The proteome of Middle Pleistocene Xujiayao and common Denisovan morphology

Late Middle Pleistocene Homo systematics in eastern Eurasia remain poorly defined compared with the better-established evolutionary scenarios in western Eurasia, with East Asian hominins variously classified as archaic Homo sapiens, archaic Homo, China archaics or archaic humans^2,3,4,5. Recent studies have revealed greater morphological diversity in this group than previously known, prompting their assignment to distinct species¹. Homo longi, defined by the Harbin cranium (about 309–146 thousand years ago (ka)) and now classified as Denisovan, is characterized by a massive, long, low cranium; a low, flat face with a shallow canine fossa, reduced alveolar prognathism; a relatively rounded occipital contour with weak torus; a robust supraorbital torus overhanging near-square orbits; and a large, forwardly and inwardly inclined mastoid process, clustering phylogenetically with Jinniushan, Dali, Hualongdong and Xiahe^6,7,8,9. However, despite some primitive traits, Hualongdong has been designated as a transitional form from archaic hominins to H. sapiens on the basis of derived facial morphologies, mainly including a small size, flattened malar region, weak prognathism, a well-developed anterior nasal spine and a parabolic dental arcade with an anteriorly displaced incisive foramen¹⁰. Another lineage, characterized by an exceptionally large cranium, has been proposed as Homo juluensis, encompassing Xujiayao/Xuchang crania and the Denisovan-affiliated Xiahe and Penghu mandibles, identified through palaeoproteomic analyses^1,11,12. Beyond the Denisovan hypothesis, competing phylogenetic hypotheses proposed these hominins as ancestral to either H. sapiens or another sister group, further obscuring the evolutionary scenarios in late Middle Pleistocene eastern Eurasia^6,13,14.

The Xujiayao assemblage, dated to around 199–138 ka through combined uranium series and electron-spin-resonance methods¹⁵, comprises 20 hominin cranial, mandibular and dental fossils excavated between 1976 and 1979 from a well-defined stratigraphic context in the Nihewan Basin, northern China^16,17. This assemblage, recovered alongside abundant artifacts (dominated by scrapers and spheroids) and faunal remains, represents one of the most extensive collections among East Asian late Middle Pleistocene archaic Homo^18,19 (Fig. 1 and Supplementary Text 1). Initial analyses identified a thick cranial vault, large cerebral fossa relative to the cerebellar fossa, moderate occipital torus, an anterior nasal spine, reduced inion–endinion distance and Neanderthal-like dental morphology, leading to classification as Homo sapiens neanderthalensis, early H. sapiens or archaic H. sapiens^3,16,17. To circumvent taxonomic disputes when sapiens lacks consensus, terms such as archaic Homo were temporarily adopted⁴. Recent analysis of Xujiayao 6, comprising parietal, temporal and occipital bones, revealed an exceptionally large endocranial capacity of 1,700 ml, designating Xujiayao as the holotype of H. juluensis^20,21. Shared dental traits between Xujiayao and Altai Denisovans, including large crown size, complex occlusal surface and highly divergent molar roots, have been proposed to support their taxonomic affinity²². Altai Denisovans and Xujiayao were then grouped under H. juluensis, alongside the Penghu and Xiahe mandibles, Xuchang crania and the Tam Ngu Hao 2 tooth due to their close morphological affinity^1,23. This lineage is thought to be distinct from H. longi (Harbin, Dali and Jinniushan), archaic Homo (Hualongdong) and a potential Neanderthal-like Maba lineage¹. As an abundant fossil assemblage with a clear stratigraphic context and linked to nearly all classifications of East Asian late Middle Pleistocene archaic Homo, a confirmed taxonomy of Xujiayao would be critical to resolving the evolutionary relationships of other Middle Pleistocene Homo in East Asia through palaeoproteomic analysis and morphological comparisons.

Here we implemented a palaeoproteomic analysis to explore Xujiayao’s taxonomic classification and assess its genetic population affinity. Subsequently, we conducted a systematic morphological comparison between Xujiayao and other members of East Asian late Middle Pleistocene within a broader evolutionary framework of genus Homo to investigate their morphological affinities to the Xujiayao population. The palaeoproteomic results revealed Xujiayao hominins as Denisovans, and their morphological similarities with other East Asian late Middle Pleistocene archaic Homo (such as Jinniushan, Dali and Hualongdong) indicate that they all belong to the Denisovan lineage.

