Origins and functional evolution of Y chromosomes across mammals

Abstract

Y chromosomes underlie sex determination in mammals, but their repeat-rich nature has hampered sequencing and associated evolutionary studies. Here we trace Y evolution across 15 representative mammals on the basis of high-throughput genome and transcriptome sequencing. We uncover three independent sex chromosome originations in mammals and birds (the outgroup). The original placental and marsupial (therian) Y, containing the sex-determining gene SRY, emerged in the therian ancestor approximately 180 million years ago, in parallel with the first of five monotreme Y chromosomes, carrying the probable sex-determining gene AMH. The avian W chromosome arose approximately 140 million years ago in the bird ancestor. The small Y/W gene repertoires, enriched in regulatory functions, were rapidly defined following stratification (recombination arrest) and erosion events and have remained considerably stable. Despite expression decreases in therians, Y/W genes show notable conservation of proto-sex chromosome expression patterns, although various Y genes evolved testis-specificities through differential regulatory decay. Thus, although some genes evolved novel functions through spatial/temporal expression shifts, most Y genes probably endured, at least initially, because of dosage constraints.

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Figure 1: Y (W) protein-coding gene repertoires and their origins.
Figure 2: Expression level evolution on amniote sex chromosomes.
Figure 3: Spatial expression pattern evolution on amniote sex chromosomes.

Accession codes

Primary accessions

Gene Expression Omnibus

Sequence Read Archive

Data deposits

RNA and DNA sequencing data as well as reconstructed Y/W sequences have been deposited in the Gene Expression Omnibus (GEO), Sequence Read Archive (SRA) and Transcriptome Shotgun Assembly (TSA) Database under the accession codes GSE50747, SRP029216, SRP026469 and PRJNA236159.

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Acknowledgements

We thank K. Harshman and the Lausanne Genomics Technology Facility for high-throughput sequencing support; I. Xenarios and the Vital-IT computational facility for computational support; S. Pääbo for great ape DNA samples; C. Roos for marmoset DNA samples; P. Jensen for chicken samples; E. Tsend-Ayush for help in determining the complete sequence of the AMHY gene in platypus; P. Gonzalez-Rubio for help with figure designs; M. Cardoso-Moreira, F. Carelli, A. Necsulea and M. Warnefors for comments on the manuscript; the Kaessmann group in general for discussions; and the Marmoset Genome Sequencing Consortium for making the marmoset genome assembly and annotation data available and for granting permission to use them for the analyses described in this study before publication. D.T.F. was supported by the Mexican National Council for Science and Technology (CONACyT). P.D.W. was supported by an ARC fellowship. F.G. was supported by an ARC fellowship. This research was supported by grants from the European Research Council (Starting Independent Grant: 242597, SexGenTransEvolution) and the Swiss National Science Foundation (Grant: 130287) to H.K.

Author information

Affiliations

Authors

Contributions

D.C. performed most data processing and biological analyses. R.M. processed platypus genomic data and assessed X identity of contigs and the most likely chromosomal location of X gametologues in this species. D.T.-F. performed FISH experiments in platypus. L.F. and A.L. prepared samples and generated RNA-seq and genomic sequencing libraries. L.F. performed the large-scale PCR/Sanger sequencing validation experiments. P.D.W. provided elephant and tammar wallaby fibroblast samples and advised on these species’ sex chromosome biology. F.G. provided platypus and echidna samples, supervised FISH experiments, and provided advice on the sex chromosome biology of these species. The project was supervised and originally designed by H.K. The paper was written by D.C. and H.K. with input from all authors.

Corresponding authors

Correspondence to Diego Cortez or Henrik Kaessmann.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Overview of subtraction approach to detect and assemble Y (W) chromosome genes/transcripts.

Extended Data Figure 2 Resampling simulations to evaluate the performance of the Y transcript reconstruction approach.

ad, Results of one simulation showing the completeness (length of reconstructed sequences with respect to annotated sequences at ≥ 99% identity) of Y protein-coding transcripts using increasing numbers of randomly sampled male reads (million reads, Mr) when performing reconstructions. Simulations were carried out for human, chimpanzee, macaque and mouse where Y chromosomes have been fully sequenced. Twenty randomly selected genes from chicken were included in the simulations to control for false positive reconstructions (the median of the completeness of these control genes is zero). e, Distribution of the completeness of Y transcripts for repeated resampling analyses for different numbers of randomly sampled male reads (50 resampling replicates for each read number). Error bars, maximum and minimum values, excluding outliers.

