Platypus and echidna genomes reveal mammalian biology and evolution

doi:10.1038/s41586-020-03039-0

. 2021 Apr;592(7856):756-762.

doi: 10.1038/s41586-020-03039-0. Epub 2021 Jan 6.

Platypus and echidna genomes reveal mammalian biology and evolution

Yang Zhou^#^{1

2}, Linda Shearwin-Whyatt^#³, Jing Li^#⁴, Zhenzhen Song^{1

5}, Takashi Hayakawa^{6

7}, David Stevens³, Jane C Fenelon⁸, Emma Peel⁹, Yuanyuan Cheng⁹, Filip Pajpach³, Natasha Bradley³, Hikoyu Suzuki¹⁰, Masato Nikaido¹¹, Joana Damas¹², Tasman Daish³, Tahlia Perry³, Zexian Zhu⁴, Yuncong Geng¹³, Arang Rhie¹⁴, Ying Sims¹⁵, Jonathan Wood¹⁵, Bettina Haase¹⁶, Jacquelyn Mountcastle¹⁶, Olivier Fedrigo¹⁶, Qiye Li¹, Huanming Yang^{1

17

18

19}, Jian Wang^{1

17}, Stephen D Johnston²⁰, Adam M Phillippy¹⁴, Kerstin Howe¹⁵, Erich D Jarvis^{21

22}, Oliver A Ryder²³, Henrik Kaessmann²⁴, Peter Donnelly²⁵, Jonas Korlach²⁶, Harris A Lewin^{12

27

28}, Jennifer Graves^{29

30

31}, Katherine Belov⁹, Marilyn B Renfree⁸, Frank Grutzner³², Qi Zhou^{33

34

35}, Guojie Zhang^{36

37

38

39}

Affiliations

¹ BGI-Shenzhen, Shenzhen, China.
² Villum Center for Biodiversity Genomics, Section for Ecology and Evolution, Department of Biology, University of Copenhagen, Copenhagen, Denmark.
³ School of Biological Sciences, The Environment Institute, The University of Adelaide, Adelaide, South Australia, Australia.
⁴ MOE Laboratory of Biosystems Homeostasis and Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China.
⁵ BGI Education Center, University of Chinese Academy of Sciences, Shenzhen, China.
⁶ Faculty of Environmental Earth Science, Hokkaido University, Sapporo, Japan.
⁷ Japan Monkey Centre, Inuyama, Japan.
⁸ School of BioSciences, The University of Melbourne, Melbourne, Victoria, Australia.
⁹ School of Life and Environmental Sciences, The University of Sydney, Sydney, New South Wales, Australia.
¹⁰ digzyme Inc, Tokyo, Japan.
¹¹ School of Life Science and Technology, Tokyo Institute of Technology, Tokyo, Japan.
¹² The Genome Center, University of California, Davis, CA, USA.
¹³ Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA.
¹⁴ Genome Informatics Section, Computational and Statistical Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA.
¹⁵ Tree of Life Programme, Wellcome Sanger Institute, Cambridge, UK.
¹⁶ The Vertebrate Genome Lab, The Rockefeller University, New York, NY, USA.
¹⁷ James D. Watson Institute of Genome Sciences, Hangzhou, China.
¹⁸ University of the Chinese Academy of Sciences, Beijing, China.
¹⁹ Guangdong Provincial Academician Workstation of BGI Synthetic Genomics, BGI-Shenzhen, Shenzhen, China.
²⁰ School of Agriculture and Food Sciences, The University of Queensland, Gatton, Queensland, Australia.
²¹ Laboratory of Neurogenetics of Language, The Rockefeller University, New York, NY, USA.
²² Howard Hughes Medical Institute, Chevy Chase, MD, USA.
²³ San Diego Zoo Global, Escondido, CA, USA.
²⁴ Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany.
²⁵ Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK.
²⁶ Pacific Biosciences, Menlo Park, CA, USA.
²⁷ Department of Evolution and Ecology, College of Biological Sciences, University of California, Davis, CA, USA.
²⁸ Department of Reproduction and Population Health, School of Veterinary Medicine, University of California, Davis, CA, USA.
²⁹ Research School of Biology, Australian National University, Canberra, Australian Capital Territory, Australia.
³⁰ Institute for Applied Ecology, University of Canberra, Canberra, Australian Capital Territory, Australia.
³¹ School of Life Sciences, La Trobe University, Melbourne, Victoria, Australia.
³² School of Biological Sciences, The Environment Institute, The University of Adelaide, Adelaide, South Australia, Australia. frank.grutzner@adelaide.edu.au.
³³ MOE Laboratory of Biosystems Homeostasis and Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China. zhouqi1982@zju.edu.cn.
³⁴ Department of Neuroscience and Developmental Biology, University of Vienna, Vienna, Austria. zhouqi1982@zju.edu.cn.
³⁵ Center for Reproductive Medicine, The 2nd Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China. zhouqi1982@zju.edu.cn.
³⁶ BGI-Shenzhen, Shenzhen, China. guojie.zhang@bio.ku.dk.
³⁷ Villum Center for Biodiversity Genomics, Section for Ecology and Evolution, Department of Biology, University of Copenhagen, Copenhagen, Denmark. guojie.zhang@bio.ku.dk.
³⁸ State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China. guojie.zhang@bio.ku.dk.
³⁹ Center for Excellence in Animal Evolution and Genetics, Chinese Academy of Sciences, Kunming, China. guojie.zhang@bio.ku.dk.

