Clément Huguet

Eleonore Real

Wei-Min Zhao

Aurélie Urbain
email: urbain@unistra.fr

Secretion of glucagon-like peptide-1 induced by Cynanchum pregnane derivatives: preliminary hypotheses regarding key structural elements

Keywords: Pregnanes, Cynanchum, Apocynaceae, diabetes, glucagon-like peptide-1, structure-activity relationships

In our continued efforts to explore the antidiabetic potential of pregnanes from Apocynaceae, especially from the Asclepiadoideae subfamily, sixteen derivatives 1-16 previously isolated from various Cynanchum species, have been evaluated for their ability to increase glucagon-like peptide-1 (GLP-1) release in a cell assay. As a result, otophylloside A (2), wilfoside C1N (13), and wilfoside C3N (16) were found to stimulate significantly the secretion of GLP-1, doubling the extracellular content of this incretin.

Caudatin 3-O--D-glucopyranosyl---D-oleandropyranosyl---Dcymaropyranosyl---D-cymaropyranoside (6) was also bioactive, but to a lesser extent (133% of secretion compared to control cells). This study confirms the potential of certain pregnane derivatives from subfamily Asclepiadoideae to stimulate the release of GLP-1, reduced in patients with type 2 diabetes. In addition, this study outlines some putative structure-activity relationships: the nature of the sugar chain at C-3, as well as the ester moiety at C-12 position appear to be key elements to stimulate GLP-1 secretion.

Introduction

Diabetes is a multifactorial metabolic disorder that has reached epidemic proportions, affecting 463 million people worldwide in 2019 (International Diabetes Federation, 2019). This disease is characterized by chronic high blood sugar levels, due to impaired ability of the body to produce or respond to insulin; the latter situation is called type 2 diabetes (T2D), which represents 90% of all diabetes cases. Often asymptomatic for years, T2D leads to several complications afterwards, both at the microvascular and macrovascular level. Two decades ago, glucagon-like peptide-1 (GLP-1), an intestinal hormone secreted after food intake, was identified as a key peptide in blood sugar regulation.

Indeed, this peptide increases glucoseglucagon production, delays gastric emptying, reduces food intake by promoting satiety, contributing thus to attenuate excessive postprandial glycemia [START_REF] Drucker | Mechanisms of Action and Therapeutic Application of Glucagon-like Peptide-1[END_REF]. This has led to the development of therapeutic strategies miming (GLP-1 receptor agonists) or prolonging (gliptins) this incretin effect. As T2D has been shown to be associated with an impairment of the intestinal secretion of GLP-1 [START_REF] Vilsboll | Reduced postprandial concentrations of intact biologically active glucagon-like peptide 1 in type 2 diabetic patients[END_REF], we have decided to develop an alternative strategy, focusing on natural products that could stimulate endogenous GLP-1 secretion, and consequently restore healthy physiology.

In this context, we previously discovered that two pregnane glycosides from Cynanchum marnierianum (Apocynaceae), marnieranosides A and B, significantly stimulate GLP-1 secretion of intestinal secretin tumour cells (STC-1) [START_REF] Tsoukalas | Pregnane Glycosides from Cynanchum marnierianum Stimulate GLP-1 Secretion in STC-1 Cells[END_REF]. We also demonstrated that P57, a polyhydroxylated pregnane glycoside from Hoodia gordonii, possesses a similar activity in this bioassay. Another cell assay revealed that the ethanolic extract of Cynanchum descoingsii also increases the secretion of GLP-1 (270 ± 25% when tested at 100 µg/mL) [START_REF] Tsoukalas | Recherche de sécrétagogues naturels du GLP-Cynanchum (Apocynaceae)[END_REF]; even if bioassay-guided fractionation has not been carried out, it is conceivable that the bioactivity could be due to pregnane glycosides, common in the Asclepiadoideae sub-family. On the other hand, we reported that some pregnane derivatives from Cynanchum menarandrense have no effect on STC-1 cells [START_REF] Tsoukalas | Pregnane glycosides from Cynanchum menarandrense[END_REF].

Therefore, it was interesting to go deeper in order to figure out some structure-activity relationships (SAR) involved in this specific bioactivity. In this purpose, we have decided to evaluate the GLP-1 secretagogue effect of sixteen other pregnanes 1-16, that we have previously isolated from various Cynanchum species (Fig. 1). These compounds share a similar 5,6 pregnene core corresponding to deacetylmetaplexigenin, but they differ from the esterification at C-12 position and from the glycosidic chain at C-3 (nature, number, and sequence of sugar units): cynotophylloside E (1), otophylloside A (2), 16). This study allows us to outline preliminary hypotheses about structural elements involved in the GLP-1 secretagogue activity of polyhydroxylated pregnane derivatives from Asclepiadoideae species.

Results and discussion

The GLP-1 secretion assays on STC-1 cells revealed that the pregnane derivatives 2, 16, and 13 are significantly active (Table 1), doubling the secretion of GLP-1 compared to control cells (208, 204, and 194% of secretion, respectively). Compound 6 also stimulate GLP-1 secretion, but to a lesser extent (133%). Furthermore, none of these compounds was found to disrupt STC-1 cells membrane integrity (data not shown). Up to date, there is no consensual positive control for GLP-1 secretion in STC-1 cell trials, so we have decided to include in our bioassay the pregnane P57 from H. gordonii, for which we had already reported secretagogue properties [START_REF] Tsoukalas | Pregnane Glycosides from Cynanchum marnierianum Stimulate GLP-1 Secretion in STC-1 Cells[END_REF]; its activity is confirmed in the present study with a GLP-1 release of 146 ± 3% compared to control cells.

Although different mechanisms involved in the release of glucagon-like peptide-1 have been already

proposed, little is known about the specific mechanisms induced by pregnane glycosides. Although it is difficult to establish accurate structure-activity relationships (SAR) in the absence of a clearly identified biological target, we have attempted to establish some SAR based on these results. This was made possible by the closely-related structures of compounds 1-16, varying only slightly regarding substitutions at C-3 and C-12 positions. Careful study of this set of results have highlighted the following hypotheses: esterification at C-12 position seems to be a key feature for GLP-1 secretagogue activity.

More than the nature of the substituent, it is its size that seem to be important; indeed, bioactivity can be observed with compounds esterified either by p-hydroxybenzoyl (2) or ikemaoyl (6, 13, 16) groups.

On the other hand, there is no stimulation of GLP-1 secretion in the absence of esterification (1, 7) or with a bigger substituent such as a cinnamoyl moiety (compounds 14 and 15). The sole presence of a suitable esterification at C-12 is however not sufficient to observe bioactivity, it has to be linked with the presence of an appropriate glycosidic linkage at position 3. For example, cells treated with the aglycones 3 and 10, although carrying a p-hydroxybenzoyl and an ikemaoyl group at C-12 respectively, secrete the same GLP-1 content than control cells (104% and 103% respectively). Results seem to indicate that, as for the substituent at position 12, the glycosidic chain should not be too short, and three or four saccharide moieties appears to be preferable. As there is no pregnane in this study with five or more sugars, it is impossible to conclude concerning long chains. Regarding more in details, all pregnane glycosides in this study contain a single linear glycosidic chain linked at C-3 position, consisting of one to four sugar units. As is often the case in the Asclepiadoideae sub-family [START_REF] Vleggaar | Toxic constituents of the Asclepiadaceae. Structure elucidation of sarcovimiside A-C, pregnane glycosides of Sarcostemma viminale[END_REF], most of these sugars are 2,6-dideoxy-or 6-deoxyhexoses, more rarely glucose (compound 6). The sugar units are al --L-diginose -L-cymarose when it is at the extremity of the glycosidic chain. The results tend to show that the presence of a -D-cymaropyranose directly linked to the genin conditions also the activity. For example, compounds 2 and 11 display almost the same structure: they are both esterified at position 12 by a p-hydroxybenzoyl group and possess a tetrasaccharidic linear chain at C-3; they just differ by the nature of the first sugar: -D-cymaropyranose for compound 2 and -D-digitopyranose for compound 11. While 2 is one of the most active pregnane derivatives in this study, compound 11 has no effect on GLP-1 secretion. In the same way, pregnane glycoside 16 (with a -D-cymarose as first sugar moiety) is bioactive while 5 (having a -D-digitose at this position) is not. Finally, the rather weak activity of compound 6 could be due to the nature of the sugar at the extremity of the moiety, i.e. a hexose and not a deoxy-or dideoxyhexose, but this hypothesis would require, to be reinforced, the evaluation of other similar compounds.

To summarize, all bioactive compounds in this study (2, 6, 13, and 16) share similar features:

i. they are esterified at C-12 by an organic monocarboxylic acid of moderate size;

ii. this esterification is completed by the presence of a glycosidic linear fragment at C-3, consisting of 3-4 sugar units, preferably 2,6-dideoxysaccharides, wit -D-cymaropyranose directly attached to the pregnane skeleton.

These observations are in accordance with previously published data, reporting the GLP-1 secretagogue activity of P57 and of marnieranosides A and B [START_REF] Tsoukalas | Pregnane Glycosides from Cynanchum marnierianum Stimulate GLP-1 Secretion in STC-1 Cells[END_REF]. Interestingly, marnieranosides and P57 display structural elements similar to those aforementioned (Supplem. S1): P57 also exhibits a 5,6 pregnene core with a high degree of hydroxylation (hoodigogenin), esterified at C-12 by a tigloyl moiety, while marnieranosides are 6,7 pregnenes esterified by a benzoyl group at C-12 (and C-20).

