Functional Diversity and Evolution of Bitter Taste Receptors in Egg-Laying Mammals

Egg-laying mammals (monotremes) are a sister clade of therians (placental mammals and marsupials) and a key clade to understand mammalian evolution. They are classified into platypus and echidna, which exhibit distinct ecological features such as habitats and diet. Chemosensory genes, which encode sensory receptors for taste and smell, are believed to adapt to the individual habitats and diet of each mammal. In this study, we focused on the molecular evolution of bitter taste receptors (TAS2Rs) in monotremes. The sense of bitter taste is important to detect potentially harmful substances. We comprehensively surveyed agonists of all TAS2Rs in platypus (Ornithorhynchus anatinus) and short-beaked echidna (Tachyglossus aculeatus) and compared their functions with orthologous TAS2Rs of marsupial and placental mammals (i.e., therians). As results, the agonist screening revealed that the deorphanized monotreme receptors were functionally diversified. Platypus TAS2Rs had broader receptive ranges of agonists than those of echidna TAS2Rs. While platypus consumes a variety of aquatic invertebrates, echidna mainly consumes subterranean social insects (ants and termites) as well as other invertebrates. This result indicates that receptive ranges of TAS2Rs could be associated with feeding habits in monotremes. Furthermore, some orthologous receptors in monotremes and therians responded to β-glucosides, which are feeding deterrents in plants and insects. These results suggest that the ability to detect β-glucosides and other substances might be shared and ancestral among mammals.     

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.     

Identification and structural analysis of four serine proteases in a monotreme, the platypus, Ornithorhynchus anatinus

To study the emergence of the major subfamilies of serine proteases during vertebrate evolution, we present here the primary structure of four serine proteases expressed in the spleen of a monotreme, the platypus, Ornithorhynchus anatinus. Partial cDNA clones for four serine proteases were isolated by a PCR-based strategy. This strategy is based on the high level of sequence identity between various members of the large gene family of trypsin-related serine proteases, over two highly conserved regions, those of the histidine and the serine of the catalytic triad. The partial cDNA clones were used to isolate full-length or almost full-length cDNA clones for three of these proteases from a platypus spleen cDNA library. By phylogenetic analysis, these three clones were identified as being the platypus homologues of human coagulation factor X, neutrophil elastase, and a protease distantly related to the T-cell granzymes. The remaining partial clone was found to represent a close homologue of human complement factor D (adipsin). The isolation of these four clones shows that several of the major subfamilies of serine proteases had evolved as separate subfamilies long before the radiation of the major mammalian lineages of today, the monotremes, the marsupials, and the placental mammals. Upon comparison of the corresponding proteases of monotremes and eutherian mammals, the coagulation and complement proteases were shown to display a higher degree of conservation compared to the hematopoietic proteases N-elastase and the T-cell granzymes. This latter finding indicates a higher evolutionary pressure to maintain specific functions in the complement and coagulation enzymes compared to many of the hematopoietic serine proteases.     

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.     

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.     

Molecular Phylogeny and Evolution of the Neurotrophins from Monotremes and Marsupials

We have investigated the phylogenetic relationships of monotremes and marsupials using nucleotide sequence data from the neurotrophins; nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3). The study included species representing monotremes, Australasian marsupials and placentals, as well as species representing birds, reptiles, and fish. PCR was used to amplify fragments encoding parts of the neurotrophin genes from echidna, platypus, and eight marsupials from four different orders. Phylogenetic trees were generated using parsimony analysis, and support for the different tree structures was evaluated by bootstrapping. The analysis was performed with NGF, BDNF, or NT-3 sequence data used individually as well as with the three neurotrophins in a combined matrix, thereby simultaneously considering phylogenetic information from three separate genes. The results showed that the monotreme neurotrophin sequences associate to either therian or bird neurotrophin sequences and suggests that the monotremes are not necessarily related closer to therians than to birds. Furthermore, the results confirmed the present classification of four Australasian marsupial orders based on morphological characters, and suggested a phylogenetic relationship where Dasyuromorphia is related closest to Peramelemorphia followed by Notoryctemorphia and Diprotodontia. These studies show that sequence data from neurotrophins are well suited for phylogenetic analysis of mammals and that neurotrophins can resolve basal relationships in the evolutionary tree. Received: 27 January 1997 / Accepted: 20 March 1997     

Phylogenetic Analysis of 18S rRNA and the Mitochondrial Genomes of the Wombat, <Emphasis Type="BoldItalic">Vombatus ursinus,</Emphasis> and the Spiny Anteater, <Emphasis Type="BoldItalic">Tachyglossus aculeatus:</Emphasis> Increased Support for the Marsupionta Hypothesis