Extended Data Figure 3 Y (W) genes previously described in the literature.

See Fig. 1 for detailed legend. Numbers indicate the references in which Y and W genes are described.

Extended Data Figure 4 Phylogenetic trees constructed on the basis of alignments of concatenated genes for a given stratum.

ag, All trees are based on coding nucleotide sequences, except for therian S1 and marsupials S1 trees, which are based on an amino acid alignment, given that coding sequences are highly diverged, leading to a tree that is less well supported at key nodes. Bootstrap values are based on 100 bootstrap replicates. In the specific case of therian S1 and eutherian S3, we included all different gene copies (where these were available from previous work or could be reconstructed) in the analyses for the multi-copy genes (RBMY, RPS4Y, HSFY, Cyorf15) by repeatedly (100 times) and randomly selecting gene copies for the concatenations, and then reconstructing phylogenetic trees (100 bootstrap replicates). Reported bootstrap values at each node thus represent the median value for 100 trees obtained with different combinations of multi-copy sequences. Trees for individual genes are available at ftp://ftp.vital-it.ch/papers/kaessmann/Nature-Cortez/Cortez_etal_Nature_YX_gene_trees_alignments.zip.

Extended Data Figure 5 Strata ages and synonymous substitution rate evolution.

af, Phylogenetic trees based on synonymous site divergences (dS) of concatenated Y (W) genes for different strata; key branch lengths (dS and inferred corresponding millions of years, Myr) as well as divergence times and inferred strata ages (in green) are indicated. Branch lengths and corresponding age ranges are given as 95% confident intervals calculated with simulated data (Methods). The following genes were used in underlying concatenated alignments (other genes were lacking orthologues in some species and were thus not included): SRY, RBMY and RPS4Y (total alignment length: 2,607 nucleotides (nt)) for therian S1; KDM5D (4,794 nt) for eutherian S2; KDM5D, HCHC1Y, MECP2Y, UBE1Y1 and HUWE1Y (24,657 nt) for marsupial S2; USP9Y, UTY and DDX3Y (12,408 nt) for eutherian S3; AMHY, FEM1CY, AKAP8LY and MED26Y (5,469 nt) for monotreme S1; HNRNPKW and KCMF1W (2,268 nt) for bird S1. g, dS values for the first (most basal) branches that follow different stratification events and lead to Y and X clades, respectively. Statistically significant differences (Mann–Whitney U-test): Benjamini–Hochberg-corrected *P < 0.05, ***P < 0.001. Error bars, maximum and minimum values, excluding outliers. h, Pairwise dS values for Y and X gametologues in platypus. Genes were grouped into strata on the basis of phylogenetic information and dS value clustering (Methods).

Extended Data Figure 6 Prediction of X contigs using male/female genomic read coverage analyses.

a, Male (M) to female (F) genomic read coverage ratio/difference for 23 unassembled contigs (red dots) containing the closest homologues of all platypus Y genes except for RREB1Y (no homologue could be traced) and JUND (low genomic read coverage), fully differentiated part of X5 (yellow), assembled autosomes (black), and autosomal contigs (green), previously experimentally mapped X-linked contigs (blue), and all other unassembled contigs (grey). The density plots describe the distribution of unassembled contigs; the major peak in the y-axis distribution reflects similar coverage in males and females, as expected for autosomal contigs and observed for assembled autosomes. The minor peak reflects a twofold higher coverage in females, as expected for X-linked sequences and observed for the assembled X5 chromosome. b, Coverage profiles for chromosome 1, X5, and the 24 contigs containing gametologues of reconstructed Y genes (RREB1Y contig is missing, as no gametologue could be identified for this gene). Plots are sorted by contig sequence lengths. Consistent with the expectation for X gametologues, 19 out of 24 contigs show an overall twofold higher coverage in females (M:F log2 ratio significantly different from 0 but not from -1; one-sample Wilcoxon signed-rank test; Benjamini–Hochberg-corrected, P < 0.05; Methods and Supplementary Tables 19, 20). The contig containing the ‘Novel Gene’, which has a particularly low M:F ratio, has low coverage but shows twofold higher coverage in females compared to males in regions with mapped reads, consistent with X-linkage. Contigs containing SDHAY and HNRNPKY homologues show a twofold higher coverage in females in the region containing the genes, suggestive of a location in a non-recombinant X region (these genes are likely located close to pseudoautosomal boundaries). The closest homologue of SYCP3Y shows a profile typical of autosomes, which indicates that SYCP3Y was recruited directly to the Y from an autosome. JUND cannot be analysed owing to the limited genomic read coverage.

