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.     

On the phylogenetic position of monotremes (Mammalia,Monotremata)

Henosferida from the Middle-Upper Jurassic of Western Gondwana is the most probable sister group for monotremes. They share the derived pretribosphenic structure of lower molars combined with the presumably absent protocone on the upper molars and the plesiomorphic retention of postdentary bones and pseudangular process of the lower jaw. In addition, the two groups share the dental formula with three molars and the position of the Meckel’s groove, which passes ventral to the mandibular foramen. In the course of subsequent evolution, monotremes acquired the mammalian middle ear with three auditory ossicles independently of therian mammals and multituberculates. Jurassic Laurasian Shuotheriidae are probably a sister group of the Gondwanian clade Henosferida + Monotremata. The Jurassic shuotheriid Pseudotribos shows a great plesiomorphic similarity to monotremes in the structure of the pectoral girdle, with a large interclavicle immovably connected to the clavicle. In the lineages leading to therian mammals and multituberculates, the pectoral girdle changed probably independently and in parallel in connection with the establishment of the parasagittal posture of the forelimbs (reduction of the interclavicle, mobile articulation of the interclavicle with clavicle, reduction of the procoracoid, and development of a supraspinous fossa of the scapula) and formation of the mammalian middle ear with three auditory ossicles.     

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.     

Monotreme ossification sequences and the riddle of mammalian skeletal development

The developmental differences between marsupials, placentals, and monotremes are thought to be reflected in differing patterns of postcranial development and diversity. However, developmental polarities remain obscured by the rarity of monotreme data. Here, I present the first postcranial ossification sequences of the monotreme echidna and platypus, and compare these with published data from other mammals and amniotes. Strikingly, monotreme stylopodia (humerus, femur) ossify after the more distal zeugopodia (radius/ulna, tibia/fibula), resembling only the European mole among all amniotes assessed. European moles also share extreme humeral adaptations to rotation digging and/or swimming with monotremes, suggesting a causal relationship between adaptation and ossification heterochrony. Late femoral ossification with respect to tibia/fibula in monotremes and moles points toward developmental integration of the serially homologous fore- and hindlimb bones. Monotreme cervical ribs and coracoids ossify later than in most amniotes but are similarly timed as homologous ossifications in therians, where they are lost as independent bones. This loss may have been facilitated by a developmental delay of coracoids and cervical ribs at the base of mammals. The monotreme sequence, although highly derived, resembles placentals more than marsupials. Thus, marsupial postcranial development, and potentially related diversity constraints, may not represent the ancestral mammalian condition.     

Distribution of keratin and associated proteins in the epidermis of monotreme,marsupial, and placental mammals

The expression of acidic and basic keratins, and of some keratinization marker proteins such as filaggrin, loricrin, involucrin, and trichohyalin, is known for the epidermis of only a few eutherian species. Using light and high-resolution immunocytochemistry, the presence of these proteins has been studied in two monotreme and five marsupial species and compared to that in eutherians. In both monotreme and marsupial epidermis lamellar bodies occur in the upper spinosus and granular layers. Development of the granular layer varies between species and regionally within species. There is great interspecific variation in the size (0.1-3.0 microm) of keratohyalin granules (KHGs) associated with production of orthokeratotic corneous tissues. Those skin regions lacking hairs (platypus web), or showing reduced pelage density (wombat) have, respectively, minute or indiscernible KHGs, associated with patchy, or total, parakeratosis. Ultrastructural analysis shows that monotreme and marsupial KHGs comprise irregular coarse filaments of 25-40 nm that contact keratin filaments. Except for parakeratotic tissues of platypus web, distribution of acidic and basic proteins in monotreme and marsupial epidermis as revealed by anti-keratin antibodies AE1, AE2, and AE3 resembles that of eutherian epidermis. Antibodies against human or rat filaggrins have little or no cross-reactivity with epidermal proteins of other mammals: only sparse areas of wombat and rabbit epidermis show a weak immunofluorescence in transitional cells and in the deepest corneous tissues. Of the available, eutherian-derived antibodies, that against involucrin shows no cross-reactivity with any monotreme and marsupial epidermal tissues and that against trichohyalin cross-reacts only with cells in the inner root sheath and medulla of hairs. These results suggest that if involucrin and trichohyalin are present throughout noneutherian epidermis, they may have species-specific molecular structures. By contrast, eutherian-derived anti-loricrin antibodies show a weak to intense cross-reactivity to KHGs and corneous tissues of both orthokeratotic and parakeratotic epidermis in monotremes and marsupials. High-resolution immunogold analysis of loricrin distribution in immature keratinocytes of platypus parakeratotic web epidermis identifies labeled areas of round or irregular, electron-pale granules within the denser keratohyalin component and keratin network. In the deepest mature tissues, loricrin-like labeling is diffuse throughout the cytoplasm, so that cells lack the preferential distribution of loricrin along the corneous envelope that characterizes mature eutherian keratinocytes. Thus, the irregular distribution of loricrin in platypus parakeratotic tissues more resembles that which has been described for reptilian and avian keratinocytes. These observations on the noneutherian epidermis show that a stratum granulosum is present to different degrees, even discontinuous within one tissue, so that parakeratotic and orthokeratotic areas may alternate: this might imply that parakeratotic monotreme epidermis reflects the primitive pattern of amniote alpha-keratogenesis. Absent from anamniote epidermis and all sauropsid beta-keratogenic tissues, the ubiquitous presence of a loricrin-like protein as a major component of other amniote corneous tissues suggests that this is a primitive feature of amniote alpha-keratogenesis. The apparent lack of specific regionalization of loricin near the plasma membranes of monotreme keratinocytes could be an artifactual result of the immunofluorescence technique employed, or there may be masking of the antigenicity of loricrin-like proteins once they are incorporated into the corneous envelope. Nevertheless, the mechanism of redistribution of such proteins during maturation of monotreme keratinocytes is different from, perhaps more primitive, or less specialized, than that in the epidermis of eutherian mammals.     