The monotremes, the duck-billed platypus and the echidnas, are characterized by a number of unique morphological characteristics, which have led to the common belief that they represent the living survivors of an ancestral stock of mammals. Analysis of new data from the complete mitochondrial (mt) genomes of a second monotreme, the spiny anteater, and another marsupial, the wombat, yielded clear support for the Marsupionta hypothesis. According to this hypothesis marsupials are more closely related to monotremes than to eutherians, consistent with a basal split between eutherians and marsupials/monotremes among extant mammals. This finding was also supported by analysis of new sequences from a nuclear gene—18S rRNA. The mt genome of the wombat shares some unique features with previously described marsupial mtDNAs (tRNA rearrangement, a missing tRNA^Lys, and evidence for RNA editing of the tRNA^Asp). Molecular estimates of genetic divergence suggest that the divergence between the platypus and the spiny anteater took place ≈34 million years before present (MYBP), and that between South American and Australian marsupials ≈72 MYBP. Received: 28 October 2000 / Accepted: 23 March 2001     

A survey of type I interferons from a marsupial and monotreme: implications for the evolution of the type I interferon gene family in mammals

Sequence data for type I interferons (IFNs) have previously only been available for birds and eutherian ('placental') mammals, but not for the other two groups of extant mammals, the marsupials and monotremes. This has left a large gap in our knowledge of the evolutionary and functional relationships of what is a complex gene family in eutherians. In this study, a PCR-based survey of type I IFN genes from a marsupial, the tammar wallaby (Macropus eugenii), and a monotreme, the short-beaked echidna (Tachyglossus aculeatus), was conducted. Along with Southern blot and phylogenetic analysis, this revealed a large number of type I IFN genes for the wallaby, rivalling that of eutherians, but relatively few type I IFN genes in the echidna. The wallaby genes include both IFNA and IFNB orthologues, indicating that the gene duplication leading to these subtypes occurred prior to the divergence of marsupials and eutherians some 130 million years ago. Results from this study support the idea that the expansion of type I IFN gene complexity in mammals coincides with a concomitant expansion in the functionality of these molecules. For example, this expansion in complexity may have, at least partially, facilitated the evolution of viviparity in marsupials and eutherians. Other evolutionary aspects of these sequences are also discussed.     

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 .     

Evolution of the Monotremes: Phylogenetic Relationship to Marsupials and Eutherians, and Estimation of Divergence Dates Based on α-Lactalbumin Amino Acid Sequences

The amino acid sequences of the -lactalbumins of the echidna, Tachyglossus aculeatus, and the platypus, Ornithorhynchus anatinus, were compared with each other and with those of 13 eutherian and 3 marsupial species. Phylogenetic parsimony analyses, in which selected mammalian lysozymes were used as outgroups, yielded trees whose consensus indicated that the two monotremes are sister taxa to marsupials and eutherians and that the latter two clades are each other's closest relatives. The data do not support the notion of a Marsupionta (monotreme–marsupial) clade. Pairwise comparison between the -lactalbumins yielded maximum-likelihood distances from which divergence dates were estimated on the basis of three calibration points. The distance data support the view that the echidna and platypus lineages diverged from their last common ancestor at least 50 to 57 Ma (million years ago) and that monotremes diverged from marsupials and eutherian mammals about 163 to 186 Ma.     

Immunoglobulin genetics of Ornithorhynchus anatinus (platypus) and Tachyglossus aculeatus (short-beaked echidna)

In this paper, we review data on the monotreme immune system focusing on the characterisation of lymphoid tissue and of antibody responses, as well the recent cloning of immunoglobulin genes. It is now known that monotremes utilise immunoglobulin isotypes that are structurally identical to those found in marsupials and eutherians, but which differ to those found in birds and reptiles. Monotremes utilise IgM, IgG, IgA and IgE. They do not use IgY. Their IgG and IgA constant regions contain three domains plus a hinge region. Preliminary analysis of monotreme heavy chain variable region diversity suggests that the platypus primarily uses a single VH clan, while the short-beaked echidna utilises at least 4 distinct VH families which segregate into all three mammalian VH clans. Phylogenetic analysis of the immunoglobulin heavy chain constant region gene sequences provides strong support for the Theria hypothesis. The constant region of IgM has proven to be a useful marker for estimating the time of divergence of mammalian lineages.     

Type I interferon genes from the egg-laying mammal, Tachyglossus aculeatus (short-beaked echidna)

The type I IFN are an important group of multifunctional cytokines that have, for whatever reason, evolved to a high level of complexity in eutherian mammals such as humans and mice. However, until recently, little was known about the type I IFN systems of the other two groups of extant mammals, the marsupials and the egg-laying monotremes. Preliminary partial type I IFN sequences from the short-beaked echidna were previously found to cluster only with the IFN-beta subtype in phylogenetic analyses, but a lack of sequence information made interpretation of these results tenuous. Here, we report cloning of the full-length genes of representatives from the two previously defined groups of echidna type I IFN by genomic walking PCR. Along with analysis of conserved cysteine placement and promoter elements, phylogenetic analysis incorporating these sequences strongly suggest that the two groups of echidna type I IFN genes are in fact homologous to IFN-alpha and IFN-beta, confirming that the duplication leading to these two major classes of type I IFN occurred prior to the divergence of eutherians and monotremes some 180 million years ago. Thus, even though there are major differences in gene copy number and heterogeneity, separate IFN-alpha and IFN-beta gene families are a feature of the cytokine networks of all three groups of living mammals.     

