Regions are not drawn to scale. Werning and the emu silhouette is created by D. Naish vectorized by T. Keesey ; all three silhouettes are reproduced under the Creative Commons Attribution 3. The nasal cavity of the platypus is closed off during diving and the size of the main olfactory bulb of the platypus is much smaller than that of the echidna 1.
Consistent with this, the number of olfactory receptors OR genes in platypus is much smaller than in echidna Fig. The difference in the large olfactory bulb and OR repertoire in echidna may contribute to the ability to search for odours of underground prey, whereas the platypus relies on electroreception to detect prey in the water. However, the size of the accessory olfactory bulb is larger in the platypus than in the echidna 1. The accessory olfactory bulb receives projections from the vomeronasal organ, and there is a marked expansion of the number of vomeronasal type-1 receptors V1R genes in the platypus compared with the echidna 28 Fig.
Vomeronasal receptors probably have important roles in courtship, parental care, induction of lactation and milk ejection in monotremes Therefore, the diversification of the olfactory bulb and accessory olfactory bulb systems in monotremes provide an interesting example of the eco-evolutionary trade-off. V1R amplification has been associated with the size of the vomeronasal organ and nocturnal activity This is also consistent with the fact that the platypus closes its eyes when diving and therefore relies entirely on other senses underwater and in the burrow.
The semi-aquatic lifestyle of the platypus is supported by particularly high haemoglobin levels and large numbers of small red blood cells The haemoglobin—haem detoxification system in mammals provides efficient clearance to minimize oxidative damage 32 in which haptoglobin is the haemoglobin chaperone 32 and free haem is bound by haemopexin and alpha-1 microglobulin Both the haemopexin and alpha-1 microglobulin genes are found in the monotreme genomes, whereas the haptoglobin gene is absent Fig.
Haptoglobin evolved in the common ancestor of vertebrates from an immune gene of the MASP family 33 but has neofunctionalized in mammals to bind to haemoglobin with a higher affinity and to bind to the CDA receptor, which is also absent in monotremes, for clearance in macrophages The absence of the haptoglobin gene and CDA in monotremes suggests that the neofunctionalization of haptoglobin happened after the divergence of monotremes from therians, not before it as previously thought 34 , and long after the evolution of enucleated red blood cells in the common ancestor of mammals Several nonmammalian vertebrates have lost haptoglobin, including chicken 34 Fig.
We confirmed the expansion of the CD family in platypus 2 ten members and found five in echidna, compared with two and three in humans and mice, respectively Extended Data Fig. As mammalian CDA can bind to haemoglobin in the absence of haptoglobin 36 and one CD family member has become the haemoglobin chaperone in chicken, the CD family protein s may have evolved this role in monotremes. Monotremes provide the key to understanding how viviparity evolved in mammals.
They are not as dependent on egg proteins as egg-laying avian and reptilian species owing to their nutrient acquisition from uterine secretions 23 , 37 , and the subsequent reliance of the young on lactation. Similar to marsupials, monotremes have an extended lactation period and the composition of the milk changes dynamically as the development progresses to match the changing needs of the young SPINT3, a major milk-specific protein that is present in early lactation of therians with a probable role in the protection of immunoincompetent young in marsupials 39 , is absent in monotremes.
Syntenic analysis confirmed that this region is conserved in platypus but contains two copies of a new protein that contains a Kunitz domain Extended Data Fig. The Kunitz family is a rapidly evolving family, and one of the new members could have a immunoprotective function similar to SPINT3 in monotremes.
The monotreme genomes contain most of the milk genes that have been identified in therian mammals 38 , Most mammals have three casein genes 41 , which encode the most abundant milk proteins secreted throughout lactation Fig. Complete and accurate reference genomes and annotations are critical for evolutionary and functional analyses.
It remains a challenge to produce a highly accurate chromosome-level assembly, particularly for differentiated sex chromosomes. We have produced a high-quality platypus genome using a combination of single-molecule sequencing technology and multiple sources of physical mapping methods to assign most of the sequences to a chromosome-scale assembly.