The monotreme genome: a patchwork of reptile, mammal and unique features?

The first specimen of platypus (Ornithorhynchus anatinus) that reached Britain in the late 18th century was regarded a scientific hoax. Over decades the anatomical characteristics of these unique mammals, such as egg laying and the existence of mammary glands, were hotly debated before they were accepted. Within the last 40 years, more and more details of monotreme physiology, histology, reproduction and genetics have been revealed. Some show similarities with birds or reptiles, some with therian mammals, but many are very specific to monotremes. The genome is no exception to monotreme uniqueness. An early opinion was that the karyotype, composed of a few large chromosomes and many small ones, resembled bird and reptile macro- and micro-chromosomes. However, the platypus genome also features characteristics that are not present in other mammals, such as a complex translocation system. The sex chromosome system is still not resolved. Nothing is known about dosage compensation and, unlike in therian mammals, there seems to be no genomic imprinting. In this article we will recount the mysteries of the monotreme genome and describe how we are using recently developed technology to identify chromosomes in mitosis, meiosis and sperm, to map genes to chromosomes, to unravel the sex chromosome system and the translocation chain and investigate X inactivation and genomic imprinting in monotremes.     

Development of the head and pectoral skeleton of Polypterus with a note on scales (Pisces: Actinopterygii)

Comparative study of the bony head and pectoral girdle skeletons of fishes requires the establishment of sound and acceptable homologies. In this paper, the bony elements seen in a number of species of Polypterus (and Erpeloichlhys ) are described in terms of several developmental stages and the mature condition. Variations in these bones are considered in a developmental sense. As a part of the problem of bone development, a summary account of lateral-line development is included.
A number of particular problems of homology are discussed in some detail. The most radical differences of view involve the endocranial bones. The ideas of Patterson (1975) are examined and an alternative interpretation offered. The main bone in the posterolateral angle of the endo-cranium is viewed as the pterotic-epiotic. The epiotic of the teleost is seen not as the homologue of the epioccipital of the pholidophorid but a part of the so-called "pterotic" of those fishes. Other endocranial problems appear to be of a similar (interpretative) nature. The suspensorium of Polypterus is peculiar in lacking a symplectic and an interhyal. The branchial skeleton is quite like that of a "palaeoniscoid". The pectoral girdle presents a number of problems such as the form of the posttemporal, the lack of a sensory canal on the supracleithrum and the lack of an interclavicle.
The subject matter of this paper, and other papers by this author on the development of bones in fishes, lead to several developmental concepts. These are summarized in 18 statements. A table (Table II) summarizes some of the anatomical information useful in taxonomy in terms of being derived, primitive or unique.     

