, thiol and disulfide containing proteins appear to play a role in the stability of sperm tail structures. It was confirmed that the fragile marsupial sperm nucleus lacked thiols and disulfides. © 1994 Wiley-Liss, Inc.     

Cone pigments in a North American marsupial, the opossum (Didelphis virginiana)

Only two of the four cone opsin gene families found in vertebrates are represented in contemporary eutherian and marsupial species. Recent genetic studies of two species of South American marsupial detected the presence of representatives from two of the classes of cone opsin genes and the structures of these genes predicted cone pigments with respective peaks in the ultraviolet and long-wavelength portions of the spectrum. The Virginia opossum (Didelphis virginiana), a profoundly nocturnal animal, is the only marsupial species found in North America. The prospects for cone-based vision in this species were examined through recordings of the electroretinogram (ERG), a commonly examined retinal response to photic stimulation. Recorded under flickering-light conditions that elicit signals from cone photoreceptors, the spectral sensitivity of the opossum eye is well accounted for by contributions from the presence of a single cone pigment having peak absorption at 561–562 nm. A series of additional experiments that employed various chromatic adaptation paradigms were conducted in a search for possible contributions from a second (short-wavelength sensitive) cone pigment. We found no evidence that such a mechanism contributes to the ERG in this marsupial.     

Modo-UG, a marsupial nonclassical MHC class I locus

Modo-UG is a class I gene located in the MHC of the marsupial Monodelphis domestica, the gray, short-tailed opossum. Modo-UG is expressed as three alternatively spliced mRNA forms, all of which encode a transmembrane form with a short cytoplasmic tail that lacks phosphorylation sites typically found in classical class I molecules. The three alternative mRNAs would encode a full-length form, an isoform lacking the α2 domain, and one lacking both α2 and α3 domains. Genotyping both captive-bred and wild M. domestica from different geographic regions revealed no variation in the residues that make up Modo-UG’s peptide-binding groove. Modo-UG’s low polymorphism is contrasting to that of a nearby class I locus, Modo-UA1, which has a highly polymorphic peptide-binding region. Absence of functional polymorphism in Modo-UG is therefore not a general feature of opossum class I genes but the result of negative selection. Modo-UG is the first MHC linked marsupial class I to be described that appears to clearly have nonclassical features.Electronic Supplementary Material Supplementary material is available for this article at     

The first comprehensive genetic linkage map of a marsupial: the tammar wallaby (Macropus eugenii)

The production of a marsupial genetic linkage map is perhaps one of the most important objectives in marsupial research. This study used a total of 353 informative meioses and 64 genetic markers to construct a framework genetic linkage map for the tammar wallaby (Macropus eugenii). Nearly all markers (93.8%) formed a significant linkage (LOD > 3.0) with at least one other marker, indicating that the majority of the genome had been mapped. In fact, when compared with chiasmata data, >70% (828 cM) of the genome has been covered. Nine linkage groups were identified, with all but one (LG7; X-linked) allocated to the autosomes. These groups ranged in size from 15.7 to 176.5 cM and have an average distance of 16.2 cM between adjacent markers. Of the autosomal linkage groups (LGs), LG2 and LG3 were assigned to chromosome 1 and LG4 localized to chromosome 3 on the basis of physical localization of genes. Significant sex-specific distortions toward reduced female recombination rates were revealed in 22% of comparisons. When comparing the X chromosome data to closely related species it is apparent that they are conserved in both synteny and gene order.     

The use of marsupial x eutherian somatic cell hybrids to study marsupial cell surface antigens

Buck and Bodmer (1976) have developed a technique for identifying an antigen on the surface of human x mouse somatic cell hybrids, specified by a gene on a particular human chromosome. We have successfully adapted this technique to a study of marsupial cell surface antigens. Somatic cell hybrids between Macropus rufus (Marsupialia) lymphocytes and the mouse cell lines PG19 and 1R were injected intraperitoneally into mice of the same inbred strain from which the above cell lines were derived (C57B16J and C3H, respectively). The only identified M. rufus chromosome present in the hybrid cells was the X chromosome. The antisera, after adsorption with PG19 or 1R, were tested using indirect immunofluorescence, against the hybrid cells, and also against sub-clones (derived from hybrids) which had apparently lost the M. rufus X chromosome, or at least its long arm. The results of these tests showed that the absorbed antisera contained reactivity against an M. rufus cell surface antigen (or antigens). The reactions of one of the antisera were most simply interpreted by supposing that it was detecting an M. rufus X-lined antigen(s).     

Direct and indirect effects of marsupial browsing on a foundation tree species

Plant‐mediated indirect effects can be important ecological drivers in plant communities, especially in systems where extended genetic effects of foundation species can shape communities and influence ecosystem dynamics. Here we investigate the direct and indirect effects of uncontrolled browsing by marsupial herbivores including the common brushtail possum Trichosurus vulpecula, Bennetts wallaby Macropus rufogriseus and the red‐bellied pademelon Thylogale billardierii, in a Eucalyptus system known to have extended community and ecosystem genetic effects. In a common garden trial containing 525 full‐sib families from an incomplete diallel crossing program located in northeastern Tasmania, Australia, we assessed the genetic basis to herbivore preferences, the impact of a single and repeated marsupial browsing event on tree fitness and morphological traits and the associated indirect plant‐mediated effects on a subsequent herbivore, autumn gum moth Mnesampela privata. Marsupial browsing was not influenced by plant genetics, but spatial components instead affected the pattern of damage across the trial. Marsupial browsing had significant impacts on tree development, morphology and survival, resulting in reductions in survival, height and basal area, an increase proportion in multiple stems, delays in flowering as well as delays in phase change from juvenile to adult foliage. Fitness impacts were minimal in response to a once‐off browsing event, but effects were exacerbated when trees suffered repeated browsing. We demonstrate clear plant‐mediated indirect effects of marsupial browsing on subsequent tree use by an invertebrate herbivore, through induced changes in plant morphology. Such indirect effects have the potential to influence biotic community structure on a foundation species host‐plant, and the evolutionary interactions that occur between organisms and the host‐plant themselves.