The evolution of hominin bipedalism in two steps

Abstract

Bipedalism is a human-defining trait^1-3. It is made possible by the familiar, bowl-shaped pelvis, whose short, wide iliac blades curve along the sides of the body to stabilize walking and support internal organs and a large-brained, broad-shouldered baby^4-6. The ilium changes compared with living primates are an evolutionary novelty⁷. However, how this evolution came about remains unknown. Here, using a multifaceted histological, comparative genomic and functional genomic approach, we identified the developmental bases of the morphogenetic shifts in the human pelvis that made bipedalism possible. First, we observe that the human ilium cartilage growth plate underwent a heterotopic shift, residing perpendicular to the orientation present in other primate (and mouse) ilia. Second, we observe heterochronic and heterotopic shifts in ossification that are unlike those in non-human primate ilia or human long bones. Ossification initiates posteriorly, resides externally with fibroblast (and perichondral) cells contributing to osteoblasts, and is delayed compared with other bones in humans and with primate ilia. Underlying these two shifts are regulatory changes in an integrated chondrocyte-perichondral-osteoblast pathway, involving complex hierarchical interactions between SOX9-ZNF521-PTH1R and RUNX2-FOXP1/2. These innovations facilitated further growth of the human pelvis and the unique formation of the ilium among primates.

Fig. 1. Comparative chondrogenesis of human, primate and mouse iliac growth plate.

a, Morphology of the chimpanzee and human adult pelvic girdles, highlighting the shorter and wider ilium in humans compared with the tall blade-like ilium in chimpanzees. A, anterior; Ac, acetabulum; Il, ilium; Is, ischium; P, posterior; Pu, pubis. b, Reconstructed μCT scans of the developing human pelvis at E54–E72, with cartilage depicted in dark grey and ossification in bone white. c, Trichrome-stained transverse sections (red lines on model) across the human ilium (n = 3) at E57 and E72, highlighting the transversely oriented growth plate. RZ, PZ and HZ chondrocytes are aligned bidirectionally along the transverse axis. d,e, Mus musculus (E14.5; n = 3) trichrome-stained (d) and Microcebus sp. (n = 11) haematoxylin and eosin (H&E)-stained (e) coronal histological sections across the pelvis, highlighting the craniocaudally oriented growth plate chondrocyte zones. Ca, caudal; Cr, cranial. f, Hylobates sp. (n = 1), Pan sp. (n = 2) and human segmented pelvic girdles (cartilage in dark grey; bone in lighter, white colour) and corresponding μCT sections (two axial sections, indicated by locations of red lines on each model). HZ chondrocytes are depicted in a darker colour than RZ and PZ chondrocytes. In Pan and Hylobates transverse cross-sections, chondrocytes are uniform in colour (all light or all dark) at each axial level. In humans, a transversely arranged RZ–PZ–HZ can be visualized owing to varying colours of the chondrocytes in each section. Scale bars, 500 µm. Human, chimpanzee, gibbon, mouse lemur and mouse schematics were created in BioRender. Senevirathne, G. (2025) https://BioRender.com/p7qcwtp.

Fig. 2. Genetic architecture of the human iliac growth plate.

a, Uniform manifold approximation and projection (UMAP) of weighted nearest neighbour analysis (WNN) for multiomics data from E54 and E57 human ilia plus adjacent tissue. For each stage: left, cell types are depicted with unique colours that remain consistent across stages; right, cell-type-specific scATAC-seq intersections with HARs per timepoint. b,c, Spatial expression of COL9A1, SOX9, ZNF521 and PTH1R in ilium cross-sections at E45 (b) and E54 (c). Fe, femur. d–g, Radiographs of right hemipelves in a healthy 10-year-old boy (d), a 3-month-old girl with campomelic dysplasia (e), a 16-year-old boy with JMC (f) and a 9-year-old girl with Eiken syndrome (ES) (g). Red arrowheads highlight width reduction and lack of flaring (e–g) compared with normal pelvis (white open arrowheads). h,i, CellChat results at E45 (h) and E54 (i). Left, chord diagrams illustrate ligand–receptor interactions among selected cell populations across spatial transcriptomic sections. Line thickness indicates number of interacting ligand–receptor pairs. h, Right, interactions between Mes2 and ChondroProg clusters involving PTN and PTH signalling pathways. The directionality of outgoing and incoming signals is indicated by arrows and mapped onto spatial transcriptomic sections, with interactions further represented using chord plots. i, Right, interactions between Chondro and Perichondral clusters involving PTH and WNT signalling pathways. The directionality of outgoing and incoming signals is indicated by arrows and mapped onto spatial transcriptomic sections, with interactions further represented using chord plots. j, Left, scATAC-seq results for SOX9 in University of California Santa Cruz (UCSC) Genome Browser, depicting accessibility signals (vertical black lines) across chondrocytes at E53 and E57 and their intersections with HARs, human-specific deletions in conserved regions (hCONDELs) and human ancestor quickly evolved regions (HAQERs). Blue bands depict HAR intersections, yellow bands depict hCONDEL intersections, and the red area indicates a 960-kb deletion interval that causes campomelic dysplasia. Right, magnified view of E54 multiomics data (accessibility is depicted as peaks with a red asterisk identifying one HAR-regulatory element overlap identified in the left plot; gene expression is depicted as violin plots on the right) for each cell type showing HAR–accessibility intersection (blue band with an asterisk) intronic to SOX9 present in resting chondrocytes, chondroprogenitors and mesenchymal cells.