Molecular evidence was sought to determine the phylogenetic placement of the Xujiayao hominin samples, on the basis of the positive result of animal samples from the same site (Supplementary Text 2). Ancient proteins were successfully extracted from two bone fragments, including one right parietal bone (PA1490, XJY-6) and one mandible (right ramus, PA1497, XJY-14). To maximize protein yields, we extracted proteins from bone samples by sequential fractionation, resulting in one acid-soluble fraction (>3 kDa) and two acid-insoluble fractions for each sample (Methods and Supplementary Table 1). The preservation of peptides in both Xujiayao hominin samples was first confirmed through the identification of collagen peptide markers^24,25,26 in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Supplementary Text 2), and the proteomic data were then further analysed using liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis.

To obtain protein sequences from the Xujiayao hominin samples, LC–MS/MS data of the samples were searched on PEAKS Online against the Harbin database⁸, which includes bone proteins of published archaic individuals and the proteome of present-day humans. The PEAKS search yielded 102,029 peptide-spectrum matches (PSMs) and 15,194 unique peptides across the two samples (Fig. 2, Supplementary Table 2 and Supplementary Fig. 1). We also searched the data on MaxQuant for confirmation, and the search yielded 3,526 unique peptides across the 2 samples (Supplementary Fig. 2).

To differentiate endogenous ancient proteins from modern contamination, Gln and Asn deamination characteristics of ancient proteins were calculated for each protein. Proteins with neither Gln nor Asn residues were excluded from the analyses, as the deamination rates of those proteins cannot be assessed. For proteins that contain at least two peptides with Gln or Asn, we calculated deamidation rates for 41 proteins containing both or one of those Gln/Asn residues. Similar to ancient proteins obtained from previous studies^12,27,28, the majority of collagens showed a high level of deamidation rates (PA1490: Asn, 25.0–94.7%; and Gln, 33.3–100.0% (Supplementary Fig. 3a); PA1497: Asn, 20.0–89.4%; and Gln, 22.2–87.5%, except for COL14A1 (Supplementary Fig. 3b); in PEAKS results, the same hereafter), and the most common collagens in ancient bones (COL1A1, COL1A2, COL2A1, COL5A1 and COL5A2) had much higher deamidation rates (PA1490: Asn, 61.4–94.7%, and Gln, 44.9–92.6%; PA1497: Asn, 62.6–89.4%, and Gln, 49.3–86.1%). By contrast, typical contaminants such as keratins and trypsin showed a low level of deamidation (PA1490: Asn, 0.0–5.3%; and Gln, 0.0–7.7% (Supplementary Fig. 3a); PA1497: Asn, 0.0–5.6%, and Gln, 0.0–10.0% (Supplementary Fig. 3b)). As a result, we obtained 31 proteins with elevated deamidation rates for PA1497, and 20 for PA1490. We further filtered out peptides that cannot be confidently assigned to a single gene (Supplementary Table 3), peptides identified in both the Xujiayao hominin samples and the extraction blanks, and proteins that could not be confirmed by MaxQuant, resulting in 18 endogenous ancient proteins for downstream analyses (13 for PA1490 and 15 for PA1497; Supplementary Table 4).

Preliminary MALDI-TOF spectral analysis and LC–MS/MS data searching against the Mammalia type I collagen database indicated that the Xujiayao hominin samples belong to the genus Homo (Supplementary Text 2); we therefore further used single amino acid polymorphism (SAP) assessment to establish a higher-resolution taxonomic classification. Of the SAPs that we obtained from the Xujiayao hominin samples using both software programs that were assigned to genera within Hominidae, the ancestral type was present at all non-Homo derived positions, confirming that the Xujiayao samples belong to Homo (Supplementary Tables 5 and 6 and Supplementary Fig. 4). Among the SAPs within Homo, COL1A2-996K is reliably identified (Supplementary Table 7 and Supplementary Text 3). This variant is specific to all the Denisovan individuals with proteomic or genomic evidence, including the two Xiahe individuals^12,28, the Penghu 1 individual¹¹, the Harbin individual⁸ and Denisova3 (ref. ²⁹), while Neanderthals and modern humans contain an arginine at this site. We further validated this SAP in the Xujiayao hominin samples using spectral data quality and deamidation rates. Abundant peptides with high-quality spectra (Fig. 3a,d and Supplementary Figs. 5 and 6) containing this SAP were observed in both Xujiayao hominin samples (57 peptides, 207 PSMs for PA1490; and 61 peptides, 244 PSMs for PA1497), with very few peptides identified by low-quality spectra showing an arginine there (1 peptide, 1 PSM for PA1490; and 1 peptide, 5 PSMs for PA1497). Moreover, increased deamidation rates of residue Asn977 or Gln1002 in COL1A2-996K-containing peptides were observed (Asn977: 69.2% in PA1490, and 69.4% in PA1497; Gln1002: 100.0% in both), confirming the endogenous origin of these peptides. With no new SAP reliably identified, we therefore determine the Xujiayao hominin samples to be from a Denisovan population.