Extended Data Figure 7 Synonymous and nonsynonymous substitution rates across Y/X (W/Z) branches and physical mapping of AMHY.

ad, dS, dN, dN/dS values for all Y/X branches (medians across branches for listed Y genes and their gametologues), or the first (most basal) branches that follow different stratification events and lead to Y and X clades, respectively. The Mann–Whitney U-test was used to compare distributions of Y and X rates. Significant (Benjamini–Hochberg-corrected) P values are shown in red. a, Eutherians, 17 Y genes (SRY/SOX3, RBMY/X, RPS4Y1/X, AMELY/X, DDX3Y/X, EIF1AY/X, PRKY/X, TSPY1/X, USP9Y/X, ZFY/X, UTY/X, EIF2S3Y/X, NLGN4Y/X, KDM5D/C, UBE1Y1/UBA, TMSB4Y/X and Cyorf15/TBL1X). b, Marsupials, 13 genes (ATRY/X, HCFC1Y/X, MECP2Y/X, HUWE1Y/X, RBM10Y/X, RPL10Y/X, TFE3Y/X, THOC2Y/X, KLF8Y/X, HMGB3Y/X, PHF6Y/X, KDM5D/C and UBE1Y1/UBA). c, Monotremes, 17 genes (AMHY/X, FEM1CY/X, AKAP8LY/X, PPP4R2Y/X, MED26Y/X, THAP11Y/X, STK11Y/X, RFX1Y/X, KHSRPY/X, ARF4Y/X, CTCFY/X, MAU2Y/X, PPP1R10Y/X, HNRNPKY/X, DAZAP1Y/X, PRPF4BY/X and SSR1Y/X). d, Chicken, 20 genes (NIPBLW/Z, ATP5A1W/Z, BTF3W/Z, C18orf25W/Z, CHD1W/Z, GOLPH3W/Z, HINT1W/Z, KCMF1W/Z, MIER3W/Z, NEDD4LW/Z, RASA1W/Z, RPL17W/Z, SMAD2W/Z, SPIN1W/Z, ST8SIA3W/Z, UBAP2W/Z, VCPW/Z, ZFRW/Z, ZNF532W/Z and HNRNPKW/Z). e, DAPI (4′,6-diamidino-2-phenylindole) staining of male platypus metaphase chromosomes. Chromosome Y5 is minute and the smallest chromosome in platypus. The next smallest chromosomes (Y3, Y4 and X4) are also indicated, for comparison. f, Localization of AMHY gene using FISH. The AMHY PCR probe (green signal) hybridized specifically to chromosome Y5. Error bars, maximum and minimum values, excluding outliers.

Extended Data Figure 8 Expression levels of X gametologues and proto-sex chromosome genes, expression evolution towards testis-specificity, and kinetics of Y gene loss.