Fig. 3. Comparative primate ilium ossification during development.

a, Ossification patterns of the ilium and femur across the representatives from the two primate suborders: Strepsirrhines and Haplorrhines. From top: primate schematics; 3D reconstructed pelves (cartilage in dark grey; bone in white); coronal cross-sections of ilium and femur, highlighting mineralization and bone in white. b, Trichrome staining of a coronal cross-section across the ilium of M. musculus at E18.5 (n = 3), highlighting blood vessels (BV; deep magenta) penetrating mature HZ chondrocytes, and ossification (Os) along the longitudinal axis (dark blue). c, H&E staining of a coronal cross-section across the ilium of Microcebus sp. (n = 11), highlighting the ossifying region in red, with blood vessels penetrating the HZ and chondrocytes visible in blue. In b,c, the magnified view (right) highlights ossification occurring in the mid-shaft. d, Trichrome-stained transverse cross-section across the POC of human ilium at E72 (n = 3). Right, magnified view highlights iliac perichondral ossification. Ossification (deep blue) is limited along the periphery of the cartilaginous anlage containing HZ chondrocytes. Blood vessels penetrate the peripheral ossifying tissue but not the HZ. e, 3D reconstructions of vascularization in the vicinity of human ilium at E57 and E72, with the external iliac artery capillaries extending along the lateral (not medial) ossifying surface. Scale bars, 500 µm. Human, mouse and all primate schematics (except Galago and Aotus) were created in BioRender. Senevirathne, G. (2025) https://BioRender.com/p7qcwtp.

Fig. 4. Molecular basis of human iliac perichondral ossification.

a, RNA velocity maps overlaid on UMAPs of human ilium and adjacent soft tissue cells at E54, E57 and E67. Arrows depict cell trajectories. Colour scheme as in Fig. 2. b, Expression of selected marker genes (log-transformed): RUNX2 (perichondrium/osteoblasts), SP7 (osteoblasts), and FOXP1, VEGFA and VEGFB (blood vessels) from Visium spatial analysis. Red dashed lines in the segmented model on the left depict the anatomical position of the transverse section. c, Single-cell multiomics data (accessibility is depicted as peaks; gene expression is depicted as violin plots on the right) at E54 and E57, focusing on the RUNX2–SUPT3H locus. The blue band highlights a putative enhancer region intersecting with a HAR. d, LacZ reporter assay in transgenic mice, showing the anatomical location of RUNX2 enhancer expression in iliac perichondrium at E14.5 (blue staining highlighted by arrow), and no detectable expression in embryos carrying the orthologous chimpanzee RUNX2 enhancer sequence or in the negative control (wild-type) pelvic girdle. Fe, femoral head; Pe, perichondrium. e, Enhancer-driven GRN for human chondrocyte populations at E53, inferred using SCENIC+ and visualized with Cytoscape. Hexagons (orange) represent eRegulons (transcription factors) and circular nodes represent genes containing transcription factor binding sites. Green circles highlight genes identified through GO enrichment analysis as involved in human pelvic girdle abnormalities. Nodes outlined in red indicate genes associated with HAR intersections.