Review of the monotreme fossil record and comparison of palaeontological and molecular data

Monotremes have traditionally been considered a remnant group of mammals descended from archaic Mesozoic stock, surviving to the present day on the relatively isolated Australian continent. Challenges to this orthodoxy have been spurred by discoveries of 'advanced' Cretaceous monotremes (Steropodon galmani, Archer, M., et al., 1985. First Mesozoic mammal from Australia-an Early Cretaceous monotreme, Nature. 318, 363-366) as well as by results from molecular data linking monotremes to therian mammals (specifically to marsupials in some studies). This paper reviews the monotreme fossil record and briefly discusses significant new information from additional Cretaceous Australian material. Mesozoic monotremes (including S. galmani) were a diverse group as evidenced by new material from the Early Cretaceous of New South Wales and Victoria currently under study. Although most of these new finds are edentulous jaws (limiting dental comparisons and determination of dietary niches), a range of sizes and forms has been determined. Some of these Cretaceous jaws exhibit archaic features-in particular evidence for the presence of a splenial bone in S. galmani-not seen in therian mammals or in post-Mesozoic (Tertiary and Quaternary) monotreme taxa. Tertiary monotremes were either archaic ornithorhynchids (toothed platypuses in the genera Monotrematum and Obdurodon) or tachyglossids (large echidnas in the genera Megalibgwilia and Zaglossus). Quaternary ornithorhynchid material is referable to the sole living platypus species Ornithorhynchus anatinus. Quaternary echidnas, however, were moderately diverse and several forms are known (Megalibgwilia species; 'Zaglossus' hacketti; Zaglossus species and Tachyglossus aculeatus).     

Did sex chromosome turnover promote divergence of the major mammal groups?

Comparative mapping and sequencing show that turnover of sex determining genes and chromosomes, and sex chromosome rearrangements, accompany speciation in many vertebrates. Here I review the evidence and propose that the evolution of therian mammals was precipitated by evolution of the male‐determining SRY gene, defining a novel XY sex chromosome pair, and interposing a reproductive barrier with the ancestral population of synapsid reptiles 190 million years ago (MYA). Divergence was reinforced by multiple translocations in monotreme sex chromosomes, the first of which supplied a novel sex determining gene. A sex chromosome‐autosome fusion may have separated eutherians (placental mammals) from marsupials 160 MYA. Another burst of sex chromosome change and speciation is occurring in rodents, precipitated by the degradation of the Y. And although primates have a more stable Y chromosome, it may be just a matter of time before the same fate overtakes our own lineage. Also watch the video abstract .     

A comparative study of covalently-bound fatty acids in keratinized tissues.

1. Covalently-bound fatty acids were characterized in keratinous tissues obtained from a wide range of animals. 2. 18-Methyleicosanoic acid was a major component in all the mammalian fur samples examined except monotreme fur. In monotreme fur 26-carbon fatty acids predominated. 3. Fatty acids from feather keratin and reptile skin had different profiles to the alpha-keratins of mammalian fur. 4. The major forms of covalently-bound fatty acids are very similar in species that diverged up to 125 million years ago.     

A new stereospondyl from the German Middle Triassic, and the origin of the Metoposauridae

Recent finds of well-preserved temnospondyl skeletons from the Lower Keuper (Ladinian, Middle Triassic) in southern Germany are assigned to a new genus and species, Callistomordax kugleri . This taxon is characterized by the following autapomorphies: (1) wide unpaired frontal; (2) vomerine fangs greatly enlarged to occupy entire width of element; (3) intercentra elongated and massive, anterior face being convex; (4) humerus semilunar with enlarged deltopectoral crest; (5) cleithrum strongly curved and bow-shaped; (6) trunk extremely elongated to reach three times the length of the skull. Callistomordax shares with the Metoposauridae the pattern of dermal ornamentation, the proportion of both posterior skull table and snout, the position of the lacrimal, the morphology of the basicranial region, and the structure of the clavicle and interclavicle. Phylogenetic analysis suggests Callistomordax to be the sister taxon of the Metoposauridae, nested within a grade formed by various trematosaurian taxa. In this assemblage, Lyrocephaliscus and a clade formed by Almasaurus , Rileymillerus , Callistomordax , and the Metoposauridae are sister taxa. In all variants of the cladistic analysis, Callistomordax and the Metoposauridae form immediate sister groups. According to the present findings, neither plagiosaurids nor brachyopoids and rhytidosteids are closely related to this 'trematosaurian' monophylum, although these taxa share a range of homoplasies.  © 2008 The Linnean Society of London, Zoological Journal of the Linnean Society , 2008, 152 , 79–113.     