^# Contributed equally.

PMID: 33408411
PMCID: PMC8081666
DOI: 10.1038/s41586-020-03039-0

Platypus and echidna genomes reveal mammalian biology and evolution

Yang Zhou et al. Nature. 2021 Apr.

. 2021 Apr;592(7856):756-762.

doi: 10.1038/s41586-020-03039-0. Epub 2021 Jan 6.

Authors

Affiliations

¹ BGI-Shenzhen, Shenzhen, China.
² Villum Center for Biodiversity Genomics, Section for Ecology and Evolution, Department of Biology, University of Copenhagen, Copenhagen, Denmark.
³ School of Biological Sciences, The Environment Institute, The University of Adelaide, Adelaide, South Australia, Australia.
⁴ MOE Laboratory of Biosystems Homeostasis and Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China.
⁵ BGI Education Center, University of Chinese Academy of Sciences, Shenzhen, China.
⁶ Faculty of Environmental Earth Science, Hokkaido University, Sapporo, Japan.
⁷ Japan Monkey Centre, Inuyama, Japan.
⁸ School of BioSciences, The University of Melbourne, Melbourne, Victoria, Australia.
⁹ School of Life and Environmental Sciences, The University of Sydney, Sydney, New South Wales, Australia.
¹⁰ digzyme Inc, Tokyo, Japan.
¹¹ School of Life Science and Technology, Tokyo Institute of Technology, Tokyo, Japan.
¹² The Genome Center, University of California, Davis, CA, USA.
¹³ Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA.
¹⁴ Genome Informatics Section, Computational and Statistical Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA.
¹⁵ Tree of Life Programme, Wellcome Sanger Institute, Cambridge, UK.
¹⁶ The Vertebrate Genome Lab, The Rockefeller University, New York, NY, USA.
¹⁷ James D. Watson Institute of Genome Sciences, Hangzhou, China.
¹⁸ University of the Chinese Academy of Sciences, Beijing, China.
¹⁹ Guangdong Provincial Academician Workstation of BGI Synthetic Genomics, BGI-Shenzhen, Shenzhen, China.
²⁰ School of Agriculture and Food Sciences, The University of Queensland, Gatton, Queensland, Australia.
²¹ Laboratory of Neurogenetics of Language, The Rockefeller University, New York, NY, USA.
²² Howard Hughes Medical Institute, Chevy Chase, MD, USA.
²³ San Diego Zoo Global, Escondido, CA, USA.
²⁴ Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany.
²⁵ Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK.
²⁶ Pacific Biosciences, Menlo Park, CA, USA.
²⁷ Department of Evolution and Ecology, College of Biological Sciences, University of California, Davis, CA, USA.
²⁸ Department of Reproduction and Population Health, School of Veterinary Medicine, University of California, Davis, CA, USA.
²⁹ Research School of Biology, Australian National University, Canberra, Australian Capital Territory, Australia.
³⁰ Institute for Applied Ecology, University of Canberra, Canberra, Australian Capital Territory, Australia.
³¹ School of Life Sciences, La Trobe University, Melbourne, Victoria, Australia.
³² School of Biological Sciences, The Environment Institute, The University of Adelaide, Adelaide, South Australia, Australia. frank.grutzner@adelaide.edu.au.
³³ MOE Laboratory of Biosystems Homeostasis and Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China. zhouqi1982@zju.edu.cn.
³⁴ Department of Neuroscience and Developmental Biology, University of Vienna, Vienna, Austria. zhouqi1982@zju.edu.cn.
³⁵ Center for Reproductive Medicine, The 2nd Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China. zhouqi1982@zju.edu.cn.
³⁶ BGI-Shenzhen, Shenzhen, China. guojie.zhang@bio.ku.dk.
³⁷ Villum Center for Biodiversity Genomics, Section for Ecology and Evolution, Department of Biology, University of Copenhagen, Copenhagen, Denmark. guojie.zhang@bio.ku.dk.
³⁸ State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China. guojie.zhang@bio.ku.dk.
³⁹ Center for Excellence in Animal Evolution and Genetics, Chinese Academy of Sciences, Kunming, China. guojie.zhang@bio.ku.dk.

^# Contributed equally.

PMID: 33408411
PMCID: PMC8081666
DOI: 10.1038/s41586-020-03039-0

Abstract

Egg-laying mammals (monotremes) are the only extant mammalian outgroup to therians (marsupial and eutherian animals) and provide key insights into mammalian evolution^1,2. 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.

PubMed Disclaimer

Conflict of interest statement

J.K. is an employee of Pacific Biosciences, a company that develops single-molecule sequencing technologies.

Figures

**Fig. 1. Chromosome assembly of monotreme and mammalian genome evolution.**
a, The contig length distribution among the three monotreme assemblies shows a large improvement in the sequence continuity of the platypus assembly, and at least equivalent quality of the echidna assembly. b, Mammalian karyotype evolution trajectory. 2n = 60 ancestral karyotypes were inferred for the common ancestor of mammals. Conserved blocks were colour-coded in accordance with their chromosomal source in the mammalian ancestor. Numbers of estimated rearrangements are shown for each branch. Silhouettes of the human and opossum are from https://www.flaticon.com/. Silhouettes of the platypus and Tasmanian devil are created by S. Werning and are reproduced under the Creative Commons Attribution 3.0 Unported licence (http://creativecommons.org/licenses/by/3.0/).

