A list of authors and their affiliations appears at the end of the paperWe present a draft genome sequence of the platypus, Ornithorhynchus anatinus. This monotreme exhibits a fascinating combination of reptilian and mammalian characters. For example, platypuses have a coat of fur adapted to an aquatic lifestyle; platypus females lactate, yet lay eggs; and males are equipped with venom similar to that of reptiles. Analysis of the first monotreme genome aligned these features with genetic innovations. We find that reptile and platypus venom proteins have been co-opted independently from the same gene families; milk protein genes are conserved despite platypuses laying eggs; and immune gene family expansions are directly related to platypus biology. Expansions of protein, non-protein-coding RNA and microRNA families, as well as repeat elements, are identified. Sequencing of this genome now provides a valuable resource for deep mammalian comparative analyses, as well as for monotreme biology and conservation.

In therian mammals (placentals and marsupials), sex is determined by an XX female: XY male system, in which a gene (SRY) on the Y affects male determination. There is no equivalent in other amniotes, although some taxa (notably birds and snakes) have differentiated sex chromosomes. Birds have a ZW female: ZZ male system with no homology with mammal sex chromosomes, in which dosage of a Z-borne gene (possibly DMRT1) affects male determination. As the most basal mammal group, the egg-laying monotremes are ideal for determining how the therian XY system evolved. The platypus has an extraordinary sex chromosome complex, in which five X and five Y chromosomes pair in a translocation chain of alternating X and Y chromosomes. We used physical mapping to identify genes on the pairing regions between adjacent X and Y chromosomes. Most significantly, comparative mapping shows that, contrary to earlier reports, there is no homology between the platypus and therian X chromosomes. Orthologs of genes in the conserved region of the human X (including SOX3, the gene from which SRY evolved) all map to platypus chromosome 6, which therefore represents the ancestral autosome from which the therian X and Y pair derived. Rather, the platypus X chromosomes have substantial homology with the bird Z chromosome (including DMRT1) and to segments syntenic with this region in the human genome. Thus, platypus sex chromosomes have strong homology with bird, but not to therian sex chromosomes, implying that the therian X and Y chromosomes (and the SRY gene) evolved from an autosomal pair after the divergence of monotremes only 166 million years ago. Therefore, the therian X and Y are more than 145 million years younger than previously thought.

The (9). The relationships between the SR Y and SOX genes are obviously of interest in considering the origin and evolution of the SRY gene in mammals.Marsupials diverged from eutherian (placental) mammals 120-150 million years ago and monotremes (egg-laying mammals) diverged even earlier, so that comparisons between these three major mammalian groups may provide information about the function and early evolution of mammalian sex chromosomes and sex-determining genes. Eutherian, marsupial, and monotreme sex chromosomes have been found to differ in size and gene content, enabling the different evolutionary origins of regions of the human sex chromosomes to be deduced. The genes on the long arm and proximal short arm of the human X chromosome are present on the X chromosome in marsupial and monotreme mammals, and this region, therefore, represents a conserved, probably original, mammalian X chromosome (10,11). The marsupial and monotreme X chromosome lacks genes borne on the short arm of the human X chromosome, suggesting that this region was originally autosomal and was added later to the eutherian X chromosome. The presence of several genes in this region with homologues on the Y chromosome implies that the region was added to both X and Y chromosomes, probably by recombination within an original pseudoautosomal region (10,12). In marsupials, as in eutherian mammals, the Y chromosome is testis determining, but at least some sexual dimorphisms are sex hormone independent and seem to be a function of X chromosome dosage, rather than the presence or absence of a Y chromosome (13).We have isolated an SR Y-related sequence from the marsupial X chromosome, which is closely homologous to the mouse and human SOX3 gene. This raises the possibility that X chromosome inactivation or gene dosage may play a role in sex determination in marsupials. However, we suggest that a more likely explanation for the presence of SR Y homologues on marsupial, mouse, and human X chromosomes is that SOX3 and SRY were originally alleles of a developmentally important gene shared by partly differentiated ancestral X and Y chromosomes. MATERIALS AND METHODSWe used two marsupial species, representing the two major Australian orders that diverged about 50 million years ago, the striped-faced dunnart Sminthopsis macroura (Order Polyprotodonta, Family Dasyuridae) and the Tammar wallaby Macropus eugenii (Order Diprotodonta, Family Macropodidae). Tissue was originally provided by

Egg-laying mammals (monotremes) are the only extant mammalian outgroup to therians (marsupial and eutherian animals) and provide key insights into mammalian evolution1,2. Here we generate and analyse reference genomes of the platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus), which represent the only two extant monotreme lineages. The nearly complete platypus genome assembly has anchored almost the entire genome onto chromosomes, markedly improving the genome continuity and gene annotation. Together with our echidna sequence, the genomes of the two species allow us to detect the ancestral and lineage-specific genomic changes that shape both monotreme and mammalian evolution. We provide evidence that the monotreme sex chromosome complex originated from an ancestral chromosome ring configuration. The formation of such a unique chromosome complex may have been facilitated by the unusually extensive interactions between the multi-X and multi-Y chromosomes that are shared by the autosomal homologues in humans. Further comparative genomic analyses unravel marked differences between monotremes and therians in haptoglobin genes, lactation genes and chemosensory receptor genes for smell and taste that underlie the ecological adaptation of monotremes.