Spatially explicit fate factors of phosphorous emissions to freshwater at the global scale

Purpose

The location of a phosphorus emission can strongly affect its expected fate in freshwater. To date, in Life Cycle Assessment (LCA), fate factors for phosphorus emissions have been derived for continents or large countries and had limited spatial resolution. These fate factors do not account sufficiently for local variations and are not applicable globally. In this paper, fate factors for freshwater eutrophication are derived for phosphorus emissions to freshwater on a global scale with a half-degree resolution.

Methods

For this purpose, a new global fate model for phosphorus has been developed on a half-degree resolution. The removal processes taken into account are grid-specific advection, phosphorus retention and water use. Aggregated fate factors based on archetypes and on administrative units are presented.

Results and discussion

The derived fate factors represent the persistence of phosphorus in the freshwater environment. The typical fate factor of phosphorus emissions to freshwater is 10?days and can vary more than 2 orders of magnitude among the grid cells (the 5th and 95th percentile are 0.8 and 310?days, respectively). Advection is the dominant removal process of phosphorus in freshwater (67.5%), followed by retention (27.6%) and water use (4.9%).

Conclusions

The results demonstrate inclusion of information on the location of phosphorus emissions to freshwater can improve the comparative power of the fate factor implementation in LCAs. The fate factors enable consistent assessment and comparison of freshwater eutrophication impacts at different locations across the globe.     

The major histocompatibility complex in monotremes: an analysis of the evolution of <Emphasis Type="Italic">Mhc</Emphasis> class I genes across all three mammalian subclasses

We report the isolation and characterization of cDNA clones of expressed, functional major histocompatibility complex class-I ( Mhc-I) genes from two species of monotremes: the duck-billed platypus and the short-beaked echidna. The cDNA clones were isolated from libraries constructed from spleen RNA, clearly establishing their expression in at least this one peripheral lymphoid organ. From the presence of conserved amino acid residues, it appears the expressed sequences encode molecules that likely function as classical Mhc-I. These clones were isolated using monotreme Mhc-I processed pseudogenes as probes. These processed pseudogenes were isolated from genomic DNA and, based on their structure, are likely independently derived in the platypus and echidna. When all the monotreme sequences were included in phylogenetic analyses, we found no apparent orthologous relationships between the platypus and echidna Mhc-I. Analyses that included a large number of Mhc-I sequences from other taxa support a separate monotreme Mhc-I clade, basal to a therian Mhc-I clade that is comprised of sequences from marsupial and placental mammals. The phylogenies also support the hypothesis that Mhc-I genes of placental mammals, marsupials, and monotremes are derived from three separate lineages of Mhc-I genes, best explained by two rounds of duplications and deletions. The first round would have occurred prior to the divergence of monotremes and therians, and the second prior to the divergence of marsupials and placental mammals. The sequences described here represent the first reported functional monotreme Mhc-I, as well as the first processed pseudogenes of any type from monotremes.     

The complexity of expressed kappa light chains in egg-laying mammals

Complementary DNAs encoding immunoglobulin light chains were isolated from two monotreme species, Ornithorhynchus anatinus (duckbill platypus) and Tachyglossus aculeatus (echidna). The sequences of both the variable and constant regions of these clones had greater similarity to IGK than to other light chain classes and phylogenetic analyses place them squarely within the mammalian IGK group, establishing them as monotreme IGK homologues. The constant region sequences of all clones were essentially identical within each species and, along with Southern blot results, the data are consistent with a single IGKC in each species. The expressed IGKV repertoires from both platypus and echidna were randomly sampled and there appear to be at least four platypus and at least nine echidna IGKV subgroups. The IGKV subgroups are highly divergent within species, in some cases sharing as little as 57% nucleotide identity. Two of the IGKV subgroups are present in both species, so there is some degree of overlap in the germline repertoires of these two monotremes. Overall the complexity seen in platypus and echidna IGK light chains is comparable with that of other mammals considered to have high levels of germline diversity and is in contrast to what has been found so far for monotreme IGL.Electronic Supplementary Material Supplementary material is available for this article at .     

The isolation and amino acid sequences of echidna (Tachyglossus aculeatus) milk lysozyme I and II.