To confirm the phylogenetic placement for the Xujiayao hominin samples, we reconstructed a Bayesian phylogenetic tree from the concatenated consensus sequences of 2,074 amino acid positions overlapping between the PEAKS and MaxQuant results (with at least 2 PSMs coverage in each software) from 8 endogenous proteins, which is 16.0% of the total protein sequence alignment (Supplementary Data 1). The protein sequences of a set of selected representative individuals from the Harbin database were used to build the phylogenetic tree (Methods). The phylogenetic tree (Fig. 3c) exhibited topological congruence with those established by genomic^29,30,31,32 and palaeoproteomic studies^8,11,12. Although incongruence might occur when using different datasets due to the relatively modest amount of proteomic data and the sequence variation among modern human population (Supplementary Text 4), the Xujiayao hominin samples, Denisova3 and other Denisovan-related individuals always formed a monophyletic group with a high posterior probability (>99%). However, within the Denisovan clade, different individuals formed unstable topology with low posterior probabilities (<50%; Supplementary Fig. 7) as no more SAPs were shared besides COL1A2-996K (Supplementary Text 5); we can therefore only determine the Xujiayao hominins to belong to a late Middle Pleistocene East Asian Denisovan population, without further grouping.

With Xujiayao classified as Denisovan, the preserved cranial, mandibular and dental materials, incorporating the newly identified Harbin Denisovan^8,9, enable a detailed morphological characterization of the Denisovan lineage (Supplementary Figs. 8–10 and Supplementary Text 6). Xujiayao hominins exhibit erectus-like features, including a low and wide neurocranium (Fig. 4 and Supplementary Fig. 11), a thick cranial vault (bregma, 7.7 mm; parietal tubercle, 12.0 mm) (Supplementary Fig. 11), a weak parasagittal depression (not in Harbin), a short (large in Harbin) and inwardly inflexed mastoid process, an asymmetrical mandibular notch (with the crest attached to the lateral third of the condyle), an open mandibular foramen, and M², M³ and M₃ complete main cusps (where the superscript and subscript values denote upper jaw and lower jaw molars, respectively)^6,18,33,34. Denisovans also display a group of derived features shared with some later Homo species (African and European Homo heidelbergensis, Neanderthals and modern humans): a strongly arched temporal squama, a moderately developed suprameatal spine and a more symmetrical crown outline with a longer buccal cusp compared with the lingual cusp, reduced buccal contour protrusion and a simplified occlusal surface lacking the bifurcated buccal essential crest in P³ (upper third premolar)^35,36,37.

Notably, the Xujiayao Denisovan displays several metric and non-metric features that are different compared with most H. erectus and modern humans (Fig. 4 and Supplementary Figs. 8–10). These include a low cranial profile with near-parallel or slightly convergent lateral walls, a nearly vertical occipital plane (with a larger occipital angle than in H. erectus; Supplementary Fig. 11) but a non-rounded occipital bone, a non-coronally oriented tympanic plate deflected to the petrosal (also present in late Neanderthals), a weak angular torus and a moderate linear occipital torus (commonly moderate in late Neanderthals but weak in Harbin)^6,18. In these features, the Xujiayao Denisovan is generally comparable to African and European H. heidelbergensis and early Neanderthals^35,36,37,38. Although retaining the moderate or pronounced labial convexity and pronounced shovelling as in H. erectus, the finger-like prolongations in the lingual side of the upper central incisor, elevation degree of the lingual central ridge and cuspule-like marginal ridge in the upper canines tend to be simplified in Denisovans. The Denisovan mandibles represented by those from Xujiayao, Xiahe and Penghu display a distinct depression within the mandibular planum triangulare, a feature that is uncommon in other members of the genus Homo^12,34,39 (Fig. 4 and Supplementary Fig. 9).

After defining the Denisovan morphology using the Xujiayao and Harbin fossils, we investigated whether this morphological suite is consistent with that of other East Asian late Middle Pleistocene archaic Homo (Fig. 4 and Supplementary Table 8). As in Xujiayao and Harbin, the other members of East Asian late Middle Pleistocene also exhibit erectus-like features (for example, parietal sagittal keeling and a weak parasagittal depression), derived characteristics (such as pronouncedly or strongly arched temporal squama) and a group of features that differ from both H. erectus and modern humans (such as a low cranial profile with near-parallel or slightly convergent lateral walls, a weak angular torus and a moderate linear occipital torus)^2,6,13,40 (Supplementary Fig. 8). Xujiayao’s mandibular features, including an asymmetrical mandibular notch, open mandibular foramen, superior medial pterygoid tubercle, planum triangulare depression and gonial eversion, are fully present in Penghu and partially so in Xiahe and Hualongdong^12,13,34,39. This unique combination of morphological traits, distinct from H. erectus, modern humans and Neanderthal-lineage hominins, supports the assignment of most members of East Asian late Middle Pleistocene archaic Homo as Denisovans, reinforced by shared cranial traits with Harbin and mandibular traits with Penghu, confirmed as Denisovan by ancient DNA analysis and/or palaeoproteomics^8,9,11,12. This suite of morphology does not appear simultaneously in individual specimens but manifests in different forms or combinations across various specimens. The inclusion of additional members of East Asian late Middle Pleistocene archaic Homo, alongside Xujiayao, expands the morphological variability of East Asian Denisovans.