Largest vertebrate vomeronasal type 1 receptor gene repertoire in the semiaquatic platypus

Vertebrate vomeronasal chemoreception plays important roles in many aspects of an organism's daily life, such as mating, territoriality, and foraging. Vomeronasal type 1 receptors (V1Rs) and vomeronasal type 2 receptors (V2Rs), 2 large families of G protein-coupled receptors, serve as vomeronasal receptors to bind to various pheromones and odorants. Contrary to the previous observations of reduced olfaction in aquatic and semiaquatic mammals, we here report the surprising finding that the platypus, a semiaquatic monotreme, has the largest V1R repertoire and nearly largest combined repertoire of V1Rs and V2Rs of all vertebrates surveyed, with 270 intact genes and 579 pseudogenes in the V1R family and 15 intact genes, 55 potentially intact genes, and 57 pseudogenes in the V2R family. Phylogenetic analysis shows a remarkable expansion of the V1R repertoire and a moderate expansion of the V2R repertoire in platypus since the separation of monotremes from placentals and marsupials. Our results challenge the view that olfaction is unimportant to aquatic mammals and call for further study into the role of vomeronasal reception in platypus physiology and behavior.     

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.     

Phylogenetic analysis of 1.5 Mbp and platypus EST data refute the Marsupionta hypothesis and unequivocally support Monotremata as sister group to Marsupialia/Placentalia

The extant mammalian groups Monotremata, Marsupialia and Placentalia are, according to the 'Theria' hypothesis, traditionally classified into two subclasses. The subclass Prototheria includes the monotremes and subclass Theria marsupials and placental mammals. Based on some morphological and molecular data, an alternative proposition, the Marsupionta hypothesis, favours a sister group relationship between monotremes and marsupials to the exclusion of placental mammals. Phylogenetic analyses of single genes and even multiple gene alignments have not yet been able to conclusively resolve this basal mammalian divergence. We have examined this problem using one data set composed of expressed sequence tags (EST) and another containing 1 510 509 nucleotide (nt) sites from 1358 inferred cDNA genomic sequences. All analyses of the concatenated sequences unambiguously supported the Theria hypothesis. The Marsupionta hypothesis was rejected with high statistical confidence from both data sets. In spite of the strong support for Theria, a non-negligible number of single genes supported either of the two alternative hypotheses. The divergence between monotremes and therian mammals was estimated to have taken place 168–178 Mya, a dating compatible with the fossil record. Considering the long common evolutionary branch of therians, it is surprising that sequence data from many thousand amino acid sites were needed to conclusively resolve their relationship to monotremes. This finding draws attention to other mammalian divergences that have been taken as unequivocally settled based on much smaller alignments. EST data provide a comprehensive random sample of protein coding sequences and an economic way to produce large amounts of data for phylogenetic analysis of species for which genomic sequences are not yet available.     

A molecular perspective on the phylogeny of placental mammals based on mitochondrial 12S rDNA sequences,with special reference to the problem of the Paenungulata

Part of the 12S rDNA gene was amplified and sequenced for 11 placental mammals, 3 marsupials, and 2 monotremes. Multiple alignments for these sequences and nine additional placental sequences taken from GenBank were obtained using CLUSTAL. Phylogenetic analyses were performed using standard parsimony, transversion parsimony, and Lake's method of invariants. All of our analyses uniteLoxodontia withDugong. Procavia, in turn, is a sister group to these taxa, thus supporting the monophyly of the Paenungulata. Perissodactyls are a sister group to paenungulates when transitions and transversions are both included but not when transitions are omitted. Likewise, cetaceans are a sister group to artiodactyls on minimum length trees under standard parsimony but not under transversion parsimony. Rodent monophyly and bat monophyly also receive mixed support, as does a putative alliance between primates and lagomorphs. Interestingly, the percentage divergence between the echidna and the platypus is less than for the rat and mouse.     

Relationships Among the Families and Orders of Marsupials and the Major Mammalian Lineages Based on Recombination Activating Gene-1

Controversies remain over the relationships among several of the marsupial families and between the three major extant lineages of mammals: Eutheria (placentals), Metatheria (marsupials), and Prototheria (monotremes). Two opposing hypotheses place the marsupials as either sister to the placental mammals (Theria hypothesis) or sister to the monotremes (Palimpsest or Marsupionta hypothesis). A nuclear gene that has proved useful for analyzing phylogenies of vertebrates is the recombination activation gene-1 (RAG1). RAG1 is a highly conserved gene in vertebrates and likely entered the genome by horizontal transfer early in the evolution of jawed vertebrates. Phylogenetic analyses were performed on RAG1 sequences from seven placentals, 28 marsupials, and all three living monotreme species. Phylogenetic analyses of RAG1 sequences support many of the traditional relationships among the marsupials and suggest a relationship between bandicoots (order Peramelina) and the marsupial mole (order Notoryctemorphia), two lineages whose position in the phylogenetic tree has been enigmatic. A sister relationship between South American shrew opossums (order Paucituberculata) and all other living marsupial orders is also suggested by RAG1. The relationship between the three major groups of mammals is consistent with the Theria hypothesis, with the monotremes as the sister group to a clade containing marsupials and placentals.