Fig. 5. Progression of human iliac perichondral ossification and the ontogeny of pelvic bipedal musculature.

a, Reconstruction of human pelvis development from gestational weeks 10 to 25 (cartilage is depicted in dark grey, and bone is depicted in white). Perichondral ossification extends radially, whereas the AGZ remains cartilaginous up to gestational week 25. b, Sites of muscle origins (attached and unattached) for the gluteal muscles, rectus femoris, vastus lateralis and iliacus. c, UMAP of WNN for multiomics data from human ilium plus soft tissue at E59, with cell types depicted using the colour scheme in Fig. 2. Muscle cell types (MyoC, MyoProg and MyoProg+Pax7) cluster together. d, H&E staining of a transverse section across E59 human ilium (n = 2), highlighting the attached dorso-lateral muscles and the AGZ (left) with corresponding Visium spatial transcriptomics (right). Visium spatial transcriptomics analysis independently recognizes 11 cell clusters. e, Visium gene expression plots (log-transformed) for E59 human ilium integrated with single-cell RNA-sequencing (scRNA-seq) data for selected cell clusters. ASIS, anterior superior iliac spine. Scale bar, 2 mm.

Extended Data Fig. 1. A putative gene regulatory network (GRN) underlying the dual structural novelties of the human ilium.

a, In EARLY-stage non-human primates (NHP) and mice, the ilium (cartilage in blue) is oriented longitudinally (top), with resting zone (RZ) marker UCMA and early proliferating marker PTH1R positioned at the cranial end, while SOX9, an early chondrocyte marker, is expressed throughout the cranio-caudal ilium (bottom, section). In the EARLY-stage human ilium, the anteroposterior (AP) axis which is initially rod-like, widens while the vertical axis shortens (top). In cross-section, SOX9 is expressed bilaterally along the AP axis, followed by PTH1R, which gets restricted to the anterior pole. ZNF521, located external to undifferentiated chondrocytes, interacts with PTH1R (bottom, section). In LATE-stage NHPs and mice, ilium ossification (white) initiates at mid-length and extends along the longitudinal axis (top); UCMA (RZ marker) is present at cranial and caudal ends, with PTH1R positioned between the hypertrophic zone (HZ, marked by COL10A1) and the RZ. RUNX2, an osteoblast marker, is expressed internally and along the perichondrium (Pe) at ossification sites in mice and NHPs (bottom). In LATE-stage human ilium, ossification initiates posteriorly and extends radially (top; white arrows); COMP, PTH1R, COL10A1 are expressed along the transversely oriented growth plate, while RUNX2 is expressed along the perichondrium, illustrating delayed internal ossification (bottom). Cartoon schematics of human, chimpanzee, mouse and, mouse lemur were created in BioRender. Senevirathne, G. (2025) https://BioRender.com/p7qcwtp. b, Ossification extends radially, and several muscles (red), including the three gluteal muscles (maximus posterior, medius intermediate, and minimus anterior) and the rectus femoris (RF), attach to the ilium early in development. c. A summary of the interactive network underlying these developmental shifts (see text). Additional abbreviations: ASIS, anterior superior iliac spine; AIIS, anterior inferior iliac spine; IL, ilium; FM, femur.

Extended Data Fig. 2. Experimental design and comparative growth plate morphology of human and primates.

a, Human sample experimental design, including both genetic and morphological components. The genetic component involves spatial transcriptomics and single cell multiomics using the 10X platform (left), and the morphology component includes histology and microCT scanning (right). b, Transverse trichrome-stained cross-sections (indicated by dashed redlines on the model) across the human ilium (n = 3 from each stage) at E54, E57, and E72, highlighting the transversely oriented growth plate (GP). At E54, there is no differentiation of chondrocytes, with only undifferentiated chondrocytes/resting zone chondrocytes (RZ). By E57 and E72, RZ, proliferating zone (PZ), and hypertrophic zone (HZ) chondrocytes are aligned bi-directionally along the transverse axis. c, Sagittal trichrome-stained cross-sections (indicated by dashed redlines on the model) across the human ilium at E72 (n = 3), highlighting the absence of a true longitudinally oriented GP. d, Microcebus sp. (n = 3) and e, Saguinus sp. (high-resolution slide photographs for Saguinus obtained from MCZ website, under special collections (Museum of Comparative Zoology, Harvard University; ©President and Fellows of Harvard College; licensed under CC BY-SA 4.0). H&E coronal histological sections (from early to late developmental stages: A-C) across the pelvis highlighting the longitudinally oriented GP chondrocytes. Abbreviations: Ac: Acetabulum; Ch: Chondrocytes; Hy: Hypertrophy; Is: Ischium. Schematics for the CT scanner, embryo, pelvis, microscope, and mouse-lemur are created using BioRender.com. Scale bars depict 500 µmm. In each ilium model, gray is cartilage, white is ossification. Cartoon schematics of embryo, pelvis CT scanner and microscope in a and mouse lemur and Saguinus in d created in BioRender. Senevirathne, G. (2025) https://BioRender.com/p7qcwtp.