These three compounds also possess a linear saccharide chain at C-3, consisting of several 2,6-dideoxyor 6-deoxyhexoses, with -D-cymaropyranose directly linked to the steroid core. On the contrary, menarandrosides A-E and carumbellosides I and II had no effect on GLP-1 secretion (Tsoukalas et al., 2017) 5,6 pregnenes, isolated from Cynanchum menarandrense, exhibit a rather rudimentary structure (Supplem. S1), with an unesterified genin and a sugar linkage at C-3 made up of one to two glucose moieties exclusively. All these data reinforce the above-mentioned SAR hypotheses.

The high degree of oxidation and esterification of the genin, as well as the peculiar nature of encountered sugars, makes pregnanes from Asclepiadoideae remarkable natural products. The present study highlights the importance of these specific features for bioactivity: both the nature of the sugar chain at C-3 and the ester at C-12 are key elements to increase the release of GLP-1, a peptide involved in blood glucose regulation. This work provides a starting point for further in-depth medicinal chemistry studies to identify a lead compound. Then, mechanistic studies and in vivo confirmation of the hypoglycemic potential of such molecule could be undertaken.

Experimental

Compounds isolation

The isolation and identification of each Each pregnane evaluated in this study was provided by the Department of Natural Product Chemistry of Shanghai Institute of Materia Medica. Their isolation and identification have been previously reported as following (> 91% of relative purity at 220 nm); cynotophyllosides C (8), D ( 12), E (1), and caudatin 3-O--D-thevetopyranosyl---Dcymaropyranosyl---D-digitoxopyranoside (5) have been obtained from Cynanchum otophyllum C.K. Schneid. [START_REF] Li | Bioactive C-21 Steroidal Glycosides from the Roots of Cynanchum otophyllum That Suppress the Seizure-like Locomotor Activity of Zebrafish Caused by Pentylenetetrazole[END_REF], as well as otophylloside A (2) [START_REF] Mu | Two new antiepilepsy compounds -otophyllosides A and B[END_REF], otophylloside T (9) [START_REF] Shen | Four new C21 steroidal glycosides from Cynanchum otophyllum Schneid[END_REF], qingyangshengenin (3) and caudatin (10) [START_REF] Ma | New pregnane glycosides from the roots of Cynanchum otophyllum[END_REF]. Caudatin 3-O--Dglucopyranosyl-(1 4)--D-oleandropyranosyl---D-cymaropyranosyl---Dcymaropyranoside (6) has been purified from Cynanchum caudatum roots [START_REF] Warashina | C21 Steroidal Glycosides from Cynanchum wilfordii[END_REF].

Roots of Cynanchum wilfordii (Maxim.) Hemsl. have afforded qingyangshengenin 3-O--Dcymaropynanosyl---D-digitoxopyranoside (4) [START_REF] Li | Identification and Evaluation of Antiepileptic Activity of C21 Steroidal Glycosides from the Roots of Cynanchum wilfordii[END_REF] and qingyangshengenin 3-O--D-oleandropyranosyl---D-cymaropyranosyl---D-digitoxopyranoside (11) (Xiang et al., 2009). Wilfosides M1N (7), C1N (13), K1N ( 14) (Tsukamoto et al., 1985a), and C3N ( 16) (Tsukamoto et al., 1985b) have been also obtained from C. wilfordii roots. Cynauricoside A (15) [START_REF] Liu | Appetite suppressing pregnane glycosides from the roots of Cynanchum auriculatum[END_REF] have been isolated from Cynanchum auriculatum Royle ex Wight roots.

Materials, chemicals and reagents

STC-1 cells (ATCC® CRL-

The cell-culture material was purchased as following: GLP-1 a medium (DMEM, Gibco), Hanks' Balanced Salt Solution (HBSS, Thermo Fisher Scientific), fetal bovine serum (FBS, Gibco Thermo Fisher Scientific), penicillin, and streptomycin (Lonza). The pregnane P57 was purchased from LGC Standards. The reading of the 96-well plates was performed with a SAFAS Monaco plate reader.

Bioassays

GLP-1 secretion assays and cytotoxicity assays were carried out as described previously [START_REF] Huguet | Glucagon-like peptide-1 secretagogues from the leaves of Oparanthus teikiteetinii[END_REF]. Briefly, compounds 1-16 and P57 were dissolved in HBSS containing 1% DMSO for a final added to the cells in 96-well plates for 2 hours. HBSS containing 1% DMSO was used as control, and P57 as positive control. Afterwards, the GLPtwo distinct experiments for a total of six replicates (n=6), except for P57 tested twice in both experiments (n=4). Results were expressed as the percentage of secreted GLP-1 compared to the basal secretion of cells treated with the vehicle only (considered as 100% of secretion). For the statistical analysis, a one-way ANOVA with post hoc Dunnett test was performed (*** padj 0.001).

  9), caudatin (10), qingyangshengenin 3-O--D-oleandropyranosyl---Dcymaropyranosyl---D-digitoxopyranoside (11), cynotophylloside D (12), wilfoside C1N (13), wilfoside K1N (14), cynauricoside A (15), and wilfoside C3N (

Table 1 .

 1 Glucagon-like peptide-1 secretagogue activity induced by compounds 1-16 and by P57, used as positive control (tested at 100 µM on STC-1 cells).

	Compound	GLP-1 secretion stimulation (%)
	control	100 ± 19
	P57	146 ± 3
	1	118 ± 16
	2 ***	208 ± 17
	3	104 ± 20
	4	95 ± 11
	5	92 ± 8
	6	133 ± 13
	7	98 ± 18
	8	87 ± 5
	9	92 ± 17
	10	104 ± 19
	11	118 ± 13
	12	85 ± 14
	13 ***	194 ± 30
	14	95 ± 12
	15	114 ± 24
	16 a	204 ± 18
		.
	*** padj	.

b Ikem: ikemaoyl; p-OH-Bz: p-hydroxybenzoyl; Cin: cinnamoyl. c Stimulation of GLP-1 secretion in STC-1 cells when tested at 100 µM.

Acknowledgments

The authors would like to thank Nicolas Humbert for his help with microplate reading, as well as Rafik Haderbache and Dr Christian D. Muller for the cytotoxicity assays and image cytometry measurements.

Conflicts of Interest


W Knafo

T D Matsuda

F Hardy

D Aoki

J Flouquet

High-field moment polarization in the itinerant ferromagnet URhSi

Keywords: numbers: 71.27.+a, 74.70.Tx, 75.30.Kz, 75.30.Mb

We report a high-magnetic-field study of the itinerant ferromagnet URhSi. Magnetization and electrical resistivity were measured under magnetic fields µ0H up to 58 T applied along the directions a, b, and c of the orthorhombic structure and temperatures T ranging from 1.5 to 50 K. For H b, pseudo-metamagnetism at µ0Hm 30 -40 T is associated with a broad step in the magnetization and a maximum in the resistivity. The properties of URhSi are discussed and compared with those of the isostructural superconducting ferromagnets URhGe and UCoGe and of the superconducting paramagnet UTe2.

I. INTRODUCTION

While superconductivity sets in at the antiferromagnetic instability of a large number of materials including heavy-f -electron, 1 Fe-based, 2 and organic 3 systems, its occurrence close to a ferromagnetic instability was reported only in a few U-based heavy-electron systems. 4 Superconductivity develops in the ferromagnets URhGe [5] and UCoGe [6] at ambient pressure and UGe 2 [7] and UIr [8] under pressure. Recently, the paramagnet UTe 2 , which is suspected to lie in the vicinity of a ferromagnetic instability, was found to become superconducting at ambient pressure too. 9,10 Under a magnetic field applied along a hard magnetic axis (H b), a reentrance of superconductivity or an anomalous S-shape of the superconducting field have been reported in URhGe [11], UCoGe (H b) [12], and UTe 2 [9,13,14] at ambient pressure. In UGe 2 under pressure, a S-shape of the superconducting field was also reported in a field applied along the easy magnetic axis (H a) [15].

A challenge in these U-based magnets is to understand the relationship between field-induced superconductivity and metamagnetism, in which changes of the magnetic fluctuations possibly couple to a Fermi surface reconstruction. In URhGe, a metamagnetic transition, evidenced as a field-induced first-order step in the magnetization at µ 0 H m = 12 T, and associated with a reorientation of the moments (rotating from the c to the b axes), coincides with the field-reentrance of superconductivity. 11 In this material, hydrostatic pressure leads to the increase of H m and the disappearance of reentrant superconductivity, 16 while uniaxial pressure applied along b reduces H m and boosts the superconducting temperature. 17 In UTe 2 , field-induced superconductivity also develops in the vicinity of a metamagnetic transition at µ 0 H m 35 T [13,18,19,14]. The situation in UCoGe is more subtle, since a fall of T C observed by magneto-transport at µ 0 H * = 15 T coincides with a S-shape of the superconducting borderline H sc [12], while a pseudo-metamagnetic crossover is observed at a much higher field µ 0 H m 50 T [20]. To understand the interplay between magnetism and superconductivity in U-based ferromagnets and nearly-ferromagnets, a target is, thus, to characterize their high-field properties, and in particular, their high-field moment polarization processes.