Denisovan morphology has been characterized through comparisons with H. erectus sensu stricto, African and European H. heidelbergensis and modern humans. As sister groups sharing a common ancestor, Denisovans and Neanderthals warrant detailed comparison to elucidate both shared and distinct features between them and clarify their morphological divergence (Supplementary Figs. 8–10). Denisovans share some features with Neanderthals: a low placement of the posterior canal in temporal labyrinth configuration (present in Xujiayao and Xuchang, but not in early Neanderthals), a suprainiac fossa (present in Xuchang; in early Neanderthals it appears as a rough and porous area), a retromolar space (present in Xujiayao and Xiahe) and a superior medial pterygoid tubercle (present in Xujiayao, Penghu and Hualongdong)^{2,12,13,34,39,41,42,43,44,45,46,47}.

However, East Asian late Middle Pleistocene archaic Homo differs from Neanderthals in several aspects. The erectus-like features (parasagittal depression, parietal sagittal keeling) are generally absent in early and late Neanderthals. M², M³ and M₃ of Xujiayao and most other members display complete main cusps, different from a number of Neanderthal specimens that lack the last-formed cusp⁴⁸. Other features that are typical in early and/or late Neanderthals, including an occipital bun, a rounded cranial profile in the posterior view, a mandibular notch crest attaching to the middle third of the condyle, a bridged mandibular foramen and an inverted gonial angle, are generally absent in East Asian late Middle Pleistocene archaic Homo^{12,13,34,35,39,43,46}. The typical Neanderthal-like dental morphologies (for example, extremely convex labial surface of incisor I¹, cuspule-like tuberculum dentale of upper canine, transverse crest of M¹ and middle trigonid crest of lower molars) are rarely seen in East Asian late Middle Pleistocene archaic Homo⁴⁸.

Molecular phylogeny and shared morphological traits classify Xujiayao, initially assigned to different lineages, as Denisovan. The abundant Xujiayao remains, comprising a large part of the skull, offer insights into the physical appearance of Denisovans through a suite of morphologies instead of less direct characterizations through molecular and fragmentary fossil evidence. Dated at around 199–138 ka, Xujiayao is among the earliest molecularly confirmed Denisovans and approximately coeval with the early confirmed Denisovans from northeastern China and southern Siberia^8,29,49.

This consistent suite of morphologies has been identified among East Asian late Middle Pleistocene archaic Homo, supporting their classification as Denisovans. Xujiayao and Xuchang were previously assigned to a single lineage (H. juluensis), distinct from the groups of Dali, Harbin, Jinniushan and Hualongdong¹. However, the main difference in endocranial capacity between H. juluensis (1,700–1,800 ml) and other members of East Asian late Middle Pleistocene Homo (1,120–1,420 ml) falls within the documented variation range of modern humans (1,090–1,880 ml), and also is approximately compatible with the spectrum of Neanderthals (1,065–1,740 ml)^6,20,50. This variation in endocranial capacity should therefore be considered an intrapopulation variation, especially considering these two groups shared a unique combination of plesiomorphic, derived and transitional features that distinguish them from other members of the genus Homo. If taking into consideration the facial bones of Harbin (confirmed as Denisovan by molecular analysis^8,9), Jinniushan, Hualongdong and Dali, we will have a nearly complete picture of the cranial morphology of Denisovans. They exhibit a low and sloping forehead, massive supraorbital torus curved backward, pronounced but not continuous supratoral sulcus, square or obliquely rectangle-like orbit, flat or moderately elevated nasal saddle, broad inferior nasal aperture, weak anterior nasal spine and subnasal fossa, coronally flat zygomaxillary region lacking obvious midfacial pneumatization and weak prognathism (also matched with the genetically predicted phenotypes)^{6,10,13,40,51}.