**Fig. 2. Origin and evolution of the sex chromosomes of the platypus.**
a, Genomic composition of the platypus sex chromosomes. From the outer to inner rings: the X chromosomes with the PARs (light colours) and SDRs (dark colours) labelled; the assembled Y chromosome fragments within SDRs showing the colour-scaled sequence divergence levels with the homologous X chromosomes; female-to-male (F/M) ratios of short sequencing-read coverage in non-overlapping 5-kb windows; F/M expression ratios (each red dot is one gene) of the adult kidney and the smoothed expression trend; and GC content in non-overlapping 2-kb windows. In addition, we labelled the positions on the X chromosome ring of the gametologue pairs that have suppressed recombination before the divergence of monotremes (‘shared’, orange triangles) or after the divergence (‘independent’, blue triangles). b, Homology between X and Y chromosomes of platypus. In particular, most of Y5 shows homology with X1 and X2, which suggests an ancestral ring conformation of the platypus sex chromosomes. We also labelled the position of the putative sex-determining gene *AMH*. The platypus silhouette is created by S. Werning and is reproduced under the Creative Commons Attribution 3.0 Unported licence (http://creativecommons.org/licenses/by/3.0/).

**Fig. 3. Interactions between the platypus sex chromosomes.**
a, Interchromosomal interactions among the platypus sex chromosomes detected by Hi-C data of liver tissue in platypus (top) and human (bottom). The bars between the Hi-C panels show the platypus sex chromosomes and their orthologues in the human genome. Grey, intrachromosomal interactions; red, interchromosomal interactions. Red lines link the regions with significantly high interchromosomal interactions. The interchromosomal interactions seem to be conserved in mammals, as indicated by the homologous chromosomal fragments of the human and platypus sex chromosomes and their Hi-C contact patterns. b, FISH with BAC probes to detect sex chromosomes Y2, Y3 or X1 and autosome chromosome17 (*WSB1*) in interphase platypus fibroblasts. Examples show no interaction between chromosomes Y2 and Y3 (top, n = 593, 3 independent experiments) and interaction (bottom, n = 56, 3 independent experiments). Scale bars, 10 μm. c, The significantly higher frequency of interaction between Y2 and Y3 than that between Y2 and X1, and between Y2 and *WSB1* (chromosome 17). n = 185, 206, 258 cells for the three independent replicate experiments of Y2–Y3, n = 258, 250, 205 cells for the three independent replicate experiments of Y2–X1, n = 298, 262, 220 cells for the three independent replicate experiments of Y2–*WSB1*. Data are mean ± s.d. ***P < 0.001 (Y2–Y3 versus Y2–X1, P = 0.0004675; Y2–Y3 versus Y2–*WSB1*, P = 6.376 × 10⁻⁵), one-sided Fisher’s exact test. d, Putative CTCF-binding-site density plot showing its enrichment among homologous regions in the platypus, human and chicken genomes.

**Fig. 4. Genomic features related to biological characteristics of the monotremes.**
a, Differences in numbers of TAS2R, OR and V1R genes between platypus and echidna. b, Phylogeny and synteny of the HP gene. Regions are not drawn to scale. c, Synteny conservation of the region surrounding caseins (CSN genes) and the ancestral teeth genes (*ODAM*, *FDCSP*, *AMTN*, *AMBN* and *ENAM*). Silhouettes of the human, opossum, koala and frog are from https://www.flaticon.com/. Silhouettes of the platypus and Tasmanian devil are created by S. Werning and the emu silhouette is created by D. Naish (vectorized by T. M. Keesey); all three silhouettes are reproduced under the Creative Commons Attribution 3.0 Unported licence (http://creativecommons.org/licenses/by/3.0/).