Extended Data Fig. 3. Comparative microCT sections of the growth plate of human and primates.

Reconstructed pelvic girdles (i.e., cartilage in dark gray; bone in a lighter, white color) on the left, with corresponding microCT sections (locations indicated by dashed redlines on each model) shown to the right. a, Human vs d, primate transverse CT scan-sections depicting the differentiation of chondrocytes in humans along the transverse axis. The hypertrophic zone (HZ) is shown in dark color and resting zone (RZ) and proliferating zone (PZ) in lighter colors. In Galago, Saguinus, and Aotus transverse cross-sections, chondrocytes at each axial level appear uniform in color (either all light or all dark). b, Sagittal CT scan cross-sections of human E54, E57, E67, and E72 highlighting the absence of a bidirectionally longitudinally oriented growth plate. c, late-stage microCT reconstructions of the human pelvic girdle where cartilage is in dark gray, and bone in white color. Reconstructed pelvic girdles are shown on the left and transverse cross sections across the blade are shown on the right. The red line on each model depicts the anatomical location of each transverse section. e, reconstructed chimp iliac blade (cartilage in dark gray; bone in a lighter, white color), with corresponding microCT sections with locations indicated by red lines on the model. e’, transverse sections. f, sagittal section. Saguinus schematic was created in BioRender. Senevirathne, G. (2025) https://BioRender.com/p7qcwtp.

Extended Data Fig. 4. Developmental changes in human single-cell RNA and ATAC-seq cell clusters over time.

a, Uniform manifold approximation and projection (UMAP) plots for RNA-seq, ATAC-seq, and integrated RNA + ATAC-seq using Weighted Nearest Neighbor (WNN) for E53, E57, E67, and E72 for human ilia + adjacent soft tissue sc-multiomics data. Cluster names and colors are consistent with Fig. 2a. The marker gene (refer to Supplementary Table 2) expression levels for each cell cluster are displayed by different shaded dots in the plots on the right side, with darker shading depicting higher average expression. The size of the dot reflects the percentage of cells expressing the gene of interest (b). c, Quality control plots (total RNA, ATAC, and mitochondrial reads) for multiomics samples. Bar plots depicting the total number of cells for each stage (d) and total number of spots that were captured by each spatial transcriptomic section (e).

Extended Data Fig. 5. Gene expression patterns underlying the transversely oriented human growth plate and perichondral ossification.

a-c, E53, d-e, E57, and f-h, E72 marker gene expression patterns for osteochondro progenitors, resting zone (RZ), proliferating zone (PZ), and hypertrophic zone (HZ). i-k, E53, l-n, E57, and o-p, E72 marker gene expression patterns for perichondrium, osteoblasts, and blood vessels. a,d,f,i,l,o, WNN UMAPs and feature-plots for each stage and marker genes. b,c,e,g,h,j,k,m,n,p, Visium gene expression plots for each marker gene, with the top-most image highlighting the H&E histomorphology of each corresponding Visium section. Anatomical location of each Visium spatial section is depicted by a red line on the reconstructed model.

Extended Data Fig. 6. Putative intrinsic and extrinsic cues underlying the growth plate shift in human E45 and E53.

Spatial gene expression patterns for selected genes from the intrinsic (a) and extrinsic (b) analysis (see Methods) during early iliac development. The top row shows the regions selected for the intrinsic/extrinsic analysis, with the anatomical locations of the spatial transverse cross sections depicted by a red line on the reconstructed model (in which gray is cartilage). The second row presents H&E images of the corresponding spatial transverse cross-sections. COMP, COL2A1, and perichondral marker THBS2 are expressed along the periphery at E45 and E53. The asterisk (“*”) represents the genes that have a HAR-intersection. Abbreviations: Fe, femur; Il, ilium.