Defined initially by DNA from remains in Siberia^29,52, Denisovans appear to have occupied an extensive geographical range across East Asia, represented by Xiahe and Harbin in the north and Penghu in the south. If both morphological and molecular evidence were considered, Denisovans could have lived in East Asia from at least 300 ka to 45 ka, parallelling the temporal range of Siberian Denisovans (250–55 ka)^13,28,53,54. The inclusion of the isolated lower molar from Cobra Cave of northern Laos, assigned to Denisovan due to its morphological similarity to Xiahe²³, further supports Denisovans as a broadly distributed Homo lineage that adapted to diverse ecological zones, including the high-altitude plateau, subtropical forest and insular environments, across Siberia, East Asia and Southeast Asia. Sporadic dispersal of modern humans into southern China (for example, Zhiren and Daoxian) might not have substantially interfered with the living niches of Denisovans until a major migration around 40 ka replaced them (with partial Denisovan introgression into modern humans), forming the basal population of regional living populations^55,56,57.

The classification of Xujiayao and most East Asian late Middle Pleistocene archaic Homo as Denisovan, which is a sister group to Neanderthals^29,30,31,32, provides insights into the morphological divergence of these two lineages. Early and late Neanderthals form a single lineage based on a series of apomorphic traits^35,45,48, but differ in such aspects as the cranial profile, mastoid process orientation, mandibular ramus breadth, suprainiac fossa form and the mandibular gonial angle. Molecular studies revealed that the late Neanderthal’s Y chromosome and mitochondrial DNA possibly were replaced with the introgression of the modern human population in the early Late Pleistocene^58,59. Alternatively, late Neanderthals may have originated from a unilineal modern human expansion into Europe 400–250 ka, accompanied by substantial introgression of local archaic genes into the autosomes⁶⁰. Except for the configuration of the temporal labyrinth and indented suprainiac fossa^2,41, East Asian late Middle Pleistocene archaic Homo aligns more closely with early Neanderthals, as well as African and European H. heidelbergensis. They share a low cranial profile with nearly parallel or slightly convergent lateral walls, wide mandibular rami, and more primitive features, such as short and inwardly inflexed mastoid process, parietal sagittal keeling, and parasagittal depression or flattening. Taken together, these findings suggest that modern humans or local archaic hominin introgression not only altered the genetic structure but also influenced the cranial morphology of later Neanderthals. This might explain the substantial morphological divergence between Denisovans and late Neanderthals.

Protein extraction

Proteins were extracted from bone powder of Xujiayao hominin samples and animal samples from the same layer (Supplementary Tables 1 and 9) using previously published methods^61,62. All sample preparations were performed using personal protective equipment in the dedicated clean room for ancient protein analysis in the Molecular Paleontology Laboratory, Institute of Vertebrate Paleontology and Paleoanthropology, Chinese Academy of Sciences. For the hominin samples, a minimal-impact sampling was conducted, which did not adversely affect macroscopic morphology analysis. A small hole in each sample was drilled to collect the bone powder, while protecting surrounding areas with aluminium foil during the drilling procedures (Supplementary Fig. 12). The holes were kept as small as possible to avoid affecting important morphological features. The 3D scan was done before protein extraction to preserve the complete morphological structure.

Samples of drilled hominin bone powder were demineralized in 0.6 M HCl and then separated. After centrifugation, the supernatant was ultrafiltered through an Amicon Ultra-4 (3 kDa) centrifugal filter unit. After washing the filtration units with 50 mM ammonium bicarbonate solution (ABC), the filtered proteins were dissolved in ABC and referred to as the acid-soluble fraction (3 kDa). The acid-insoluble pellet was washed with ultrapure water until neutral and then incubated with 50 mM ABC at 65 °C for 3 h. After centrifugation, the supernatant was collected as acid-insoluble fraction 1 (pellet). The new acid-insoluble pellet was rinsed in the same way as the first pellet, and then incubated with 50 mM ABC at 65°C for 3 h. The supernatant with pellet was regarded as acid-insoluble fraction 2 (pellet 2). For the hominin samples, three fractions were collected for further digestion and mass spectrometry analysis, and, for the animal samples, only the conventional acid-insoluble fraction 1 (pellet) fraction was used.

All of these fractions were digested with 3 μl of 0.4 μg μl⁻¹ porcine trypsin (Promega) overnight at 37 °C, then stopped by adding trifluoroacetic acid to a final concentration of 0.1%. Peptides were desalted and eluted using Pierce C18 Pipette Tips (Thermo Fisher Scientific). A small aliquot of the elution was used for MALDI-TOF analysis. The other elution of the hominin samples was dried for the LC–MS/MS analysis. For PA1490, neither the ‘pellet’ nor the ‘pellet 2’ elution showed an obvious collagen signal, so they were combined for further LC–MS/MS analysis. Extraction blanks following the identical procedure were used to estimate protein contamination during protein extraction and analysis. A bone sample from a modern camel (Camelus bactrianus) was also extracted concurrently with the samples as a positive control.