This permits better-resolved analyses of the origin and diversification of the complex sex chromosome system that evolved specifically in monotremes. We delineate ancient and lineage-specific changes in the sensory system, haemoglobin degradation and reproduction that represent some of the most fascinating biology of platypus and echidna.
The new genomes of both species will enable further insights into therian innovations and the biology and evolution of these extraordinary egg-laying mammals. No statistical methods were used to predetermine sample size.
The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment.
Heart muscle of Emale01 was used for a variety of library construction and Illumina sequencing analyses. Echidna RNA was extracted from brain, cerebellum, kidney, liver, testis and ovary and sequenced using a previously published procedure Platypus Y chromosome BAC isolation via hybridization was performed using a previous published procedure 4 and sequenced with PacBio. The platypus genome was assembled following VGP assembly pipeline v.
The echidna genome was assembled using Platanus 44 v. Manual curation was performed for both assemblies. Details are available in the Supplementary Methods. The read depth of each sex was calculated in 5-kb non-overlapping windows to identify X-borne sequences and 2-kb non-overlapping windows to identify Y-borne sequences, normalized against the median depth. Parameter evaluation details are available in the Supplementary Methods. We collected 75 BAC and marker genes Supplementary Table 3 and ordered them according to their relative order from those papers.
We also used the anchored sequences of OANA5 except for the sequence of chromosome 14 to anchor the scaffolds into chromosomes. All identified PARs were included in chromosome X. Classified Y-borne scaffolds failed to anchor and orient due to the lack of information. We also curated and anchored some echidna X-borne scaffolds to chromosome X based on Mashmap 49 v.
We identified repetitive elements in both assemblies using the same pipeline, which included homologue-based and de novo prediction. For the homology-based method, we used default repeat library from Repbase v. For the de novo method, we first ran RepeatModeler v. Gene annotation was performed by merging the homology, de novo prediction and transcriptome analyses to build a consensus gene set of each species.
Protein sequences from human, mouse, opossum, platypus, chicken and green lizard Anolis carolinensis from Ensembl 54 release 87 were aligned to the genome using TBLASTN 55 v. Candidate gene regions were refined using GeneWise for more accurate gene models.
We randomly selected 1, high score homology-based genes to train Augustus 56 v. Results from these three methods were merged into a nonredundant gene set. Iprscan was used to annotate the GO of genes.
Detailed descriptions of the manual annotation, curation and phylogenetic analysis of genes related to imprinting, immune system, reproduction and haemoglobin degradation can be found in the Supplementary Methods.
We identified gap-filling regions using an alignment-based strategy similar to a previously published study We considered gaps for which both flanking regions mapped to mOrnAna1 as closed gaps. Only properly closed gaps defined by 1 both flanking regions were aligned but did not overlap and 2 closed gap size were within times the estimated gap size in OANA5 were considered for repeat and gene improvement analysis.
A one-to-one relationship was obtained in the first round of Mashmap. In the second round of Mashmap, those OANA5 sequences that were unmapped in the first round of mapping were used as query. Candidate redundant sequences were obtained from the second round Mashmap result, but excluded regions that were gaps in OANA5.
A one-to-one gene pair between the two gene sets was defined as the liftover of the OANA5 gene when it overlapped with only one mOrnAna1 gene. Only one-to-one pairs were used for the comparison of open-reading frame completeness. We defined a gene as having a complete open-reading frame if its first codon is a start codon and the last codon is a stop codon. We defined one-to-one orthologues between the human sequence and the sequences of other species by considering both reciprocal best BLASTP hits RBH and synteny, taking the human sequence as reference, as previously described Next, we identified RBH orthologues between human and every other species on the basis of the following parameters: alignment score, alignment rate and identity.
From these RBH orthologues, we retained those pairs with conserved synteny across species. Synteny was determined based on their flanking genes. If RBH orthologous gene pairs shared the same flanking genes, we retained the genes for downstream analyses.