We present here a high-magnetic field study of the itinerant ferromagnet URhSi, which is isostructural to the ferromagnetic superconductors URhGe and UCoGe (Pnma orthorhombic structure), but where superconductivity has not been observed yet 21-23 (see Table I). The Curie temperature T C = 10.5 K of URhSi is quite comparable to that (T C = 9.5 K) of URhGe. For the three materials, magnetic susceptibility measurements indicate similar Ising anisotropies, where c is the easy magnetic axis, a is the hardest magnetic axis, and b is an intermediate hard magnetic axis. 20,24,25 In this study, we have studied the field-induced properties of URhSi in fields applied along the three crystallographic directions a, b, and c. In a magnetic field applied along b, we observe and characterize a broad pseudo-metamagnetic crossover in URhSi. In the discussion, comparison is made with similar metamagnetic phenomena observed in the superconducting ferromagnets URhGe, UCoGe and paramagnet UTe 2 .

II. EXPERIMENTAL TECHNIQUES

Single crystals were grown by the Czochralski-pulling method in a tetra-arc furnace with a stoichiometry composition under argon-gas atmosphere (see Ref. [25] for more details). Crystal orientation was checked by x-ray diffraction. Pulsed magnetic fields were generated using standard 60-T magnets at the LNCMI-Toulouse pulsed field facility (France). Magnetization was measured us- ing compensated coils on a 1.3 × 1 × 0.9 mm 3 sample under pulsed magnetic fields up to 53 T. Pulsed-field magnetization data were adjusted (to eliminate an offset) to low-field magnetization data measured up to 5 T using a commercial 'MPMS' magnetometer from Quantum Design. Electrical resistivity under pulsed magnetic fields up to 58 T was measured by the four-contacts technique, and extracted using a numerical lock-in, on a 1 × 0.3 × 0.1 mm 3 sample with a current of 10 mA, with a 40 -70 kHz, applied along the a direction.

III. HIGH MAGNETIC-FIELD PROPERTIES

Figure 1 presents the low-temperature (T = 1.5 K) magnetization of URhSi in a magnetic field applied along its three main crystallographic directions a, b, and c. Similarly to the recent report on URhSi in Ref. [23], and to the cases of URhGe [24] and UCoGe [20], a is found to be the hardest magnetic axis, while c is the easy magnetic axis. However, a different magnetic anisotropy, with b being the hardest magnetic axis instead of a, was reported from magnetization data on URhSi in Refs. [22,26]. A sensibility of the physical properties to sample mis-orientation combined with crystal mosaicity may explain the differences between the different sets of data. In a field H applied along c, the spontaneous magnetization M s = 0.5 µ B /U is reached at very low fields, indicating the alignment of the magnetic domains; beyond the domains alignment, the magnetization M continues to increase with H, reaching 1.1 µ B /U at µ 0 H = 53 T, and is probably associated with a field-induced quenching of magnetic fluctuations. In a magnetic field applied along a, an almost linear magnetization is observed in the whole field range, with a slope similar to that of M (H) in high fields along c. In a field applied along the intermediate magnetic axis b, a broad crossover can be seen in the M (H) data, leading to a kink of M/H at µ 0 H kink M = 24 T and to a maximum of slope at µ 0 H max ∂M/∂H = 32 T (at T = 1.5 K). This anomaly is identified as the signature of a pseudo-metamagnetic crossover, i.e., a similar but broader phenomenon than a first-order metamagnetic transition.

Figure 2 shows the resistivity ρ versus magnetic field data measured for H b, where a broad maximum is observed at µ 0 H max ρ , which equals 42 T at T = 1.5 K. A maximum of the slope of ρ(H) at H max ∂ρ/∂H precedes the maximum of ρ(H). The fields H max ρ and H max ∂ρ/∂H both decrease when the temperature is raised, before disappearing at temperatures T > T C = 10.5 K. H max ρ and H max ∂ρ/∂H , as well as H kink M and H max ∂M/∂H , were defined using different criteria, but they all characterize the same broad moment polarization process. These different field and temperature scales are summarized in the magnetic phase diagram plotted in Figure 3. In the following, we will associate the 'mean' characteristic field µ 0 H m = 30-40 T to the pseudo-metamagnetic crossover occurring in URhSi for H b.

From ρ versus T 2 plots extracted at different magnetic fields and shown in Figure 4(a), a Fermi-liquidlike fit ρ = ρ 0 + AT 2 to the data under temperatures T ≤ 4 K permits to extract the quadratic coefficient A. Its magnetic-field-variation is shown in Figure 4(b). After a small decrease in fields up to 5 T, the coefficient A increases and passes through a maximum at 38 T, before decreasing in higher fields. Within a crude Fermi-liquid picture assuming a Kadowaki-Woods behavior, which is often followed in heavy-fermion compounds, A is proportional to the square m * 2 of the effective mass 27 and is driven by electronic correlations associated with quantum magnetic fluctuations (see the Ce 1-x La x Ru 2 Si 2 and URhGe cases [28,29,30,24]). In URhSi, the broad step in M (H) and maximum in ρ(H) coincide with the

IV. DISCUSSION

We compare here the high-magnetic-field properties of URhSi with those of the isostructural itinerant ferromagnets URhGe and UCoGe, and of the paramagnet UTe 2 .

Although they all have the same Ising-type anisotropy characterized by M c > M b > M a , the three materials URhSi, URhGe, and UCoGe present different initial slopes (∂M/∂H) i hierarchies in their low-temperature magnetization. While (∂M/∂H) b > (∂M/∂H) c > (∂M/∂H) a is found in URhGe [24], we observe here the hierarchy (∂M/∂H) c > (∂M/∂H) b > (∂M/∂H) a in URhSi, which is similar to observed in UCoGe. 20 These slopes are related to the field-quenching of the magnetic fluctuations and indicate that the magnetic fluctuations continue to subsist in high magnetic field, where a large polarization is achieved. However, little is known about the magnetic fluctuations of these systems in high magnetic field. At zero field, ferromagnetic fluctuations were observed in the ferromagnets UGe 2 and UCoGe and their magnetic anisotropy was suggested to be an important parameter for the appearance of superconductivity 31-33 .

A key action of a magnetic field applied along the hard magnetic axis b is, by reducing the Curie temperature T C , to drive to a ferromagnetic quantum instability. Remarkably, metamagnetism or pseudo-metamagnetism occurs in the four compounds URhSi, URhGe, UCoGe, and UTe 2 in a magnetic field applied along their hard magnetic axis (H b) and its onset coincides with a lowtemperature magnetization reaching M 0.3-0.4 µ B /U [see Figure 5 scales, only the kink at the onset of the step is observed, and the transition to the high-field regime is not completed at 53 T. Figure 5(b) shows the resistivity versus field of the four materials in a magnetic field H b, at the temperatures T = 1.5 K for URhGe and URhSi, T = 1.4 K for UTe 2 , and T = 3 K for UCoGe. Here also, the maximum in ρ(H) delimiting the high-field regime is much less marked in URhSi than in UTe 2 and URhGe, UCoGe being in an intermediate situation. The large low-temperature and zero-field resistivity [in relation with the small residual resistivity ratio ρ(300K)/ρ(T → 0) 2.5] indicates the low quality of our URhSi single crystals, in comparison with the URhGe, UTe 2 , and UCoGe crystals whose resistivity is presented (residual resistivity ratios of 11, 25, and 45, respectively; see also Refs. [18,20]). In these systems, due to a sensibility of the physical properties to the field direction, a strong crystal mosaicity can lead to broader anomalies in the field variations of A and M . However, it is difficult to infer whether the quality of the URhSi crystals is related -or not -with the broad nature of the pseudo-metamagnetic crossover reported here. Further investigations are needed to clarify this point.

A comparison of the magnetic-field-temperature phase diagrams of URhSi, URhGe, UTe 2 , and UCoGe in a field H b is shown in Figure 6(a) (see also Ref. [20]). For all samples, the metamagnetic field decreases with increasing temperature, before vanishing in temperatures higher than T C in URhGe and URhSi. In UCoGe, the situation is different since anomalies are observed at the metamagnetic field under temperatures up to 20 K, i.e., much higher than T C = 3 K, and seem to disappear at temperatures higher than T max χ = 38 K. Interestingly, in URhGe the magnetic susceptibility measured for H b is maximal at T C = T max χ = 9.5 K and this borderline is connected with the metamagnetic field H m . In UTe 2 , which is paramagnetic, contrary to the three other samples, a sharp first-order transition is observed at H m at low temperature and ends into a critical endpoint at T CEP = 7 K, above which a pseudo-metamagnetic crossover is established 18 . In URhGe and UTe 2 , the sharpness of the maximum in χ(T ) at T max χ is related with the sharpness of the anomalies at H m . Oppositely, in URhSi the broad nature of the crossover at H m may be related with the non-observation of a maximum in χ(T ) (see Ref. [25]). Figure 6 (b) shows that, similarly to the URhSi case, a maximum of A at H m is observed in URhGe and UTe 2 under H b [18,30]. The maximum of A is sharper and more intense in URhGe and UTe 2 than in URhSi, indicating stronger magnetic fluctuations in these two materials. A study of URhGe further showed that, the higher the residual resistivity ratio is, the higher the A coefficient and the reentrant superconducting temperature T sc are. 30 The critical magnetic fluctuations responsible for the enhancement of A are also suspected to drive to fieldreentrant superconductivity in URhGe and UTe 2 . On one side, the relatively small enhancement of A at H m in URhSi suggests that field-induced superconductivity may not be expected at H m . On the other side, its similarities with URhGe, and to a lesser extend with UCoGe and UTe 2 , stress that URhSi could be a candidate for superconductivity, once the condition of higher-quality single crystals (i.e., with a residual resistivity ratio at least > 10) would be fulfilled. Indeed, crystal imperfections are known to destroy unconventional superconductivity.