For preliminary collagen assessment, the peptides were screened on MALDI-TOF MS after protein digestion and peptide purification. All samples were analysed in triplicate on the Bruker autoflex maX MALDI-TOF mass spectrometer. Specifically, 1 μl of sample was spotted onto an MTP384 Bruker ground-steel MALDI target plate, and 1 μl of α-cyano-4-hydroxycinnamic acid matrix solution (1% in 50% ACN/0.1% trifluoroacetic acid (v/v/v)) was added to and mixed with the sample. Mass spectra were obtained over 700–3,500 m/z, and the raw files were then processed using mMass (v.5.5.0)⁶³. Peak picking parameters in mMass were S/N threshold=3.0, Abs. intensity threshold=0.0, Rel. intensity threshold=0.5%, and picking height=90(%). Baseline, smoothing and deisotoping were applied and no shoulder peaks were removed.

The LC–MS/MS experiments were performed in two independent laboratories. For the LC–MS/MS analysis at Capital Medical University (labelled as SY), the eluted peptides from the bone powder samples were analysed under DDA mode on an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) coupled to an Easy nLC 1200 system (Thermo Fisher Scientific). The peptides were initially loaded onto a trap column (100 μm inner diameter × 2 cm, C18) and then separated on an analytical column (150 μm inner diameter × 15 cm). Both columns were packed in-house with reversed-phase silica of 3 μm (Reprosil-Pur C18 AQ, Dr. Maisch). The elution of peptides was carried out using a 120 min linear gradient, increasing from 2 to 100% solvent B (where solvent A is 0.1% formic acid in water, and solvent B is 0.1% formic acid in 80% acetonitrile) at a flow rate of 500 nl min⁻¹. MS1 data were acquired across 375–1,400 m/z with a resolution of 120,000, a 250% AGC target and a maximum injection time of 50 ms. MS2 scans were performed at a resolution of 15,000 with an AGC target of 100%, a 35% normalized collision energy and a maximum injection time of 22 ms. Two runs were conducted to obtain more peptide identification from three fractions from PA1497 and the acid-soluble fraction from PA1490. Blank extractions were processed concurrently to monitor the exogenous contaminants during the procedure.

The LC–MS/MS analyses were processed on an Orbitrap Exploris 480 mass spectrometer with a FAIMS Pro device (Thermo Fisher Scientific) coupled to the Easy nLC 1200 system (Thermo Scientific) at Fudan University, Shanghai (labelled as FD). Then, 4.00 μl of sample solution was injected for each fraction, and the peptides in solution were separated on a home-packed analytical column (75 μm inner diameter × ~25 cm; 1.9 μm, ReproSil-Pur 120 C18-AQ, Dr. Maisch). The LC flow rate was 0.80 μl min⁻¹ under a 500.00 Bar maximum pressure. Mobile phase A consisted of 0.1% formic acid, and mobile phase B consisted of 80% acetonitrile and 0.1% formic acid. A 80 min gradient was used with the following profile: 5–8% B, 2 min, at a flow rate of 200 nl min⁻¹; 8–44% B, 38 min, 200 nl min⁻¹; 44–70% B, 8 min, 200 nl min⁻¹; 70–100% B, 2 min, 200 nl min⁻¹; 100% B, 10 min, 200 nl min⁻¹; 100–5% B, 2 min, 200 nl min⁻¹; 5% B, 2 min, 300 nl min⁻¹; 5–100% B, 6 min, 300 nl min⁻¹; 100% B, 10 min, 300 nl min⁻¹. Full MS scans were acquired for the first 65 min, across the range of m/z 350–1,600 with a resolution of 60,000 and an isolation window of 1.6 m/z. Standard AGC target and auto maximum injection time mode were set. The MS/MS spectra were acquired at a resolution of 15,000 with standard AGC target, a maximum injection time of 30 ms and a normalized collision energy of 30%. The column was then washed and re-equilibrated without data acquisition for 15 min.

Proteomic data analysis

To verify the taxonomic identification (Homo), the MS/MS data files from each LC–MS/MS run were searched against a custom Mammalia type I collagen database, comprising type I collagens of mammals on GenBank and UniProt. Next, raw files were searched against our previously published database⁸, which included the modern human proteome and common bone and dentine proteins translated from archaic hominin genomes (hereafter, the Harbin database). This search was conducted to obtain accurate sequences for the bone proteome of the Xujiayao hominin samples. A common contaminant database was also included in each database search, which comprised the cRAP database (https://www.thegpm.org/crap/), the contaminant database from MaxQuant⁶⁴ and a published contaminant database⁶⁵.