Extended Data Fig. 7. Decoding upstream targets of PTH1R/ZNF521 pathway and effects of PTH1R in chondrification.

a, (Top) scATAC-seq results for ZNF521 locus in this hg38 human UCSC Genome browser window depicting E53, E57, E67, and E72 accessibility signals (vertical black lines) across cell-types and their intersections with HARs, hCONDELs, and HAQERs. Numbered blue highlighted areas depict intersections with HARs and numbered yellow highlighted areas depict intersections with hCONDELs. (Below) Zoom-in view at E53 and E57 multiomics data (accessibility on left as peaks; gene expression as horizontal violin plots on right) per cell type showing one HAR/accessibility signal intersection (broad blue highlighted area) in an intronic area of ZNF521 and three overlaps with hCONDELs. Region #2: Zoom-in view at E54 multiomics data (accessibility on left as peaks; gene expression as horizontal violin plots on right) per cell type showing HAR/accessibility signal intersection (blue highlighted area with an asterisk) in the intron of ZNF521 present in smooth muscle progenitors and mesenchymal cells. b, CT scans and histological differences of Wildtype vs PTH1R humanized mouse pelves at P21 and E15.5 (bottom row). c, (Left) scATAC-seq results for upstream targets of PTH1R in the hg38 human UCSC Genome browser window depicting E53, E57, E67, and E72 accessibility signals (vertical black lines) across cell-types and their intersections with HARs, hCONDELs, and HAQERs. Blue highlighted areas depict intersections with HARs. (Right) Zoom-in view at E53 and E57 multiomics data (accessibility on left as peaks; gene expression as horizontal violin plots on right) per cell type showing HAR/accessibility signal intersection (broad blue highlighted area) in IGF1 and no HAR/hCONDEL/HAQER intersections at IHH and WNT7A. Abbreviations: AC, acetabulum; HZ, hypertrophic zone; IL, ilium; PZ, proliferative zone.

Extended Data Fig. 8. Co-expression profiles for SOX9 against PTN and MK pathways and SOX9’s downstream targets at E45, E53.

Anatomical location of each Visium spatial section is depicted by a red line on the reconstructed model. Top row for E45 and E53 are gene expression profiles for each target gene, and the bottom row depicts the co-expression plots. The heatmap beneath each co-expression plot highlights spatial spots, with SOX9 expression shown in dark red, expression of the compared gene in yellow, and co-expression of both genes in orange. Abbreviations: Fe, femur; il, ilium.

Extended Data Fig. 9. The SOX9 locus, its evolutionary signals and Hi-C heatmap.

a, (Top) scATAC-seq results for SOX9 locus in this hg38 human UCSC Genome browser window depicting E53 and E57 accessibility signals (vertical black lines) across cell-types and their intersections with HARs, hCONDELs, and HAQERs. Numbered blue highlighted areas depict intersections with HARs and yellow highlighted areas depict intersections with hCONDELs. (Below) Zoom-in view at E53 multiomics data (accessibility on left as peaks; gene expression as horizontal violin plots on right) per cell type showing at least one HAR/accessibility signal intersection (broad blue highlighted area). b, Hi-C heatmap visualization depicting chromatin interactions within the genomic region closer to SOX9. Topologically associating domains (TADs) are highlighted by triangles along the diagonal, representing areas of increased intradomain interactions. Chromatin loops connecting active promoter and distal anchors (enhancer) regions are indicated by arcs below the heatmap. Interaction intensity, shown as a color gradient from light (low interaction frequency) to dark red (high interaction frequency), shows the strength of spatial contacts. RestingChondro and Perichondro peaks that intersect with HARs at E53 also fall within this active chromatin interaction area.

Extended Data Fig. 10. The RUNX2, FOXP1, and FOXP2 loci and their evolutionary signals.

a, A highly conserved iliac-specific RUNX2 open chromatin region amongst bulk ATAC-seq results for RUNX2 locus in this hg19 UCSC browser plot depicting accessibility signals from various tissues across the skeleton. The highlighted red rectangle focuses on the open chromatin region that is only present in bulk iliac data at E53 and overlaps with a HAR. b,c,d (Top) scATAC-seq results for RUNX2(b), FOXP1(c), FOXP2(d) gene loci displayed in each hg38 human UCSC Genome browser window, depicting accessibility signals at E53, E57, E67, and E72 (vertical black lines) across cell-types and their intersections with HARs, HCONDELs, and HAQERs. (Below) A zoomed-in view of multiomics data at E53, E57, and E67 (accessibility on left as peaks; gene expression as horizontal violin plots on right) per cell type. At each locus, numbered blue highlighted areas indicate intersections with HARs, and numbered yellow highlighted areas depict intersections with hCONDELs.