In heavy-fermion Ising paramagnets in a field applied along their easy magnetic axis, a correlatedparamagnetic (CPM) regime is delimited by the temperature T max χ , where a maximum occurs in χ(T ), and by the pseudo-metamagnetic field H m , where a moment polarization occurs. 34 In these systems, the CPM regime is characterized by strong antiferromagnetic fluctuations and often indicates the proximity of an antiferromagnetic instability. In the case of U-based ferromagnets in a field applied along a hard magnetic axis, no microscopic picture has been proposed yet to describe the maximum in the magnetic susceptibility and its relation with metamagnetism. Interestingly, the ferromagnet URhGe is in the vicinity of an antiferromagnetic instability, which can be induced by Ir-doping on the Rh-site [35,36]). Further studies are now needed to identify how the physics, including superconductivity, of the U-based ferromagnets or nearly-ferromagnets is affected by their proximity to ferromagnetic and antiferromagnetic quantum phase transitions.

V. CONCLUSION

In summary, we have shown that for H b a pseudometamagnetic crossover occurs at µ 0 H m 30 -40 T in the ferromagnet URhSi. Similar moment polarization processes have been reported in the isostructural ferromagnetic superconductors URhGe and UCoGe for H b. Its strong similarities with URhGe, UCoGe and UTe 2 suggest that URhSi is a potential candidate for zero-field and field-induced superconductivity, with the condition that high-quality single crystals could be grown. Future experimental and theoretical works are needed to achieve a microscopic description of the magnetic interactions, with the aim to describe the transitions and crossovers occurring at T C , T max χ and H m . Another challenge will be to determine whether the magnetic fluctuations are coupled or not to a Fermi surface instability.

(

  20] [6,12,20] [21,22] [9,13,18,19] † In UCoGe, a S-shape of Hc,2 is observed at 10-15 T [12].

FIG. 1 .

 1 FIG. 1. Magnetization M versus magnetic field H of URhSi at the temperature T = 1.5 K, for H a, b, c. Open-symbol data were measured under steady fields, full-line data were measured under pulsed fields. Some oscillations in the data correspond to non-physical noise. The inset shows a zoom on M/H versus H data for H b.

FIG. 2 .

 2 FIG. 2. Electrical resistivity ρ versus magnetic field H, at different temperatures T from 1.5 to 50 K, for H b.

FIG. 4 .

 4 FIG. 4. (a) Electrical resistivity ρ versus the square of temperature T 2 , at different magnetic field µ0H from 1 to 50 T, for H b. (b) Magnetic field variation of the quadratic coefficient A of the resistivity.

  (a)]. While a sharp step-like anomaly is observed at µ 0 H m 35 T in UTe 2 and at µ 0 H m 12 T in URhGe, a broad anomaly is reported at µ 0 H m 30 -40 T in URhSi. In UCoGe, due to higher field

FIG. 5 .

 5 FIG. 5. (a) Magnetization M versus magnetic field H of URhSi and UCoGe at T = 1.5 K, of URhGe at T = 1.8 K, and of UTe2 at T = 1.4 K. (b) Electrical resistivity ρ versus magnetic field H of URhSi, URhGe at T = 1.5 K, of UTe2 at T = 1.4 K and of UCoGe at T = 3 K. (data on UCoGe, URhGe, and UTe2 were taken from Refs. [20,30])

FIG. 6 .

 6 FIG. 6. (a) Magnetic-field-temperature phase diagram of URhSi, URhGe, UCoGe, and UTe2 for H b, (b) Quadratic coefficient A of the electrical resistivity versus magnetic field of URhSi, URhGe, and UTe2 for H b. (data on UCoGe, URhGe, and UTe2 were taken from Refs. [19,20])

TABLE I .

 I Curie temperature TC , spontaneous moment Ms, superconductive temperature TSC at H = 0 and, for H b, temperature T max χ at the maximum of the magnetic susceptibility, metamagnetic field Hm, and maximum T max RSC of the reentrant superconductive temperature, in URhGe, UCoGe, URhSi, and UTe2.

ACKNOWLEDGEMENTS

We acknowledge Atsushi Miyake for sending us his data on the magnetization of UTe 2 and on the A coefficient of URhGe. This work at the LNCMI was supported by the Programme Investissements d'Avenir under the project ANR-11-IDEX-0002-02 (reference ANR-10-LABX-0037-NEXT). We acknowledge the financial support of the Cross-Disciplinary Program on Instrumentation and Detection of CEA, the French Alternative Energies and Atomic Energy Commission, and KAKENHI (JP15H05882, JP15H05884, JP15K21732, JP16H04006, JP15H05745, JP19H00646).


Frido Welker
email: frido.welker@bio.ku.dk

Jazmín Ramos-Madrigal

Martin Kuhlwilm

Wei Liao

Petra Gutenbrunner

Marc De Manuel

Diana Samodova

Meaghan Mackie

Morten E Allentoft

Anne-Marie Bacon

Matthew J Collins

Jürgen Cox

Carles Lalueza-Fox

Jesper V Olsen

Fabrice Demeter

Wei Wang
email: wangw@sdu.edu.cn

Tomas Marques-Bonet

Enrico Cappellini
email: ecappellini@bio.ku.dk

Enamel proteome shows that Gigantopithecus was an early diverging pongine

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We identified no endogenous proteins from the dentine, but instead recovered an ancient enamel proteome composed of 409 unique peptides matching 6 endogenous proteins: amelogenin X (AMELX), ameloblastin (AMBN), amelotin (AMTN), enamelin (ENAM), matrix metalloproteinase-20 (MMP20) and alpha-2-HS-glycoprotein (AHSG, also known as FETUA; Extended Data Table 1). This observation extends the survival of ancient mammalian proteins to a thermal age, obtained by normalizing the chronological age to a constant temperature of 10 °C, of approximately 11.8 million years (Myr)@10 °C (Extended Data Table 2). Such a thermal age is well beyond the thermally oldest DNA (0.25 Myr@10 °C, Sima de los Huesos, Spain [START_REF] Meyer | Nuclear DNA sequences from the Middle Pleistocene Sima de los Huesos hominins[END_REF] ), collagen (0.22 Myr@10 °C, Happisburgh, UK [START_REF] Wadsworth | Proteome degradation in fossils: investigating the longevity of protein survival in ancient bone[END_REF] ) and enamel proteome (2.2 Myr@10 °C, Dmanisi, Georgia [START_REF] Cappellini | Early Pleistocene enamel proteome from Dmanisi resolves Stephanorhinus phylogeny[END_REF] ) reported to date. The Chuifeng Cave enamel proteome is thus, to our knowledge, the oldest Cenozoic skeletal proteome currently reported (Fig. 1). The survival of a subtropical proteome from approximately 2 Ma suggests that chronologically older specimens from higher latitudes are likely to contain preserved ancient proteomes as well.

The content of the recovered enamel proteome is consistent with previously reported ancient enamel proteomes [START_REF] Cappellini | Early Pleistocene enamel proteome from Dmanisi resolves Stephanorhinus phylogeny[END_REF][START_REF] Stewart | The identification of peptides by nanoLC-MS/MS from human surface tooth enamel following a simple acid etch extraction[END_REF][START_REF] Castiblanco | Identification of proteins from human permanent erupted enamel[END_REF] , with the addition of several peptides deriving from a single region of AHSG. Peptide matches to these proteins covered a minimum of 43 informative single amino acid polymorphisms (SAPs; Supplementary Table 3). In addition, the retrieved protein regions fell largely within areas previously recovered from an Early Pleistocene Stephanorhinus enamel proteome from Dmanisi 17 (Supplementary Fig. 1). The absence of peptides specific to amelogenin Y (AMELY) suggests that the sampled molar might have belonged to a female Gigantopithecus. Alternatively, male-diagnostic AMELY-specific peptides were not observed due to their degradation beyond the limit of detection of the instrument. The endogenous enamel proteome sequence coverage of 456 amino acids is lower than the previously recovered sequence coverage for a Dmanisi Stephanorhinus specimen (875 amino acids [START_REF] Cappellini | Early Pleistocene enamel proteome from Dmanisi resolves Stephanorhinus phylogeny[END_REF] ; Supplementary Table 1). This observation is consistent with the older thermal age for Chuifeng Cave, compared to Dmanisi [START_REF] Cappellini | Early Pleistocene enamel proteome from Dmanisi resolves Stephanorhinus phylogeny[END_REF] .

We replicated enamel demineralization using two acids (TFA and HCl). The chromatograms of these two extracts showed that different peptide populations were released (Extended Data Fig. 3). Owing to the partial acidic hydrolysis [START_REF] Cristobal | Toward an optimized workflow for middle-down proteomics[END_REF] , which potentially occurs alongside demineralization, peptide populations with a wider range of acidity (Extended Data Fig. 4a) and hydrophobicity (Extended Data Fig. 4c) are generated using TFA. Our TFA-based demineralization returned 127 more unique non-overlapping peptide sequences than did the HCl-based demineralization (Extended Data Fig. 4e). The TFA extract, therefore, outperformed the HCl-based extraction, despite being carried out on a smaller amount of starting material [START_REF] Cappellini | Early Pleistocene enamel proteome from Dmanisi resolves Stephanorhinus phylogeny[END_REF] . Ultimately, the extended coverage of TFA-based demineralization increased the identification rate of informative SAPs, enhancing the phylogenetic information obtained (Extended Data Fig. 4d). Finally, the HCl-and TFA-demineralized samples had similar deamidation rates and average peptide lengths (Extended Data Fig. 5), which indicates that the two acids released peptide populations that were modified to the same extent.