All PEAKS Online (v.12)⁶⁶ searches, including de novo, DB Search, PTM and SPIDER, were conducted using a precursor ion (MS1) mass tolerance of 10 ppm and a fragment ion (MS2) mass tolerance of 0.02 Da. Semispecific trypsin cleavage was applied, allowing a maximum of two missed cleavages in the searches. We included deamidation (NQ), oxidation (M), hydroxylation (P), Pyro-Glu from E and Pyro-Glu from Q as variable modifications, with no fixed modifications, allowing up to six modifications per peptide. The length range of peptides was 8–45 amino acids. A 1% false-discovery rate (FDR) at the peptide level was set, and proteins were filtered using the criteria of −10log₁₀[P] ≥ 20. Other parameters were the default.

To validate our analysis result, we also conducted a search with MaxQuant (v.2.6.0.0). Digestion and modification parameters were the same as those used in PEAKS. Proteins were filtered with 10% FDR level to identify potential low-abundance proteins^{8,11,67,68,69}, and the PSM FDR was 1%. Other parameters were set to the default. For monitoring the contaminant proteins from the experiment and comparing with other published individuals, we also searched our extraction blank data and the published Xiahe 1 and Xiahe 2 data^12,28 using the same strategy.

To assess the endogeneity of the identified proteins, overall Asn and Gln deamidation rates for each sample were first calculated on the basis of peptide intensities⁷⁰. Then, peptides associated with multiple genes were excluded, and the Asn and Gln deamidation rates were calculated for each identified protein on the basis of PSM counts. Proteins containing one or fewer Gln/Asn-containing peptides were excluded. Proteins with low deamidation rates were considered exogenous, while those with relatively high deamidation rates (Asn or Gln) in both PEAKS and MaxQuant results were classified as endogenous ancient proteins (Supplementary Table 4). Additional attempts to find other proxies to classify and confirm endogenous proteins were in Supplementary Text 7, Supplementary Tables 10–16 and Supplementary Figs. 13–19. We did not use those additional proxies here as they were not well examined with enough data.

The number of peptides and PSMs covering phylogenetically informative sites, along with their y or b ion conditions, was evaluated to assess their reliability. After removing peptides which could match bacteria or fungi, or could be attributed to other proteins from plants or animals possibly distributed around Xujiayao site by BLASTp (https://ngdc.cncb.ac.cn/blast/blastp/), only the sites with at least two remaining peptides from both PEAKS and MaxQuant were retained. The y and b ions of each spectrum were then checked. The sites were considered reliable only if at least one related spectrum contained enough y and b ions. If the sites were located at the terminals of the peptides, at least 1 of the 2 possible y or b ions was required. If not, at least 2 of the 4 possible y or b ions was required. An additional test of Penghu-specific SAP COL5A2-E1211V was also conducted, but no reliable evidence supporting E or V was obtained (Supplementary Text 5 and Supplementary Fig. 20).

Construction of consensus protein sequences and phylogenetic analysis

For each endogenous protein, peptides with abnormal or artificial post-translational modifications were excluded. Only alleles that met the strict criteria of PSM count ≥ 2, PSM ratio ≥ 25% and intensity ratio ≥ 10% were considered reliable and were retained, regardless of whether known or novel. For sites with both H/D (an oxidation product of His), P/E (di-oxidation product of Pro), N/D (deamidation product of Asn), or Q/E (deamidation product of Gln) residues, we changed them to single fixed His, Pro, Asn or Gln, respectively, as the alternative amino acids were probably derived from common taphonomic post-translational modifications. We then converted all leucine to isoleucine as they are isobaric. Consensus sequences were subsequently filtered by multisoftware confirmation, and only the amino acid positions that had overlapping coverage in both PEAKS and MaxQuant results were retained. This strategy resulted in 9 proteins with consensus sequences out of the 18 endogenous proteins (Supplementary Data 1). COL4A4 was excluded from further phylogenetic analysis as it was identified only in PA1490 with very low coverage (13 amino acids, 0.77% of the total sequence). We selected two Xujiayao hominin samples, Denisova3 (ref. ²⁹), Altai (Neanderthal)³² and Vindija33.19 (Neanderthal)³¹ in the Harbin database, and 50 randomly selected present-day human sequences from the Human Genome Diversity Project (HGDP)⁷¹ to build the comparative dataset. For genome data retaining heterozygous amino acids, we randomly chose one protein sequence per individual. P. troglodytes sequences translated from a published high-coverage genome (Clint)⁷² were used as the outgroup. We used PartitionFinder2 (v.2.1.1)⁷³ to find the optimal partitioning schemes and amino acid substitution models. Subsequently, we constructed a consensus Bayesian phylogenetic tree using the software MrBayes (v.3.2.6)⁷⁴, running for 10 million iterations in 2 independent runs, sampling every 500 generations, with the first 2 million iterations discarded as a burn-in. Additional phylogenetic trees based on different datasets were also conducted using the same procedure (Supplementary Text 4 and Supplementary Figs. 7 and 21–23).