**Extended Data Fig. 1. Platypus genome assembly and evaluation.**
a, b, Hi-C two-dimensional juicebox maps of mOrnAna1 before (a) and after (b) manual assembly curation. The grey lines depict scaffold boundaries. The off-diagonal matches between scaffolds indicate potential missed joins, whereas ‘empty’ areas within scaffold boundaries indicate misjoins. The gEVAL-supported manual assembly curation led to a notably improved arrangement with >96% of the assembly sequence inside chromosome-scale scaffolds. c, d, The Super_Scaffold_40 was misassigned to chromosome 15 in OANA5 but FISH on metaphase spreads from platypus fibroblasts map it to chromosome 13. c, Co-hybridization of the BAC of chromosome 15 (green, top arrow) and Super_Scaffold_40 probe (red, bottom arrow) showing an absence of co-localization (14 nuclei scored, 2 independent experiments). Inset, interphase example. d, Co-hybridization of the BAC of chromosome 13 (green) and Super_Scaffold_40 probe (red) showing co-localization (arrows, 40 nuclei scored, 5 independent experiments). Scale bars, 10 μm. e, An example of scaffold chromosome misassignment in OANA5. Female-to-male (F/M) depth ratio, normalized female depth and normalized male depth along OANA5 chromosome 14 in 5-kb non-overlapping windows. Depth ratio, normalized female depth and normalized male depth all suggest that OANA5 chromosome 14 should be an X-borne rather than autosomal sequence. f, g, Normalized depth distribution of redundant sequences and one-to-one sequences in male (f) and female (g). Redundant sequences (red) in OANA5 are probably assembly artefacts due to heterozygotes of the sequenced individual of OANA5, and are therefore featured with 0.5× normalized depth in OANA5 but 1× normalized depth in mOrnAna1 in both male and female. One-to-one sequences in OANA5 (black) have 1× normalized depth in both OANA5 and mOrnAna1 in reads that are mapped from both sexes as expected. Each dot represents one mapping region between OANA5 and mOrnAna1 by Mashmap, and the normalized depth values of each dot are calculated as the mean depth across the mapping region in OANA5 and mOrnAna1. The small peak in one-to-one sequence density plot in the male indicates candidate X-linked sequences. h, Example redundant sequences Contig40802, Contig44497 and Contig35847 in OANA5 that could be interpreted as false duplications. Dot plot is generated between the target region of mOrnAna1 chromosome 1 and OANA5 Contig1255, Contig40802, Contig44497 and Contig35847 by FlexiDot. Candidate redundant sequences are those mapped to the same region in mOrnAna1 chromosome 1, highlighted by dashed lines in the dot plot and grey in the normalized depth plot. Normalized male and female read depths along each sequence are calculated in 500-bp windows, and plotted along each sequence. Although the normalized depth is always around 1 in the region of mOrnAna1 chromosome 1, normalized depth drops half in Contig40802, Contig44497, Contig35847 and the aligned regions in Contig1255, indicating that Contig40802, Contig44497 and Contig35847 are probably redundant sequences in OANA5. i, j, Examples of gene annotation artefacts in OANA5: *CIT* (i) and *PBRM1* (j) have been fragmented into multiple small artificial genes in OANA5 (purple) but have now been fully recovered in mOrnAna1 (orange). Orthologous human genes (grey) are also shown to indicate that the mOrnAna1 rather than OANA5 annotation has a similar gene structure to that of the human genes.

**Extended Data Fig. 2. Mammalian genome evolution.**
a, Phylogenetic tree constructed using fourfold degenerate sites from 7,946 one-to-one orthologues among seven representative species (human, mouse, opossum, platypus, echidna, chicken and green lizard). The fossil time calibration of the nodes marked by circles were obtained from a previously published study. The numbers of gene families that have undergone significant (Viterbi P < 0.05) lineage-specific expansions (green) and contractions (red) are marked on each branch. Exact P values are available in Supplementary Table 29. No multiple-testing correction was applied. b, Examples of some imprinting gene clusters improved in mOrnAna1 compared to OANA5. The first line of each synteny plot represents mOrnAna1 and the second line represents OANA5. Names of genes that have been found to be imprinted in human and mouse are highlighted in black, and non-imprinting genes in red. Fragmented genes with alignment rate lower than 70% are marked by triangles. The double slash represents the intermediate region longer than 100 kb. c, Distribution of MSHCEs on genomic elements. d, Enriched GO terms in the top-300 MSHCE-associated genes. P values of enrichment are calculated using a χ² test, and FDRs are computed to adjust for multiple testing. GO terms are clustered based on semantic similarity. GO terms related to nervous system development are highlighted in bold. e, A case of one MSHCE in *BCL11A* that overlaps with the enhancer signals inferred from H3K27ac ChIP-seq experiments at 8.5 and 12 weeks after conception (p.c.w.). f, Evolution highway comparative chromosome browser visualization of reconstructed MACs at a 500-kb resolution. Blocks overlaid on each MAC represent human syntenic fragments. Numbers within blocks indicate the homologous human chromosome. g, Evolution highway comparative chromosome browser visualization of the human genome at a 500-kb resolution, with each block overlaid on each human chromosome representing putative chromosome fragments of the ancestral mammalian genome. Numbers within blocks depict the ancestral mammal chromosome numbers. Silhouettes of the human and opossum are from https://www.flaticon.com/. The silhouette of the platypus is created by S. Werning and is reproduced under a Creative Commons Attribution 3.0 Unported licence (http://creativecommons.org/licenses/by/3.0/).

**Extended Data Fig. 3. Evolution of immune gene family in monotremes.**
a, MHC genes in platypus and echidna are located on two different chromosomes, but the classical class I and II genes involved in antigen presentation are located within a single cluster in each genome. b, Phylogenetic relationship of class I genes in representative mammals and chicken. Classical class I genes (red) in monotremes exhibit high similarity, which is rarely observed in other species. Only bootstrap values with >50% support are shown. c, d, Phylogenetic relationship of MHC class II alpha (c) and beta (d) genes. Genes with prefix ‘HLA’, ‘Modo’, ‘Phci’, ‘Oran’, ‘Taac’ and ‘Gaga’ indicate genes in human, opossum, koala, platypus, echidna and chicken, respectively. Only bootstrap values with >50% support are shown. e, Phylogenetic relationship among putative functional Vγ sequences from platypus (yellow), echidna (purple), koala (green), mouse (orange), human (red), sheep (grey), cow (dark red) and chicken (dark yellow). Groups according to a previous study are displayed around the outside of the tree, with the putative marsupial–monotreme-specific group denoted by a ‘?’. Only bootstrap values with greater than 50% support are shown. f, Synteny conservation of beta-defensin genes in monotremes and loss of functional venom defensins in echidna. Venom defensins (OavDLP genes) and venom-like defensin (DEFB-VL genes) are shown in red. Only putative functional defensins are shown. g, Putative OavDLP loss in echidna. OavDLP genes and *DEFB-VL* each contain two exons (indicated by a box and triangle) in platypus. Both exons of platypus *DEFB-VL* can be mapped to echidna chromosome X2. A single platypus OavDLP exon can be mapped to echidna chromosome X2 while the second exons cannot. Grey links indicate platypus–echidna LASTZ alignment. h, Phylogenetic relationship of *DEFB-VL* and OavDLP genes suggested that ancestral monotremes had all three OavDLP genes but that echidna has lost the two of them (*OavDLP-B* and *OavDLP-C*). Branch length is not shown. ta, echidna; oa, platypus. Silhouettes of the human, opossum, koala and frog are from https://www.flaticon.com/. The silhouette of the platypus is created by S. Werning and is reproduced under a Creative Commons Attribution 3.0 Unported licence (http://creativecommons.org/licenses/by/3.0/).