a, Expression level characteristics (somatic tissues) of gametologues on current X and proto-sex chromosomes. Expression level distributions for current X gametologues (X), precursors of current X/Y genes on proto-sex chromosomes as inferred from 1:1 autosomal orthologues in outgroup species, all current X-linked genes, all proto-sex chromosomal genes. Similar distributions for chicken Z-linked genes and proto-sex chromosome precursors. Statistically significant differences (Mann–Whitney U-test): Benjamini–Hochberg-corrected *P < 0.05, ***P < 0.001. b, Current (Y) and inferred ancestral (proto-sex) expression levels of Y genes that gained testis-specific expression during evolution (right) and those that did not (left) in somatic tissues and testis (as indicated on x axis). Note: for proto-sex chromosome plots, inferred expression output values were calculated per single gene copy (see Fig. 2 legend). c, Kinetics of ancestral gene decay on the therian Y. Gene numbers are plotted on the y axis (ancestral gene numbers are indicated at the top), and time (in Myr) is plotted on the x axis. Dots indicate minimum inferred or observed gene numbers at different time points of mammalian evolution based on our dS inferences (see Fig. 1 and Extended Data Fig. 5) and known lineage divergence times: 181 Myr (S1 formation), 152 Myr (S2a formation), 115 Myr (S2b and S3), 105 Myr (afrotherian split from other placentals), 80 Myr (American–Australian marsupial split), 25 Myr (Old-World monkey–ape split) and 6 Myr (human–chimp split). Dashed lines represent best-fit curves to data points using each of the decay models as indicated. Ancestral gene numbers and decay rates (K, Myr − 1) were taken from Hughes et al.10 except where alternative rates are indicated. Error bars, maximum and minimum values, excluding outliers.

Extended Data Figure 9 Spermatogenic expression patterns of Y protein-coding genes and along the mouse Y chromosome.

a, Left, expression levels of 4 mouse Y genes with ubiquitous spatial expression profiles across different organs and individual spermatogenic cell types. Right, expression levels of 7 mouse Y genes with testis-specific expression across organs and cell types. b, Top, spermatogenic expression of protein-coding genes located in the Y-conserved region (YCR). Lower panel: Expression of 31 reconstructed Y-linked transcript contigs (including unknown presumably noncoding sequences, the known protein-coding genes SLY and SSTY; all with more than 10 copies) along the ampliconic region of the mouse Y chromosome. Genes and contigs were mapped to 1,452 positions using BLASTn to the assembled mouse Y chromosome from genome version GRCm38 (ref. 65).

Extended Data Figure 10 Nonsynonymous over synonymous substitution rates across Y/X (W/Z) branches and simulated multi-copy genes.

a, Median dN/dS values for all Y/X branches and branches leading to autosomal orthologues in outgroup species with different sex chromosome systems (A). Statistically significant differences (Mann–Whitney U-test): Benjamini–Hochberg-corrected *P < 0.05, **P < 0.01, ***P < 0.001. b, Mean coverage values from 1,000 different complementary DNAs from human (>1 kb <10 kb) that were introduced to a mock genome having different copy-numbers (CN), that is, from 1 copy to 20 identical copies. Error bars, maximum and minimum values, excluding outliers.

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Supplementary information

Supplementary Dataset 1

This zipped file contains all newly established Y and W sequences, CDS sequences of XY and ZW gametologs, the alignments of former pseudogenes in chimpanzee and relevant scripts. See the "SI Guide.txt" file in the folder for further details. (ZIP 474 kb)

Supplementary Tables 1-3

Supplementary Tables 1-3 contain information regarding the RNA-seq and genomic data used in this study. The original file uploaded was corrupt and was replaced on 2 May 2014 (XLS 74 kb)

Supplementary Table 4

This Supplementary Table contains information regarding the Y transcript reconstruction pipeline. The original file uploaded was corrupt and was replaced on 2 May 2014 (XLS 23 kb)

Supplementary Tables 5-18

Supplementary Tables 5-18 contain information regarding Y and W genes in 13 mammals and chicken. The original file uploaded was corrupt and was replaced on 2 May 2014 (XLS 658 kb)

Supplementary Tables 19-20

Supplementary Tables 19 and 20 contain information regarding predicted X chromosome localization for X gametologs and statistical information about X-linked contigs in platypus. The original file uploaded was corrupt and was replaced on 2 May 2014 (XLS 42 kb)

Supplementary Tables 21-23

Supplementary Tables 21-23 contain information regarding GO terms enrichment among Y (W) genes and tissue-specificity analyses (statistics). The original file uploaded was corrupt and was replaced on 2 May 2014 (XLS 80 kb)

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Cortez, D., Marin, R., Toledo-Flores, D. et al. Origins and functional evolution of Y chromosomes across mammals. Nature 508, 488–493 (2014). https://doi.org/10.1038/nature13151

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