Measurements and quantitative analysis of the hominin crania

The Xujiayao hominin fossils were scanned using high-resolution computed tomography with the following parameters: 190 kV, 130 μA and a resolution of 37.56 μm. Three-dimensional (3D) reconstruction and cranial measurements were conducted in VGSTUDIO MAX 2023.1 (Volume Graphics). A total of 26 linear distances, 3 cranial angles and 2 indices were obtained (Supplementary Table 17). The definitions and measurement follow standard procedures described in the published literature^75,76.

Comparative fossils or models were digitized using a 3D surface Scanner (Artec 3D) at a point accuracy of 50 μm. Metric data for these specimens were collected and further supplemented as needed. The comparative samples include East and Southeast Asian H. erectus, African, European and South Asian H. heidelbergensis/early Neanderthals, East Asian late Middle/Late Pleistocene archaic Homo, Neanderthals and modern humans (Supplementary Table 18).

Principal component analysis was performed on key linear distances, angles and indices, including maximum cranial breadth (XCB), biporionic breadth (POB), porion-bregmatic height (PBH), parietal sagittal chord (PAC), parietal subtense (PAS), occipital sagittal chord (OCC), occipital subtense (OCS), parietal angle (PAA), occipital angle (OCA), po-b height-cranial breadth index (PBH/XCB) and parietal curvature index (PAC/PSA). A correlation coefficient matrix was applied for variable standardization in principal component analysis, which effectively reduced biases caused by inconsistent scales among different types of indicators. Box plots were used to visualize the parameters, showing significant variation across taxonomic groups (PAA, OCA, PBH/XCB and parietal bone thickness). One-way analysis of variance was used to detect differences among groups; with P values are displayed on the box plots. All statistical graphics were generated using R (v.4.1.1)⁷⁷ using the packages ggplot2 (ref. ⁷⁸), dplyr⁷⁹ and ggpubr⁸⁰.

Ethics statement

The Institute of Vertebrate Paleontology and Paleoanthropology, Chinese Academy of Sciences has authorized the protein analyses of Xujiayao hominin fossils.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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Author notes

1. These authors contributed equally: Song Xing, Zhongyou Wu, Qingfeng Shao, Junyi Ge

Authors and Affiliations

1. New Cornerstone Science Laboratory, Key Laboratory of Vertebrate Evolution and Human Origins, Institute of Vertebrate Paleontology and Paleoanthropology, Chinese Academy of Sciences, Beijing, China

   Song Xing, Zhongyou Wu, Junyi Ge, E. Andrew Bennett, Huiyun Rao, Sungui Lin, Feng Liu, Yichen Liu, Kai Zhou, Xiaotian Feng, Qigao Jiangzuo & Qiaomei Fu

2. Centro Nacional de Investigación sobre la Evolución Humana (CENIEH), Burgos, Spain

   Song Xing

3. College of Earth and Planetary Sciences, University of Chinese Academy of Sciences, Beijing, China

   Zhongyou Wu, Junyi Ge, Sungui Lin & Qiaomei Fu

4. School of Geography, Nanjing Normal University, Nanjing, China

   Qingfeng Shao

5. Key Laboratory of Virtual Geographic Environment, Ministry of Education, Nanjing Normal University, Nanjing, China

   Qingfeng Shao

6. Center for the Study of Chinese Archaeology, Peking University, Beijing, China

   Yuhao Zhao

7. School of Archaeology and Museology, Peking University, Beijing, China

   Yuhao Zhao

Contributions

Q.F. designed and supervised the research project. S.X., Z.W., S.L., Y.Z., X.F. and Q.F. analysed data. S.X., Z.W., E.A.B., S.L., Y.Z. and Q.F. prepared the database. H.R., F.L., K.Z. and Q.F. performed or supervised the laboratory work. S.X., Z.W., Q.S., J.G. and Q.F. wrote the manuscript. E.A.B., H.R., Y.L. and Q.F. revised the manuscript. S.X., Z.W., Q.S., J.G., H.R., Q.J. and Q.F. wrote and prepared the Supplementary Information. All of authors discussed, critically revised and approved the final version of the manuscript.

Corresponding author

Correspondence to Qiaomei Fu.

Competing interests

The authors declare no competing interests.

Peer review information

Nature thanks Fabrice Demeter, Daniel Green, Ryan Paterson, Antonio Rosas, Yumeko Tarusawa and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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