The Gigantopithecus enamel proteome is characterized by extensive diagenetic modifications, such as high rates of deamidation (Fig. 2a) and Fig. 1 | Thermal age of Chuifeng Cave, China, in the subtropics of Southeast Asia. Chronological (circles) and thermal (coloured wedges, @10 °C) ages of other Cenozoic ancient genomes and proteomes are given for comparison. For Chuifeng Cave, dark red shading indicates thermal age calculated at a chronological age of 1.9 Ma and lighter shading indicates 95% confidence intervals based on a chronological age of 1.7 and 2.1 Ma, respectively. Inset, geographical location of Chuifeng Cave in the subtropics of Southeast Asia (red asterisk). Base map was generated using public domain data from http://www. naturalearthdata.com/. The red asterisk also encloses the entire known geographical range of G. blacki fossils. a high degree of degradation, as indicated by relatively short peptide lengths (Fig. 2b); this is expected for an ancient proteome preserved in subtropical conditions. When quantifying peptide intensities using labelfree quantification (LFQ) implemented in MaxQuant [START_REF] Cox | MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification[END_REF] , we observed that summed and normalized MS1 spectral intensities were higher for shorter peptides than for longer peptides (Extended Data Fig. 4b). Finally, the peptide lengths of the Chuifeng Cave enamel proteome were shorter than those identified in thermally younger enamel proteomes (Fig. 2b).

Enamel-specific proteins are modified in vivo through protein phosphorylation, alternative splicing of AMELX, and MMP20-and KLK4mediated proteolysis. Such modifications could survive in ancient proteomes. We detected evidence of surviving in vivo post-translational modifications, such as serine phosphorylation in the S-x-E/phS motif, which is recognized by the secreted kinase FAM20C (Fig. 2c). The FAM20C kinase regulates the phosphorylation of extracellular proteins involved in biomineralization [START_REF] Tagliabracci | Secreted kinase phosphorylates extracellular proteins that regulate biomineralization[END_REF] . We also observed two alternative-splicing-derived isoforms of AMELX (Fig. 2d). These observations are similar to those from other Early Pleistocene enamel proteomes [START_REF] Cappellini | Early Pleistocene enamel proteome from Dmanisi resolves Stephanorhinus phylogeny[END_REF] . The Gigantopithecus enamel proteome therefore demonstrates that such in vivo modifications can be recovered from hominid samples across the Pleistocene.

To achieve a protein-based phylogenetic placement of Gigantopithecus, we compared the enamel proteome sequences we retrieved with those of extant apes (Hominoidea). We used publicly available whole-genome sequence data to predict enamel protein sequences from relevant species [START_REF] Prado-Martinez | Great ape genetic diversity and population history[END_REF][START_REF] Nater | Morphometric, behavioral, and genomic evidence for a new orangutan species[END_REF] (Supplementary Table 2, Supplementary Figs. 23456789101112). Our results show that Gigantopithecus represents a sister taxon to all extant orangutans (Pongo spp.) and forms a monophyletic group with extant pongines (Fig. 3a, Extended Data Figs. 6,7). We then attempted to estimate the divergence time between Gigantopithecus and Pongo species using two approaches: (1) a pairwise distance approach and (2) a Bayesian approach using MrBayes (see Methods). Although confidence intervals obtained for the divergence estimates of the Pongo-Gigantopithecus split are large, our results indicate that Gigantopithecus diverged from the extant Pongo species in the Middle or Late Miocene (approximately 10 Ma or 12 Ma using the Bayesian or pairwise distance approaches, respectively; Fig. 3b). This suggests that, despite an exclusively Pleistocene fossil record, Gigantopithecus is a member of an early radiation of pongines, whose diversity peaked during the Middle and Late Miocene (Fig. 3b). Our results thereby resolve not only the phylogenetic position of Gigantopithecus, but also renew the debate on the evolutionary relationships between extant hominids and early hominids present in the fossil record [START_REF] Harrison | Apes among the tangled branches of human origins[END_REF] .

The presence of AHSG in the Gigantopithecus proteome is intriguing, as this protein is not commonly observed in (modern) hominid enamel proteomes. All retrieved peptides derive from a single, highly conserved region that is bordered by disulfide cysteine bonds on either side (Extended Data Fig. 8). AHSG is highly glycosylated in vivo, but we observed no glycosylation during our bioinformatics analysis. The observed sequence contains regularly spaced aspartic acid residues that provide a suitable motif for binding to basic calcium phosphate lattices [START_REF] Tang | Calcium binding to amino acids and small glycine peptides in aqueous solution: toward peptide design for better calcium bioavailability[END_REF] . The notion that this specific peptide sequence is involved in biomineral binding is supported by three observations: (1) this region is presented on the external surface of AHSG [START_REF] Heiss | Structural basis of calcification inhibition by alpha 2-HS glycoprotein/ fetuin-A. Formation of colloidal calciprotein particles[END_REF] been demonstrated to bind biominerals in other systems as well [START_REF] Demarchi | Protein sequences bound to mineral surfaces persist into deep time[END_REF] ; and

(3) this type of binding enhances peptide preservation [START_REF] Demarchi | Protein sequences bound to mineral surfaces persist into deep time[END_REF] . AHSG acts as a key component of bone and dentine mineralization by inhibiting the extrafibrillar mineralization of collagen type I helices [START_REF] Price | Mineralization by inhibitor exclusion: the calcification of collagen with fetuin[END_REF] and has previously been hypothesized to have a role in amelogenesis [START_REF] Bartlett | Protein-protein interactions of the developing enamel matrix[END_REF] . Our extracts contained no endogenous plasma proteins, such as human serum albumin, or other common dentine proteins, such as collagen type I. We also did not identify any AHSG peptides in our dentine sample. We therefore exclude the possibility that the AHSG peptides derived from dentine. Gigantopithecus is known to have relatively long enamel formation times and thick enamel compared to several extant and extinct hominids, including its phylogenetically closest relatives [START_REF] Dean | Enamel thickness and development in a third permanent molar of Gigantopithecus blacki[END_REF][START_REF] Kono | A 3-dimensional assessment of molar enamel thickness and distribution pattern in Gigantopithecus blacki[END_REF] . We therefore hypothesize that Gigantopithecus recruited AHSG as an additional molecular component to favour enamel biomineralization during prolonged amelogenesis, ultimately playing a part comparable to the one it has in bone and dentine mineralization [START_REF] Bartlett | Protein-protein interactions of the developing enamel matrix[END_REF] .

Our results reveal the long-debated phylogenetic position of Gigantopithecus as an early diverging pongine. We demonstrate that ancient enamel proteomes can be retrieved from Early Pleistocene samples preserved in subtropical conditions, well beyond the current limitations of biomolecular research in hominid and hominin evolution. In addition, the survival of an Early Pleistocene Gigantopithecus enamel proteome enables us to assess the presence of multiple forms of in vivo modification. Finally, palaeoproteomic analysis allowed us to identify a hitherto unknown biological component of tooth formation in an extinct hominid. These findings suggest that the palaeoproteomic analysis of hominid enamel has the potential to provide a molecular perspective on human and great ape evolution.

Online content

Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/s41586-019-1728-8. Grey bars represent the 95% HPD interval obtained from the Bayesian approach and the 95% confidence interval obtained from the pairwise-distance-based approach, of the Gigantopithecus-Pongo divergence.

Publisher's note

Methods

Chuifeng Cave

The Chuifeng Cave (23° 34′ 27″ N, 107° 00′ 22″ E) is one of the most representative sites for the Early Pleistocene G. blacki fauna [START_REF] Wang | New discoveries of Gigantopithecus blacki teeth from Chuifeng Cave in the Bubing Basin, Guangxi, south China[END_REF] . The site is located in the Bubing Basin in the northwestern part of the Guangxi Zhuang Autonomous Region, south China (Extended Data Fig. 1). The cave is 19 m long, 0.5-2 m wide and 1.5-5 m high, penetrating the limestone from southeast to northwest at a height of ~77 m above the local valley floor. A fossiliferous sandy clay with a few limestone breccias fills most of the cave, with an average depth of 1.3 m (Extended Data Fig. 2). Four excavation areas (A, B, C and D) were excavated down to limestone bedrock at 10-cm intervals. Twenty-four large mammalian species, including 92 G. blacki teeth, were unearthed from the cave [START_REF] Wang | New discoveries of Gigantopithecus blacki teeth from Chuifeng Cave in the Bubing Basin, Guangxi, south China[END_REF] . The Chuifeng Cave mammalian fauna is characterized by the occurrence of typical Early Pleistocene species, such as Hystrix magna, Sinomastodon sp., Stegodon preorientalis, Ailuropoda microta, Pachycrocuta licenti, Tapirus sanyuanensis and Sus peii [START_REF] Wang | New discoveries of Gigantopithecus blacki teeth from Chuifeng Cave in the Bubing Basin, Guangxi, south China[END_REF] . This mammalian fauna is comparable with other Gigantopithecus-containing faunas of the Early Pleistocene in southern China, such as Baikong [START_REF] Jin | Chronological sequence of the early Pleistocene Gigantopithecus faunas from cave sites in the Chongzuo, Zuojiang River area, South China[END_REF] , Longgupo [START_REF] Huang | Early Homo and associated artefacts from Asia[END_REF] and Liucheng [START_REF] Pei | Discovery of Gigantopithecus mandibles and other material in Liucheng district of central Kwangsi in South China[END_REF] . The mammalian fauna composition is consistent with the results of combined electron spin resonance (ESR)/U-series dating and sediment palaeomagnetic studies (~1.9 Ma) [START_REF] Sun | Magnetochronological sequence of the Early Pleistocene Gigantopithecus faunas in Chongzuo, Guangxi, southern China[END_REF] . In the present study, we collected one well-preserved G. blacki tooth (excavation number CF-B-16) for palaeoproteomic analysis. This tooth was excavated from area B at a depth of 90 cm from the sediment surface and, based on its stratigraphic position, dated to ~1.9 Ma. No other specimens were tested before CF-B-16, and no specific selection was made as to which Gigantopithecus tooth would be analysed.