**Extended Data Fig. 4. Genomic composition of monotreme sex chromosomes.**
a, Composition of the echidna sex chromosomes. The circos plot (from outer to inner rings) shows: X chromosomes with PARs shown as light colours and SDRs as dark colours; assembled Y chromosome fragments showing the colour-scaled sequence similarity levels with homologous X chromosomes; normalized F/M ratios of Illumina DNA-sequencing depth in non-overlapping 5-kb windows; F/M expression ratios (each red dot is one gene) of adult kidney and smoothed expression ratio trend; and GC content in non-overlapping 2-kb windows. In addition, Y-linked fragments with a similar level of sequence divergence from the X chromosome indicate a pattern of evolutionary strata. As expected, F/M DNA depth ratio is centred at 1 at PARs, but is around 2 at SDRs. Some PARs show significantly higher GC content than the regions that suppressed recombination between X and Y. b, Partial dosage compensation in monotremes. The four point range plots show log₂-transformed values of the male-to-female (M/F) expression ratio in the brain, kidney, heart and liver of platypus and echidna. As expected, log₂-transformed values of the M/F expression ratio is close to 0 for genes on autosomes (A) and PARs, whereas for genes on SDRs, the expression is female-biased in all tissues, which suggests that monotremes have partial dosage compensation. Whiskers indicate the 25– 75th percentiles and circles are the median value. c, Some PARs show significantly higher GC content than SDRs. For platypus, some PARs (X2-PAR-S, X3-PAR-S, X4-PAR-L, X5-PAR-S and X5-PAR-L (where -S is the shorter PAR of the chromosome and -L the longer PAR)) show significantly (P < 0.01) higher GC content (1-kb non-overlapping windows) than the SDRs of the same chromosome, which are labelled as asterisks in the heat map. We also checked their orthologous sequences in chicken, as a proxy for the ancestral status before the chromosome became a sex chromosome, and found similarly higher GC content in the orthologous region of PARs than those of SDRs in chicken. ***P < 0.01 (all P < 2.2 × 10⁻¹⁶), one-sided Wilcoxon rank-sum test. d, Atlas of orthologous chicken fragments along each platypus sex chromosome. The PARs between the platypus X and Y chromosomes are indicated by crosses. We also labelled the position of the putative sex-determining gene *AMH*.

**Extended Data Fig. 5. Evolution of PARs after the platypus and echidna divergence.**
a, The distribution of pairwise dS values of platypus and echidna sex chromosomes. In both platypus and echidna, gametologue pairs in the X1 S0 region (Fig. 2), which is largely homologous to chicken chromosome 28, have a higher dS value than those of any other sex-linked regions. This suggests that X1 S0 is the oldest evolutionary stratum. Therefore, we also show platypus genes of X2 with an orthologue on chicken chromosome 28 separately from others (X1_S0_chr28). Following the order of dS values of different chromosome regions, we inferred the time order of formation of evolutionary strata, called S0–S6. For platypus, n = 5, 5, 2, 2, 1, 1, 4, 2 and 6 XY gametologue pairs are plotted, from left to right. For echidna, n = 7, 2, 1, 1, 4, 2 and 1 XY gametologue pairs are plotted, from left to right. Box plots show median, quartiles (boxes) and range (whiskers). b, Phylogenetic tree examples of gametologues that evolved in the common ancestor of monotremes (*EF2* in X2) and independently in two monotreme species (*IRF4* in X3). c, Alignments of platypus and echidna X chromosomes (PAR, light colours; SDR, dark colours; the top chromosomes are from platypus and the bottom chromosomes are from echidna) were used to infer X2-PAR-S and X5-PAR-L of platypus evolved independently from echidna after their divergence, given their different lengths. This is supported by the Venn diagrams of PAR genes between platypus and echidna, in which most genes are not shared within independently evolved PARs. d, Alignments of PAR–SDR boundaries between platypus and echidna. Alignments of genes (±1 Mb around the boundaries) support independent evolution of X2-PAR-S and X5-PAR-L in platypus and echidna, as most of their genes are not homologous at the PAR–SDR boundaries (blue, PAR genes; red, SDR genes; platypus, top chromosome, echidna, bottom chromosome). We used lines to connect the genes of the two species, whenever they are orthologous to each other. For each X chromosome, we also labelled their repeat information. Six repeat tracks between each X pair are shown, from top to bottom: the overall repeat content of platypus; LINE/L2 elements of platypus; SINE/MIR elements of platypus; SINE/MIR elements of echidna; LINE/L2 elements of echidna and overall repeat content of echidna. We did not find obvious repeat enrichment at PAR–SDR boundaries, as shown previously in cow.