Finally, we merged pairwise orthologue lists according to the human coordinates. In this way, we produced the final one-to-one orthologue set across species. Only alignments larger than 10 kb were kept. The synteny blocks were further cleaned of overlapping genes. N50 and the total length of the synteny block inferred from each human—species pair were calculated based on the human coordinates. The phylogenetic tree was constructed using concatenated four-degenerated sites from the 7, one-to-one orthologues using RaxML 65 v.
Points and time range included the most recent common ancestor of human—mouse, 85—94 million years ago; human—opossum — million years ago; human—platypus, — million years ago, human—chicken, — million years ago, anole—chicken, — million years ago.
The seed used for MCMC was The four-degenerated site alignment was extracted based on the human gene set Ensembl release 87 , concatenated and fed to phyloFit in the PHAST package 69 v. The substitution rate was calculated by dividing the branch length to the mammalian common ancestor to the mammal—reptile divergence time.
Gene families across the seven species were generated using orthoMCL 70 v. We first estimated the assembly error by excluding families with more than members. Then the estimated rate was used to infer the family size at every node for each family.
The ancestral node gene number of families with more than members among extant species were inferred separately. A false-discovery rate FDR adjustment was used for multiple-test corrections in GO enrichment analyses. We used pairwise LASTZ alignments of the opossum, Tasmanian devil, platypus, chicken and common wall lizard Podarcis muralis genomes to the human genome as input. Echidna was not used here as most of the sequences were not anchored to chromosomes, which would lead to a more fragmented reconstruction.
With the net and chain results, conserved segments that were uniquely and universally presented in all six species were obtained using inferCARs 74 release Jun We replaced the conserved segments of the human, opossum and Tasmanian devil genomes with those of the reconstructed therian ancestral karyotype and reconstructed marsupial ancestral karyotype using ANGES with the same parameters except setting the target reconstruction node to mammalian ancestor. We reorganized CARs on the basis of gene synteny among ingroups and outgroups inferred using MCScanX 77 release , requiring that there is synteny across CARs in at least one ingroup—outgroup pair Supplementary Tables 22 , The breakpoint number in each lineage was calculated on the basis of the output of GRIMM using an in-house-generated script, in which one breakpoint was counted in fission, two breakpoints were counted in translocation, and one or two breakpoints were counted in inversion, depending on whether the inversion happened at the end of the chromosome.
Calculations were done using resolutions of kb and kb, and using the raw ANGES output and reorganized output, respectively Supplementary Table Differences in breakpoint rates compared to the average of all branches were tested as previously described Candidate gametologue pairs were further confirmed if both of the genes were mapped to the same gene in NCBI or the SwissProt database. Translated genes were aligned using PRANK 80 , filtered using Gblock 81 , and converted back into the alignment of the coding sequence.
Identity along X chromosomes was colour-coded for visualization. Uniquely mapped reads were used in the calculation and normalization of the reads per kilobase per million reads RPKM using DESeq 82 v.
We used the median expression value in each tissue to calculate the tissue specificity index TAU 83 for each gene. Genome-wide interaction maps at a kb resolution were generated for platypus, echidna and human SRX with HiC-Pro 84 v. The normalized sex chromosomes submatrix was extracted for quantification and plotting with ggplot2 v. For human, we used the scaled homologous sequences of platypus for quantification and plotting.
CTCF densities in every kb non-overlapping sliding window along the platypus sex chromosomes or scaled homologous sequences of echidna, human and chicken were compared. The 3. The FISH protocol was carried out on cultured fibroblasts from platypus authenticated by karyotype, not mycoplasma tested obtained from animals captured at the Upper Barnard River New South Wales, Australia during the breeding season AEC permits S, S and S as previously described 88 with the following exceptions.
Sample size was determined according to ref. No blinding nor randomization was performed. Further information on research design is available in the Nature Research Reporting Summary linked to this paper. Accession codes of genes are available in Supplementary Tables 31 , 33 , 37 , 49 , Ashwell, K.