Thermal age

Thermal age was calculated to allow comparison with previously published ancient genomes, ancient proteomes and collagen peptide mass fingerprinting studies, from other temporal and geographical localities. Temperature estimates for the hominin occupation of Dmanisi based on herpetological fauna suggest a temperature about 3.1 °C above the current MAT [START_REF] Blain | Paleoclimatic and paleoenvironmental context of the Early Pleistocene hominins from Dmanisi (Georgia, Lesser Caucasus) inferred from the herpetofaunal assemblage[END_REF] , while the sea surface temperature record used [START_REF] Demarchi | Protein sequences bound to mineral surfaces persist into deep time[END_REF] predicts a negative ΔT at the time of hominin occupation. Given this discrepancy and the widely different temperature estimates for the Last Glacial Maximum (LGM) in the Caucasus, we conservatively use a scale factor of 0, correlating with a ΔT of approximately -0.2 °C and a current MAT of 11.2 °C. Our thermal age prediction for Dmanisi (2.2 Myr@10 °C) should therefore be seen as conservative. The thermal age for Chuifeng Cave was calculated with a general lapse rate between mean annual temperature (MAT) and altitude of 5.0 °C/km, a scale factor of 0.7 and a ΔT at LGM of -3 °C. The actual ΔT at LGM might have been more pronounced, again leading to a conservative estimate for the thermal age of Chuifeng Cave also. MAT was estimated based on the ten closest weather stations listed in publicly accessible World Meteorological Organization (WMO) data (Extended Data Table 2). Thermal age calculations are, among other factors, altitude dependent, but only five out of these ten weather stations have altitudes directly associated with them. We therefore estimated the altitude of the other five weather stations through an online resource (https://www.advancedconverter. com/map-tools/find-altitude-by-coordinates). The correlation between WMO altitude and estimated altitude was R 2 = 0.99, providing sufficient validity to our estimated altitudes. The MATs for all weather stations were then averaged to obtain an approximate MAT for Chuifeng Cave. Next, thermal age was calculated for chronological ages of 1.7 Myr, 1.9 Myr and 2.1 Myr, giving estimates of the minimum (9.2 Myr@10 °C), maximum (15.0 Myr@10 °C) and mean (11.8 Myr@10 °C) thermal ages associated with the Chuifeng Cave fauna within a 95% confidence interval (Fig. 1). The Chuifeng Cave proteome is therefore, to our knowledge, substantially older than the oldest collagen peptide mass fingerprint (Ellesmere Island, 0.003 Myr@10 °C), oldest mammalian genome (Thistle Creek, 0.03 Myr@10 °C), oldest hominin genome (Sima de los Huesos, 0.25 Myr@10 °C) and oldest enamel proteome (Dmanisi, 2.2 Myr@10 °C) published to date [START_REF] Demarchi | Protein sequences bound to mineral surfaces persist into deep time[END_REF] . Full thermal age calculations can be found in Supplementary Data 2.

Protein extraction

Extraction of ancient proteins took place in facilities at the Natural History Museum of Denmark dedicated to extracting ancient DNA and ancient proteins. These laboratories include clean rooms fitted with filtered ventilation and positive air pressure [START_REF] Hendy | A guide to ancient protein studies[END_REF] . A negative extraction blank was processed alongside the ancient extractions, with the additional inclusion of injection blanks during tandem mass spectrometry (MS/MS) analysis to monitor potential protein contamination during all stages of analysis. Two samples of enamel (185 and 118 mg) and one of dentine (192 mg) were removed from the same molar (CF-B-16) using a sterilized drill and crushed to a rough powder. One enamel sample and the dentine sample were demineralized in 1.2 M HCl at 3 °C for 24 h, and the other enamel sample was demineralized at the same temperature and duration using 10% TFA. Subsequently, solubilized protein residues were cleaned, concentrated and immobilized on C18 Stage-Tips using previously published methods [START_REF] Cappellini | Early Pleistocene enamel proteome from Dmanisi resolves Stephanorhinus phylogeny[END_REF] . No other samples from Chuifeng Cave were analysed before or during the analysis of CF-B-16.

LC-MS/MS analysis

The extracts were analysed by nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) using a 15-cm capillary column (75 μm inner diameter, packed with 1.9 μm C18 beads (Reprosil-AQ Pur, Dr. Maisch)) on an EASY-nLC 1200 system (Proxeon) connected to a Q-Exactive HF-X mass spectrometer (Thermo Scientific). The nanoLC gradient and MS parameters followed a previously published Q-Exactive HF-X method [START_REF] Mackie | Palaeoproteomic profiling of conservation layers on a 14th century Italian wall painting[END_REF] . System wash blanks were performed before and after every sample to hinder cross-contamination.

Database construction

We constructed a protein sequence database for Hominoidea proteins known to be present in enamel proteomes (Supplementary Table 2), to which we added the homologous sequences from one Cercopithecoid (Macaca mulatta) as an outgroup for phylogenetic analysis. As few protein sequences are publicly available for Pongo pygmaeus, we predicted those sequences from publicly available genomic sequence data using the known gene coordinates of Pongo abelii homologues. Similarly, we generated de novo AMELY sequences for P. abelii and P. pygmaeus. Finally, we added common laboratory contaminants to allow spectra from such proteins to be confidently identified [START_REF] Hendy | A guide to ancient protein studies[END_REF] .

Ancestral sequence reconstruction. Previous research indicates

that cross-species proteomic effects, observed during spectral identification, substantially reduce the identification of phylogenetically informative amino acid positions at large evolutionary distances [START_REF] Welker | Elucidation of cross-species proteomic effects in human and hominin bone proteome identification through a bioinformatics experiment[END_REF] . We reasoned that this was likely to occur in the case of Gigantopithecus proteins [START_REF] Welker | Palaeoproteomics for human evolution studies[END_REF] , and therefore reconstructed the ancestral protein sequences of enamel-specific proteins. Ancestral sequence recontruction (ASR) was conducted across the entire Hominoidea phylogeny using PhyloBot [START_REF] Hanson-Smith | PhyloBot: a web portal for automated phylogenetics, ancestral sequence reconstruction, and exploration of mutational trajectories[END_REF] . Input sequences were constrained phylogenetically to (Macaca,(Nomascus,((Pongo abelii, Pongo pygmaeus),Gorilla,(Homo,((Pan paniscus, Pan troglodytes)))))). We added those sequences to the reference protein database to account for them in the database search of PEAKS and MaxQuant. Isoform variation. After obtaining complete protein sequences for all extant hominids, we added isoforms not present in UniProt or GenBank for the proteins AMELX, AMELY, AMBN, AMTN, KLK4 and TUFT1, including the reconstructed ASR sequences of these proteins, to the database. We assumed that the isoforms for these non-human hominids would result from identically placed alternative splicing across species and ancestral nodes (as also supported by all UniProt isoforms present for the studied proteins). Thus, we copied these alternative splicing sites onto the available reference sequences to create the missing isoforms. Database sequence names for these proteins were appended with '_ManIso2' or '_ManIso3'.

Proteomic data analysis

Raw mass spectrometry data were searched per sample type (enamel, dentine, extraction blank and injection blanks) against a sequence database containing all common enamel proteins for all extant hominids (see above). We used PEAKS [START_REF] Zhang | De novo sequencing assisted database search for sensitive and accurate peptide identification[END_REF] (v.7.5) and MaxQuant 23 (v.1.6.2.6) software. The de novo and error-tolerant implementations of PEAKS, and the dependent peptide algorithm implemented in MaxQuant, were used to generate possible, additional, SAP variation in enamel protein sequences. Such novel SAPs could represent unique amino acid substitutions on the Gigantopithecus lineage, which are not relevant to its phylogenetic placement but are relevant for dating the Pongo-Gigantopithecus divergence. Next, these potential sequence variants were added to a newly constructed sequence database and verified in separate searches in PEAKS and MaxQuant. We defined as variable modifications methionine oxidation, proline hydroxylation, glutamine and asparagine deamidation, pyro-glutamic acid from glutamic acid, pyro-glutamic acid from glutamine and phosphorylation (STY). No fixed modifications were selected. We did not use an enzymatic protease during sample preparation, therefore the digestion mode was set to 'unspecific'. For PEAKS, peptide spectrum matches were only accepted with a false-discovery rate (FDR) ≤ 1.0%, and precursor mass tolerance was set to 10 ppm and fragment mass tolerance to 0.05 Da. For MaxQuant, peptide spectrum matches and protein FDR were set at ≤ 1.0%, with a minimum Andromeda score of 40 for all peptides. Protein matches were accepted with a minimum of two unique peptide sequences in at least one of the MaxQuant or PEAKS searches, including the removal of non-specific peptides after BLASTp searches of peptides matching non-enamel proteins against UniProt and GenBank databases. Proteins that are retained after applying these criteria are listed in Extended Data Table 1. Examples of annotated MS/MS spectra after MaxQuant analysis can be found in Supplementary Figs. 3-12. Assessment of protein damage and degradation followed protocols explained elsewhere [START_REF] Cappellini | Early Pleistocene enamel proteome from Dmanisi resolves Stephanorhinus phylogeny[END_REF][START_REF] Mackie | Palaeoproteomic profiling of conservation layers on a 14th century Italian wall painting[END_REF][START_REF] Welker | Palaeoproteomic evidence identifies archaic hominins associated with the Châtelperronian at the Grotte du Renne[END_REF] and included rates of deamidation and a comparison of observed peptide lengths. GRAVY index scores of peptide hydrophobicity were calculated using the R package Peptides, with the scale set to 'KyteDoolittle'.