**Extended Data Fig. 6. Sex chromosome evolution in monotremes.**
a, Mummerplot showing homology between platypus (x axis) and echidna (y axis) X chromosomes. Blue lines: forward alignment; red lines: reverse alignment. For echidna, X1, X2 and X3 are homologous to platypus X1, X2 and X3, respectively. Echidna X4 is homologous to platypus X5. And for echidna X5, it is not homologous to any platypus sex chromosome, and instead it is homologous to platypus chromosome 12. b, Homology between platypus X chromosomes (x axis) and human chromosomes (y axis). c, Homologous relationships between platypus sex chromosomes and chicken. d, Alignment between platypus and chicken showing the alternating pairing pattern of the platypus sex chromosome chain. e, X/Y pairwise dS comparison between gametologues on X1–Y5 pair (n = 18) and other sex chromosome pairs (n = 10). Box plots show median, quartiles (boxes) and range (whiskers). ***P < 0.001 (P = 0.0002954), one-sided Wilcoxon rank-sum test.

**Extended Data Fig. 7. Chromatin conformation of monotreme sex chromosomes.**
a, Hi-C interactions between platypus sex chromosomes, with chromosome 1 shown as control. a, b, There are unexpected interchromosomal interactions (shown in red) between platypus sex chromosomes detected by Hi-C data (a), whereas most interactions are within the same chromosomes (shown in red in b) for the other chromosomes (b). c, The Hi-C interchromosomal interactions among platypus sex chromosome (inter_XY, n = 2,711 100-kb windows) is significantly higher than that among autosomes (inter_A, n = 14,342,930 100-kb window). Box plots show median, quartiles (boxes) and range (whiskers). ***P < 0.0001 (P < 2.2 × 10⁻¹⁶), one-sided Wilcoxon rank-sum test. d, The interaction strength is higher between Y2 and Y3 than the interaction strengths between Y2 and other chromosomes. n = 1,002, 228, 5,025, 67,313 and 6,904,867 100-kb windows are shown in Y2-Y2, Y2-Y3, Y2-other.sex.chr, Y2-A and A-A, respectively. Box plots show median, quartiles (boxes) and range (whiskers). ***P < 0.0001 (P < 2.2 × 10⁻¹⁶), one-sided Wilcoxon rank-sum test. e, Inferred three-dimensional structure of the platypus sex chromosome system. X chromosomes are shown in red and Y chromosomes in blue, with PARs in light colour. Interchromosomal interactions inferred from Hi-C are shown by dashed lines. f, Hi-C interactions reveal unexpected interchromosomal interactions between the echidna sex chromosomes. g, Putative CTCF-binding sites are enriched at TAD boundaries in platypus and echidna sex chromosomes. For each X chromosome of platypus, we calculated their putative CTCF-binding-site density per 10 kb and plotted them along the ±500 kb of TAD boundaries. Platypus X4 and echidna X5 are not shown because less than 10 TAD boundaries are detected. h, Putative CTCF-binding-site density plot showing its enrichment among the homologous regions of platypus, echidna, human and chicken.

**Extended Data Fig. 8. Loss of dietary-related genes in monotremes.**
a, Tooth-related gene loss in representative mammals and reptiles. b–f, Potential loss of digestion-related genes in both monotremes shown by whole-genome alignment and read mapping. In each panel there are three lines in the synteny plot, representing the orthologous region of the genes in platypus, human and echidna from top to bottom, respectively. Grey links indicate human–platypus and human–echidna LASTZ alignments. Each rectangle or triangle represents an exon. Fragmented genes are marked by dashed lines. Illumina reads of platypus and echidna are aligned to the platypus or human genome (Ensembl release 87) and the flanking region of each gene is visualized by pyGenomicTrack. *GAPDH* region is also plotted as a control. g, RT–PCR expression analysis shows expression of *NGN3* in brain, stomach, intestine and pancreas of both platypus and echidna. These results are similar to other mammals. This, together with sequencing results, shows that *NGN3* in monotremes is present and is likely to be functioning normally. NGN3, NGN3 primers; b-actin, β-actin primers; -ve, negative control, no template; gDNA, genomic DNA template; brain, brain cDNA template; stom, stomach cDNA template; int, intestine cDNA template; panc, pancreas cDNA template. Lanes 1 (top), 1, 8 (middle) and 9 (bottom) are a 100-bp DNA ladder: 1,517, 1,200, 1,000, 900, 800, 700, 600, 500/517, 400, 300, 200 and 100 bp. Expected sizes of PCR products for *NGN3* in platypus is 157 bp and for echidna 145 bp, and the PCR product for the β-actin genomic region is 597 bp and cDNA is 348 bp. Silhouettes of human and opossum are from https://www.flaticon.com/. The silhouette of the platypus is created by S. Werning and is reproduced under a Creative Commons Attribution 3.0 Unported licence (http://creativecommons.org/licenses/by/3.0/).