Warren, W. Genome analysis of the platypus reveals unique signatures of evolution. Nature , — In the platypus a meiotic chain of ten sex chromosomes shares genes with the bird Z and mammal X chromosomes. Kortschak, R. Boissinot, S. The evolution of LINE-1 in vertebrates. Genome Biol. Phillips, M. Molecules, morphology, and ecology indicate a recent, amphibious ancestry for echidnas. Natl Acad. USA , — Bellott, D. Mammalian Y chromosomes retain widely expressed dosage-sensitive regulators.
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Comparative genomic analysis of the pheromone receptor class 1 family V1R reveals extreme complexity in mouse lemurs genus, Microcebus and a chromosomal hotspot across mammals. Johansen, K. Respiratory properties of blood and responses to diving of platypus Ornithorhynchus anatinus Shaw. Alayash, A. Haptoglobin: old protein with new functions. Acta , — Des Cooper, an evolutionary biologist at the University of New South Wales who did not take part in the research, said it represented a big step forward in the world's knowledge of mammals.
Graves said the research contained some surprises, such as the conclusion that genes which determine sex in a platypus are similar to those of a bird, not a mammal. Researchers also found genes that indicate platypuses — which rely on electrosensory receptors in their bills to navigate as they rummage with closed eyes in waterways — may also be able to smell underwater. Unique to Australia, the platypus has confounded observers for centuries.
Aboriginal legend explained it as the offspring of a duck and an amorous water rat. When the British Museum received its first specimen in , zoologist George Shaw was so dubious he tried to cut the pelt with scissors to make sure the bill had not been stitched on by a taxidermist. Platypuses live in the wild along most of Australia's east coast. Their numbers are not accurately known because they are notoriously shy.
Hunted for years for their pelts, they have been protected since the early s and are not considered to be endangered, though scientists say their habitat is vulnerable to human development. The platypus belongs to an ancient group of mammals — monotremes — which existed millions of years prior to the emergence of any modern-day mammal. But genetically, it is a mixture of mammals, birds and reptiles.
During our own evolution, we humans lost all three so-called vitellogenin genes, each of which is important for the production of egg yolks. Chickens on the other hand, continue to have all three.
The study demonstrates that platypuses still carry one of these three vitellogenin genes, despite having lost the other two roughly million years ago. The platypus continues to lay eggs by virtue of this one remaining gene. This is probably because it is not as dependent on creating yolk proteins as birds and reptiles are, as platypuses produce milk for their young.
In all other mammals, vitellogenin genes have been replaced with casein genes, which are responsible for our ability to produce casein protein, a major component in mammalian milk. The new research demonstrates that the platypus carries casein genes as well, and that the composition of their milk is thereby quite similar to that of cows, humans and other mammals.
Another trait that makes the platypus so unique is that, unlike the vast majority of mammals, it is toothless. The study reveals that the platypus lost its teeth roughly million years ago, when four of the eight genes responsible for tooth development disappeared.
Yet another platypus oddity investigated by the researchers was how their sex is determined. Both humans and every other mammal on Earth have two sex chromosomes that determine sex — the X and Y chromosome system in which XX is female and XY is male.
The monotremes, however, including our duck-billed friends from Down Under, have 10 sex chromosomes, with five Y and five X chromosomes. Thanks to the near-complete chromosomal level genomes, researchers can now suggest that these 10 sex chromosomes in the ancestors of the monotremes were organized in a ring form which was later broken away into many small pieces of X and Y chromosomes.
At the same time, the genome mapping reveals that the majority of monotreme sex chromosomes have more in common with chickens than with humans. But what it shows, is an evolutionary link between mammals and birds. HeritageDaily is an independent publisher of the latest scientific discoveries, research, and travel news. Email : [email protected]. We treat all information as private and confidential, any information we do collect is kept in a secure location.
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