Phylogenetic and divergence analysis

Comparative reference dataset. We assembled a reference dataset with five protein sequences retrieved from the ancient sample (AMBN, AMELX, AMTN, ENAM and MMP20) and relevant extant species (Supplementary Table 2). Protein sequences for human (Homo sapiens), common chimpanzee (P. troglodytes), bonobo (P. paniscus), Sumatran orangutan (P. abelii), Western gorilla (Gorilla gorilla), rhesus macaque (M. mulatta) and the white-cheeked gibbon (Nomascus leucogenys) were obtained from the UniProt database. Additionally, we expanded our dataset with protein sequences from publicly available wholegenome sequence data from present-day great apes (in total 27 orangutans, 42 gorillas, 11 bonobos and 61 chimpanzees [START_REF] Prado-Martinez | Great ape genetic diversity and population history[END_REF][START_REF] Nater | Morphometric, behavioral, and genomic evidence for a new orangutan species[END_REF][START_REF] De Manuel | Chimpanzee genomic diversity reveals ancient admixture with bonobos[END_REF] ), as well as 19 human individuals from the Simons Genome Diversity Project (SGDP) [START_REF] Mallick | The Simons Genome Diversity Project: 300 genomes from 142 diverse populations[END_REF] . See Supplementary Information for the human sample numbers taken from the SGDP dataset.

Reconstruction of protein sequences from whole-genome sequencing data. DNA sequence reads for reference samples used were mapped to the human genome (version hg19) using BWA-MEM v.0.7.5a-r405 (http://bio-bwa.sourceforge.net/bwa.shtml) with default parameters. PCR and optical duplicates were identified and removed using PICARD v.1.91 (https://sourceforge.net/projects/picard/files/picard-tools/1.91/). Single-nucleotide polymorphisms were called on the read alignments using the GATK UnifiedGenotyper (https://software. broadinstitute.org/gatk/documentation/tooldocs/current/org_broa-dinstitute_gatk_tools_walkers_genotyper_UnifiedGenotyper.php).

To reconstruct the protein sequences from the genotype calls, we first created a consensus sequence for each of the five genes of interest and for each sample. Indels were not considered and a random allele was chosen at heterozygous positions. Next, we removed the intron sequences from each gene using the annotation of the reference human genome (hg19) available in the ENSEMBL database. For each of the in silico spliced genes, we performed a tblastn search [START_REF] Altschul | Basic local alignment search tool[END_REF] using the human reference protein as the query. Finally, we obtained the translated protein sequences from the resulting alignments.

Assessing the phylogenetic position of G. blacki. We compared the G. blacki protein sequences with the corresponding homologues of the species in the reference panel. For each gene, we built two multiple sequence alignments using mafft [START_REF] Katoh | Adding unaligned sequences into an existing alignment using MAFFT and LAST[END_REF] . The first incorporated all samples in the reference panel (n = 164). The second incorporated only a single sample per species (Supplementary Table 3). To account for isobaric amino acids (leucines and isoleucines), which cannot be distinguished in the ancient protein data, we changed all isoleucines to leucines at positions where the ancient sample carried either of those amino acids. To assess the phylogenetic position of the ancient sample, two inference approaches were used: a maximum-likelihood and a Bayesian inference.

Maximum-likelihood approach. PhyML v.3.1 [START_REF] Guindon | New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0[END_REF] was used to infer a maximum-likelihood tree, branch lengths and substitutions rates for each individual protein alignment (Supplementary Fig. 2) and for the concatenated alignment. For each alignment, we started from three random trees (--n_rand_starts 3 -s BEST --rand_start), used the JTT model (-m JTT -f m), and obtained maximum likelihood estimates for the gamma distribution shape parameter (-a e) and the proportion of invariable sites (-v e). Support values were obtained for each bipartition based on 100 non-parametric bootstrap replicates. The bootstrap results per branch split are shown in Extended Data Fig. 6b.

Bayesian approach. As a complementary approach, we used MrBayes [START_REF] Ronquist | MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space[END_REF] and the concatenated alignment to infer the phylogenetic position of the ancient sample (Fig. 3, Extended Data Fig. 6b). We set an independent bipartition for each gene and estimated: substitution rates, across-site rate variation and the proportion of invariable sites (unlink Statefreq = (all) Ratemultiplier = (all) Aamodel = (all) Shape = (all) Pinvar = (all)). MrBayes was executed using the CIPRES portal [START_REF] Miller | Gateway Computing Environments Workshop (GCE)[END_REF] . The MCMC algorithm was set to 5,000,000 cycles with 4 chains and a temperature parameter of 0.2. The convergence of the algorithm was assessed using Tracer v.1.6.0 after discarding 25% of the iterations as burn-in. MrBayes was run against the reference sequence for each species (Extended Data Fig. 6c) or against 162 great ape individuals, one hylobatid and one cercopithecid (Extended Data Fig. 7). Both of these analyses, as well as the PHyML maximum likelihood approach, resulted in the same topology. The analysis using a large number of individuals shows, however, that resolution within the genus Pongo is limited (Extended Data Fig. 7). Nevertheless, the placement of Gigantopithecus is fully supported.

Divergence time of Gigantopithecus. We estimated the divergence time between Gigantopithecus and the Pongo branch first by using a distance-based approach. We used the alignment of the amino acid sequences of reference genome sequences for each species as well as diversity data (see above). A distance matrix was created from the concatenated protein sequences of all individuals using the function dist.ml from the R package phangorn [START_REF] Schliep | phangorn: phylogenetic analysis in R[END_REF] under the LG amino acid substitution model [START_REF] Le | An improved general amino acid replacement matrix[END_REF] . We used pairwise exclusion to increase the amount of data for the present-day branches. We then calculated the mean difference of all orangutan sequences to all sequences from Homo, Pan and Gorilla, and the mean difference of all orangutan sequences to Gigantopithecus (Extended Data Fig. 6a). We used the average distance between orangutan and the other extant great apes as a scaling factor, assuming a divergence time between these branches 53 of 23.8 Ma. Under this assumption, the molecular divergence of Gigantopithecus from the Pongo branch is 9.98 Ma. However, because Chuifeng Cave is dated to 1.9 Ma, this branch is likely to be underestimated and its age needs to be corrected to 11.88 Ma. We combine the 95% confidence interval of the distance matrix with the 95% confidence interval of the mutation rate estimate [START_REF] Besenbacher | Direct estimation of mutations in great apes reconciles phylogenetic dating[END_REF] , and add the upper and lower values of the 95% confidence interval for the Chuifeng Cave dating (1.7-2.1 Ma), and therefore suggest conservative upper and lower boundaries for the divergence of 8.91 and 15.65 Ma, respectively.

If mutation rates did not substantially differ between extant Pongo and Gigantopithecus, this estimate should reflect the molecular evolution of their common branch. We calculated the divergence between the other great apes, taking into account the mutation rate differences on these lineages as scaling factors [START_REF] Besenbacher | Direct estimation of mutations in great apes reconciles phylogenetic dating[END_REF] . The resulting divergence time between Gorilla and the Homo/Pan branch is estimated at 10.27 Ma (7.9-13.25 Ma, 95% confidence interval), and the divergence between Homo and Pan at 8.72 Ma (8.06-13.81 Ma, 95% confidence interval). These values are in strong agreement with previous estimates [START_REF] Besenbacher | Direct estimation of mutations in great apes reconciles phylogenetic dating[END_REF] , suggesting that these protein sequences represent well the known phylogeny of the great apes. Clearly, all divergence time estimates scale with assumptions of mutation rates. We also caution that the small number of mutations in the peptide fragments in Gigantopithecus constitutes a severe limitation on the precision of these estimates on this branch. However, the phylogenetic position of Gigantopithecus as a sister clade to orangutans is also well supported in this analysis: a phylogenetic tree from a distance matrix of the reference sequences for these species (neighbour joining tree in phangorn; maximum likelihood computed with the pml function; 1,000 bootstrap replicates) separates Gigantopithecus from orangutans with 100% bootstrap support.