Comparative studies of monotreme proteins are of particular value in gaining an understanding of the origin of mammals and their interrelationships. The presence of two lysozyme variants, echidna lysozyme I and II, has been confirmed in mature milk samples of Tachyglossus aculeatus multiaculeatus and Tachyglossus aculeatus aculeatus respectively. A simplified procedure is described for their isolation. Their amino acid sequences, the first determined for a monotreme secretory protein, are unusual. They are shown to be c-type lysozymes, each consisting of a single chain of 125 residues (terminating at Cys 125). The only other known c-type lysozyme with this termination is that of pigeon eggwhite. Echidna lysozyme is unique in having no Cys at position 6, but at position 9. It has precisely the residues relevant to the binding of Ca(II), and most of the residues implicated in the galactosyl transferase modifier action of alpha-lactalbumin. However, the weak modifier action previously observed for variant I, prepared by a different method, was not found for the present preparation. The evolutionary significance of the results is discussed.     

Hibernation in a monotreme, the echidna (Tachyglossus aculeatus)

The body temperatures of five echidnas in Australia's Southern Alps were monitored by radio telemetry from February to December 1987. All five hibernated throughout the winter, showing very low body temperatures (4-9 degrees C, close to ambient) when torpid, compared with 28-33 degrees C in a typical day during the active season. Spontaneous arousals from hibernation occurred every 2-3 weeks, during which body temperatures rose rapidly to over 30 degrees C for several hours before dropping to be close to ambient again. The identification of "classical" hibernation in a monotreme, with a similar pattern to that seen in Eutheria and in an animal as large as the largest eutherian hibernator, has important implications for current ideas about the evolution of endothermy.     

Revision of<Emphasis Type="Italic">Cheliderpeton vranyi</Emphasis><Emphasis Type="SmallCaps">Fritsch</Emphasis>, 1877 (Amphibia,Temnospondyli) from the Lower Permian of Bohemia (Czech Republic)

The temnospondylCheliderpeton vranyi Fritsch, 1877 from the Lower Permian Ruprechtice horizon (Rotliegend) of the Intrasudetic Basin (Bohemia, Czech Republic) is redescribed. Many features of the skeleton permit a new understanding of the type species and consequently of the genus. Diagnostic characters are the narrow and round-tipped snout, straight to convex outline of the skull roof, narrow and long otic notch, posteriorly expanded quadratojugal, and the relatively wide and short rhombic interclavicle. The ilium with a short, expanded dorsal branch and the missing contact of nasal/maxilla are features shared with the related Upper PermianIntasuchus from Russia and the Eryopidae.Actinodon germanicus is a junior synonym ofCheliderpeton vranyi.      

A Tachyglossid-Like Humerus from the Early Cretaceous of South-Eastern Australia

A partial right humerus has been recovered from the Early Cretaceous (Albian) Eumeralla Formation at Dinosaur Cove in south-eastern Australia. General morphology, size and the presence of a single epicondylar foramen (the entepicondylar) suggest that the bone is from a mammal or an advanced therapsid reptile. The humerus is similar in size, shape and torsion to the equivalent bone of extant and late Neogene echidnas (Tachyglossidae) but, contrary to the situation in extant monotremes, in which the ulna and radius articulate with a single, largely bulbous condyle, it bears a shallow, pulley-shaped (i.e. trochlear-form) ulnar articulation that is confluent ventro-laterally with the bulbous radial condyle. This form of ulnar articulation distinguishes this bone from the humeri of most advanced therapsids and members of several major groups of Mesozoic mammals, which have a condylar ulnar articulation, but parallels the situation found in therian mammals and in some other lineages of Mesozoic mammals. As in extant monotremes the distal humerus is greatly expanded transversely and humeral torsion is strong. Transverse expansion of the distal humerus is evident in the humeri of the fossorial docodont Haldanodon, highly-fossorial talpids and some clearly fossorial dicynodont therapsids, but the fossil shows greatest overall similarity to extant monotremes and it is possible that the peculiar elbow joint of extant monotremes evolved from a condition approximating that of the fossil. On the basis of comparisons with Mesozoic and Cainozoic mammalian taxa in which humeral morphology is known, the Dinosaur Cove humerus is tentatively attributed to a monotreme. However, several apparently primitive features of the bone exclude the animal concerned from the extant families Tachyglossidae and Ornithorhynchidae and suggest that, if it is a monotreme, it is a stem-group monotreme. Whatever, the animal's true affinity, the gross morphology of its humerus indicates considerable capacity for rotation-thrust digging.