**Extended Data Fig. 9. Taste-receptor evolution and olfactory-receptor organization in monotremes.**
a, Maximum-likelihood mammalian-wide gene tree of the bitter taste receptors (TAS2R genes). There are 28 eutherian (Eu), 27 marsupial (Ma) and 7 monotreme-specific (Mo) orthologous gene groups (supported by ≥95% bootstrap values), where the nodes of orthologous gene group clades are indicated by white open circles. Bootstrap values of ≥70% in the nodes connecting orthologous gene group clades are indicated by asterisks. There are 3 therian (I, II and III), 2 eutherian (I and II), 3 marsupial (I, II and III) and one monotreme-specific clusters in which massive expansion events occurred in the common ancestor of each taxon after the split from its previous ancestors. b, Genomic organization of the intact class I olfactory receptor (OR) cluster spanning over 1.2 Mb on platypus chromosome 2 (138,375,798–139,616,970 bp). The vertical lines indicate the 48 intact class I OR genes. The white open box indicates the J element, a presumable *cis*-regulatory element (enhancer) for the mammalian class I OR cluster (chromosome 2: 139,639,465–139,639,907 bp). Silhouettes of human, opossum and koala are from https://www.flaticon.com/. Silhouettes of the platypus and Tasmanian devil are created by S. Werning and are reproduced under a Creative Commons Attribution 3.0 Unported licence (http://creativecommons.org/licenses/by/3.0/).

**Extended Data Fig. 10. Genomic features related to haemoglobin clearance and reproduction in monotremes.**
a, b, Confirmation of HP absence in monotremes by whole-genome alignment (a) and read mapping (b). Grey links indicate human–platypus and human–echidna LASTZ alignments. Illumina reads of platypus and echidna are aligned to the human genome (Ensembl release 87) and coding regions of HP are visualized by pyGenomicTrack. Limited coverage is found at the exons of HP, suggesting the absence of HP in monotremes. c, Phylogenetic tree of HP and related proteases across different species using the maximum-likelihood method. Node IDs are in format of ‘species geneID’. Branch length is not shown here. d, Gene synteny plot of the *PIT54* region between chicken and platypus. Echidna is not shown in the figure as the flanking orthologues of *PIT54* are on different scaffolds, preventing us from determining the presence of the gene by synteny. e, Phylogenetic tree of members of the group B scavenger receptor cysteine-rich family across different species using the neighbour-joining method. Gene IDs are formatted as ‘species geneID’. Branch length is not shown here. f, Confirmation of *SCART1* number difference by dot plot and mapping depth of *SCART1* orthologous regions between platypus and echidna. The region of the *SCART1* cluster in platypus is plotted along the x axis while the sequence of echidna is plotted along the y axis. Lines in dot plot are visualized according to LASTZ alignment between the two species. Normalized male and female read depths along each sequence is calculated in 500-bp windows, and plotted along each sequence. Normalized depth of both sexes, especially those in the shading region, is centred at 1 along both species, confirming the *SCART1* number difference between the two species is true and is not due to assembly issues. g, Synteny conservation of vitellogenin genes. Synteny conservation of the region surrounding the vitellogenin (*VTG*) genes *VTG1*, *VTG2* and *VTG3*. Pseudogenes are marked by a dashed outline. Monotremes have pseudogene *VTG1*, functional *VTG2* and no *VTG3*; and there is a pseudogene *VTG2* in koala. Syntenic maps are shown for human (*Homo sapiens*), koala (*Phascolarctos cinereus*), chicken (*Gallus gallus*), platypus (*O. anatinus*) and echidna (*T. aculeatus*). Koala scaffold 1, NW_018343984.1; koala scaffold 2, NW_018344134.1. Gene distances are not to scale. h, Synteny conservation of regions containing *SPINT3*. Synteny conservation of the region surrounding serine peptidase inhibitor, Kunitz-type, 3 (*SPINT3*). No copy of *SPINT3* is detected in platypus but many of the other flanking genes in the region are conserved. Other members with a WFDC domain are detected including two Kunitz-domain members that did not align to any known gene (labelled *KDCP1*). Syntenic maps are reported for human (*H. sapiens*), cow (*B. taurus*), grey short-tailed opossum (*Monodelphis domestica*), koala (*P. cinereus*) and platypus (*O. anatinus*). Koala scaffold 1, NW_018343967.1; koala scaffold 2, NW_018344098.1. Gene distances are not to scale. i, Casein 3 (CSN3) protein sequence alignment in monotremes. All three CSN3 proteins identified in the monotremes have the classic five-exon structure of CSN3 with the untranslated exons I and IV (not shown), the signal peptide in exon II, a small exon III coding for 11 residues, a pSER cluster (S**) at the 5′ end of exon IV and a relatively large P/Q-rich exon IV. OA, *O. anatinus* (platypus); TA, *T. aculeatus* (short-beaked echidna). Silhouettes of human, opossum and koala are from https://www.flaticon.com/. The silhouette of the platypus is created by S. Werning and is reproduced under a Creative Commons Attribution 3.0 Unported licence (http://creativecommons.org/licenses/by/3.0/).