We used the program MrBayes [START_REF] Ronquist | MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space[END_REF] to estimate divergence time estimates in a Bayesian framework using the reference genome sequences. We defined M. mulatta as outgroup, grouped Pan, Homo and Gorilla together as well as Pongo and Gigantopithecus, and set the divergence time of the two groups with a uniform distribution of 17.739-26.061 Ma, using a previously published estimate [START_REF] Besenbacher | Direct estimation of mutations in great apes reconciles phylogenetic dating[END_REF] . Furthermore, we set the divergence time of the macaques and apes at 26.061-39.9 Ma (from the maximum divergence time of the hominids to a very high divergence time of the apes). We used a variable mutation rate and the VT amino acid substitution model [START_REF] Müller | Modeling amino acid replacement[END_REF] in five million iterations. This results in a divergence time for Gigantopithecus-Pongo of 10.14 Ma (4.76-15.79 Ma, 95% HPD interval). The divergence of Gorilla from the Homo/Pan branch is estimated at 8.59 Ma (4.62-13.56 Ma, 95% HPD interval), and the divergence of Homo and Pan at 5.78 Ma (2.64-9.53 Ma, 95% HPD interval). These are largely consistent with, but slightly younger than, previous estimates [START_REF] Besenbacher | Direct estimation of mutations in great apes reconciles phylogenetic dating[END_REF][START_REF] Langergraber | Generation times in wild chimpanzees and gorillas suggest earlier divergence times in great ape and human evolution[END_REF] , possibly owing to a mutation slowdown in these lineages compared to the Pongo lineage, which is not taken into account here. However, they seem in agreement with the fossil record indicating the origin of hominins around 6-8 Ma and the dating of a possible early Gorillini (Chororapithecus) around 7-9 Ma [START_REF] Langergraber | Generation times in wild chimpanzees and gorillas suggest earlier divergence times in great ape and human evolution[END_REF][START_REF] Katoh | New geological and palaeontological age constraint for the gorilla-human lineage split[END_REF][START_REF] Senut | First hominid from the Miocene (Lukeino Formation, Kenya)[END_REF][START_REF] Brunet | A new hominid from the Upper Miocene of Chad, central Africa[END_REF][START_REF] Haile-Selassie | Late Miocene hominids from the Middle Awash, Ethiopia[END_REF] . Therefore, we conclude that the relative branch lengths of the tree (Fig. 3b) are concordant with the overall phylogeny and the estimates presented above.

Fig. 2 |

 2 Fig.2| Modifications and degradation of Gigantopithecus enamel proteome. a, Violin plots of asparagine (N) and glutamine (Q) deamidation for selected proteins (n = 1,000 bootstrap replicates of intensity-based peptide deamidation[START_REF] Mackie | Palaeoproteomic profiling of conservation layers on a 14th century Italian wall painting[END_REF] ). Human dermcidin (DCD) is included as an example of a nondeamidated contaminant. For AMBN, all observed asparagines and glutamines are deamidated. For AMELX, all asparagines are deamidated. For DCD, no observed asparagines and glutamines are deamidated. The number of peptides used for the calculation are shown at the bottom. b, Violin plots of peptide lengths for Gigantopithecus, an Early Pleistocene rhinoceros from Dmanisi and a Medieval control sample[START_REF] Cappellini | Early Pleistocene enamel proteome from Dmanisi resolves Stephanorhinus phylogeny[END_REF] . c, Sequence-motif analysis of the over-representation of specific amino acids around the phosphorylated amino acid (position 0; n = 14). d, Peptide coverage of AMELX protein isoforms. Matching peptides are indicated by black bars for isoform 1 (n = 21) and isoform 3 (n = 7). The latter includes an insertion due to alternative splicing between isoform 1, amino acid positions 34 and 35 (coordinates in reference to UniProt Accession number: Q99217-1 [AMELX_HUMAN]). b includes data on AMELX, AMBN, ENAM, AMTN and MMP20 only. For a and b, box plots define the range of the data (whiskers extending to 1.5× the interquartile range), 25th and 75th percentiles (boxes) and medians (dots/lines). For a, outliers are indicated as black dots (beyond 1.5× the interquartile range).

Fig. 3 |

 3 Fig.3| Bayesian phylogeny and divergence of Gigantopithecus among extant apes (Hominoidea). a, Time-calibrated Bayesian phylogeny of Gigantopithecus. Circled nodes were fixed for topology (see Methods). Grey error bars represent the 95% highest posterior density (HPD) intervals for the divergence estimates. b, Distribution of probable and possible extinct pongines known from the fossil record, including Gigantopithecus (black bars). Grey bars represent the 95% HPD interval obtained from the Bayesian approach and the 95% confidence interval obtained from the pairwise-distance-based approach, of the Gigantopithecus-Pongo divergence.

Extended Data Fig. 1 |

 1 The current environment of Chuifeng Cave. a, Landscape outside Chuifeng Cave. b, Elevated altitude of Chuifeng Cave (arrow points to the entrance). Photo credit: W.W. Extended Data Fig. 2 | Excavations in Chuifeng Cave. a, Main entrance of Chuifeng Cave. b, Well-preserved deposits before excavation. c, The stratigraphic profile (1.3 m in height) of area D. d, W.W. excavating in area D. e, Excavated channel. f, g, In situ G. blacki teeth (scale bars, 3 cm). Photo credit: W.W. Extended Data Fig. 3 | Total ion current chromatograms of the analysed samples. a, HCl extract. b, TFA extract. Note differences in maximum total ion currents on the y axes. Each extract was analysed only once. Extended Data Fig. 4 | Comparison of HCl and TFA extraction protocols. a, Summed and normalized peptide intensities for each combination of peptide length and number of acidic residues (aspartic acid, glutamic acid, deamidated asparagine, deamidated glutamine). Circle sizes are proportional to the percentage of the total intensity, for each combination of peptide length and number of acidic residues. b, Summed and normalized intensities by peptide length. c, Summed and normalized peptide intensities across peptide hydrophobicity (GRAVY index values calculated using the R package Peptides, scale 'KyteDoolittle'). Insets show peptide count distribution across peptide hydrophobicity. d, Extraction performance for various data categories. Values scaled to one and compared to the best-performing extraction method for each category independently. SAPs refer to those SAPs informative within Hominoidea. e, Proportional Venn diagram of unique peptide sequences identified in the two demineralization methods. All comparisons based on MaxQuant LFQ data only. N, number of peptides. Extended Data Fig. 8 | Sequence conservation and structural relevance of retrieved AHSG peptides. All AHSG-specific peptides, identified by PEAKS and MaxQuant, derive from a single sequence region bridging cystatin domains 1 and 2. The surviving sequence region is evolutionarily conserved across Catarrhini. It contains a regular repeat of acidic amino acid residues (aspartic acid, D, on positions 133, 137 and 141) that enable binding of basic calcium phosphate (residues highlighted in green), similarly to a conserved region just N-terminal (glutamic acid, E, on position 111, and aspartic acid, D, on position 113 and 115). At the bottom, a fragment ion alignment is given of MaxQuant-identified AHSG peptides. The serine is phosphorylated in all matching spectra. The majority-based amino acid consensus sequence for the alignment of Catarrhini is shown at the top for amino acid positions 100-149 (amino acid coordinates following UniProt accession P02765 [FETUA_HUMAN]).

Data availability

All the mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium (http://proteomecentral. proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD013838. Generated ancient protein consensus sequences for G. blacki can be found in Supplementary Data 1. Full thermal age calculations can be found in Supplementary Data 2.

Extended Data Table 1 | Enamel proteome sequence coverage

Only proteins with two unique peptides in at least either MaxQuant or PEAKS searches were accepted. No protein matches in the dentine or blank extractions fulfilled this criterion. Primary entries for proteins used for phylogenetic reconstruction refer to the P. abelii accessions in UniProt for reference purposes. Protein sequence coverage in the final column indicates the coverage obtained after combining PEAKS and MaxQuant peptide recovery and is only reported for proteins considered endogenous. ALB, DCD and K1C9 are considered to be contaminants. For amino acid columns, numbers in brackets refer to amino acid positions uniquely identified in PEAKS or MaxQuant searches.

Article

Extended Data Table 2 | MAT estimation at Chuifeng Cave

The ten geographically closest meteorological weather stations included in publicly available WMO data were used to estimate current MAT at Chuifeng Cave. Correlation between online altitude estimation and WMO provided altitude is R 2 = 0.99 (Pearson correlation).

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper. [START_REF] Mackie | Palaeoproteomic profiling of conservation layers on a 14th century Italian wall painting[END_REF] . The number of peptides used for the calculation are shown at the bottom. For some proteins, only deamidated asparagines or glutamines were observed (for example, AMBN), while DCD is included as an example of a non-deamidated contaminant. All comparisons based on MaxQuant data only. For a-c, box plots define the range of the data (whiskers extending to 1.5× the interquartile range), outliers (beyond 1.5× the interquartile range), 25th and 75th percentiles (boxes) and medians (dots).

Article

Extended Data Fig. 6 | Unconstrained phylogenetic analysis of G. blacki. a, Pairwise distances between groups of selected Hominoids and Pongo estimated using the concatenated protein alignments and the phangorn R package. n shows number of pairwise comparisons. b, Maximum-likelihood tree computed on a distance matrix using pml R function. Support values were obtained from 1,000 bootstrap replicates. c, Rooted phylogenetic tree obtained using MrBayes. For each bipartition, we show the posterior probability (0-1) obtained from the Bayesian approach and the support values obtained from 100 non-parametric bootstrap replicates in a PHyML maximumlikelihood (0-100) tree. PHyML and MrBayes recover the same topology. b and c are based on the same concatenated alignment of the five proteins retrieved from Gigantopithecus, and resulted in the same tree topology.

Extended Data Fig. 7 | Bayesian phylogenetic tree of G. blacki including 162 modern genomes of great apes and a single hylobatid (N. leucogenys).

Tree obtained from the concatenated alignment using MrBayes. M. mulatta was used as an outgroup. The internal nodes corresponding to the Gorilla, Pan and Homo clades are collapsed for visualization purposes. The number of individuals in each of these nodes is indicated in parentheses.

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All the mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the data set identifier PXD013838. Protein sequences generated as part of this study are provided as supplementary files.

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