See this image and copyright information in PMC

Cited by

Monotreme-specific conserved putative proteins derived from retroviral reverse transcriptase.
Kitao K, Miyazawa T, Nakagawa S. Kitao K, et al. Virus Evol. 2022 Sep 3;8(2):veac084. doi: 10.1093/ve/veac084. eCollection 2022. Virus Evol. 2022. PMID: 36176487 Free PMC article.
A brief review of vertebrate sex evolution with a pledge for integrative research: towards 'sexomics'.
Stöck M, Kratochvíl L, Kuhl H, Rovatsos M, Evans BJ, Suh A, Valenzuela N, Veyrunes F, Zhou Q, Gamble T, Capel B, Schartl M, Guiguen Y. Stöck M, et al. Philos Trans R Soc Lond B Biol Sci. 2021 Aug 30;376(1832):20200426. doi: 10.1098/rstb.2020.0426. Epub 2021 Jul 12. Philos Trans R Soc Lond B Biol Sci. 2021. PMID: 34247497 Free PMC article. Review.
A family of olfactory receptors uniquely expanded in marsupial and monotreme genomes are expressed by a T cell subset also unique to marsupials and monotremes.
Sampson JM, Morrissey KA, Douek DC, Miller RD. Sampson JM, et al. Dev Comp Immunol. 2024 May;154:105149. doi: 10.1016/j.dci.2024.105149. Epub 2024 Feb 8. Dev Comp Immunol. 2024. PMID: 38340883 Free PMC article.
Functional Diversity and Evolution of Bitter Taste Receptors in Egg-Laying Mammals.
Itoigawa A, Hayakawa T, Zhou Y, Manning AD, Zhang G, Grutzner F, Imai H. Itoigawa A, et al. Mol Biol Evol. 2022 Jun 2;39(6):msac107. doi: 10.1093/molbev/msac107. Mol Biol Evol. 2022. PMID: 35652727 Free PMC article.
Dynamic Evolution of Retroviral Envelope Genes in Egg-Laying Mammalian Genomes.
Kitao K, Shoji H, Miyazawa T, Nakagawa S. Kitao K, et al. Mol Biol Evol. 2023 May 2;40(5):msad090. doi: 10.1093/molbev/msad090. Mol Biol Evol. 2023. PMID: 37062963 Free PMC article.

See all "Cited by" articles

References

1. Ashwell, K. Neurobiology of Monotremes: Brain Evolution in Our Distant Mammalian Cousins (CSIRO PUBLISHING, 2013).
1. Warren WC, et al. Genome analysis of the platypus reveals unique signatures of evolution. Nature. 2008;453:175–183. - PMC - PubMed
1. Grützner F, et al. In the platypus a meiotic chain of ten sex chromosomes shares genes with the bird Z and mammal X chromosomes. Nature. 2004;432:913–917. - PubMed
1. Kortschak RD, Tsend-Ayush E, Grützner F. Analysis of SINE and LINE repeat content of Y chromosomes in the platypus, Ornithorhynchus anatinus. Reprod. Fertil. Dev. 2009;21:964–975. - PubMed
1. Boissinot S, Sookdeo A. The evolution of LINE-1 in vertebrates. Genome Biol. Evol. 2016;8:3485–3507. - PMC - PubMed

Publication types

Actions

MeSH terms

Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions

Grants and funding

LinkOut - more resources

Full Text Sources
Other Literature Sources
- scite Smart Citations

[1] Ashwell, K. Neurobiology of Monotremes: Brain Evolution in Our Distant Mammalian Cousins (CSIRO PUBLISHING, 2013).

[2] Ashwell, K. Neurobiology of Monotremes: Brain Evolution in Our Distant Mammalian Cousins (CSIRO PUBLISHING, 2013).

[3] Warren WC, et al. Genome analysis of the platypus reveals unique signatures of evolution. Nature. 2008;453:175–183. - PMC - PubMed

[4] Warren WC, et al. Genome analysis of the platypus reveals unique signatures of evolution. Nature. 2008;453:175–183. - PMC - PubMed

[5] Grützner F, et al. In the platypus a meiotic chain of ten sex chromosomes shares genes with the bird Z and mammal X chromosomes. Nature. 2004;432:913–917. - PubMed

[6] Grützner F, et al. In the platypus a meiotic chain of ten sex chromosomes shares genes with the bird Z and mammal X chromosomes. Nature. 2004;432:913–917. - PubMed

[7] Kortschak RD, Tsend-Ayush E, Grützner F. Analysis of SINE and LINE repeat content of Y chromosomes in the platypus, Ornithorhynchus anatinus. Reprod. Fertil. Dev. 2009;21:964–975. - PubMed

[8] Kortschak RD, Tsend-Ayush E, Grützner F. Analysis of SINE and LINE repeat content of Y chromosomes in the platypus, Ornithorhynchus anatinus. Reprod. Fertil. Dev. 2009;21:964–975. - PubMed

[9] Boissinot S, Sookdeo A. The evolution of LINE-1 in vertebrates. Genome Biol. Evol. 2016;8:3485–3507. - PMC - PubMed

[10] Boissinot S, Sookdeo A. The evolution of LINE-1 in vertebrates. Genome Biol. Evol. 2016;8:3485–3507. - PMC - PubMed

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

Platypus and echidna genomes reveal mammalian biology and evolution

Affiliations

Platypus and echidna genomes reveal mammalian biology and evolution

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

Similar articles

Cited by

References

Publication types

MeSH terms

Grants and funding

LinkOut - more resources

Full Text Sources

Other Literature Sources

Abstract

Conflict of interest statement

Figures

Similar articles

Cited by

References

Publication types

MeSH terms

Related information

Grants and funding

LinkOut - more resources

Full Text Sources

Other Literature Sources