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Micro-destructive palaeoproteomic analysis of dental enamel from 23 Homo naledi specimens shows no evidence of conventional biological male markers

ABSTRACT: Understanding intraspecific variation within the hominin fossil record is important and challenging. The limited number and condition of fossils we recover from the Pleistocene compounds this challenge. Unusually, the Rising Star Cave system excavations resulted in a high number of well-preserved specimens of Homo naledi, making this species suitable for an in-depth analysis of variation. The evident lack of intraspecific morphological variation in H. naledi is puzzling, and recently Delezene et al. (2024) showed that the dental morphology observed within the H. naledi collection is compatible with a single sex population. Here we report the mass spectrometric (MS) sequencing of enamel proteomes from twenty-three ca. 335 thousand year old (Ka) dental specimens morphologically attributed to H. naledi. We demonstrate that protein recoveries, when sampling Pleistocene specimens via a minimally-destructive etching method or via the conventional destructive one, are similarly effective. All 23 H. naledi individuals lack confidently identified AMELY specific peptides used to recognise male individuals. By using a semi-quantitative approach, we also show that the lack of AMELY detection is not due to the sensitivity limits of the MS instrument used. Finally, the short enamel proteome sequences reconstructed so far show no variation among the analysed H. naledi individuals and, compared to extinct and extant hominins, there is a substituted position in AMELX. This study demonstrates the feasibility of recovering enamel proteins from Middle Pleistocene specimens via a minimally destructive method that in the future could also be used as a first screening approach to survey ancient protein content and to determine sex.

INSTRUMENT(S):

ORGANISM(S): Homininae

TISSUE(S): Enamel, Tooth Enamel

SUBMITTER: Palesa Madupe

LAB HEAD: Jesper Velgaard

PROVIDER: PXD054549 | Pride | 2026-06-29

REPOSITORIES: Pride

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Project description:Pleistocene Pongo teeth show substantial variation in size and morphology, fueling taxonomic debates about the paleodiversity of the genus. We investigated prominent features of the enamel-dentine-junction junction (EDJ) – phylogenetically informative internal structures – of 71 fossil Pongo lower molars from various sites by applying geometric morphometrics and conducted paleoproteomic analyses from enamel proteins to attempt to identify extinct orangutan species. Forty-three orangutan lower molars representing Pongo pygmaeus and Pongo abelii were included for comparison. The shape of the EDJ was analyzed by placing five landmarks on the tip of the main dentine horns, and 142 semilandmarks along the marginal ridges connecting the dentine horns. Paleoproteomic analyses were conducted on 15 teeth of Late Pleistocene Pongo using high-resolution tandem mass spectrometry. The geometric morphometric results show variations in EDJ shape regarding aspects of the height and position of the dentine horns and connecting ridges. Despite the issue of molar position and sample size, modern molars are distinguished from fossil counterparts by their elongated tooth outline and narrowly positioned dentine horns. Proteomic results show that neither a distinction of P. pygmaeus and P. abelii, nor a consistent allocation of fossil specimens to extant species is feasible. Based on the EDJ shape, the (late) Middle to Late Pleistocene Pongo samples from Vietnam share the same morphospace, supporting the previous allocation to P. devosi, although substantial overlap with Chinese fossils could also indicate close affinities with P. weidenreichi. The hypothesis that both species represent one chronospecies cannot be ruled out. Two fossil specimens, one from Tam Hay Marklot (Laos, Late Pleistocene), and another from Sangiran (Java, Early to Middle Pleistocene), along with some specimens within the Punung sample (Java), exhibit affinities with Pongo abelii. The Punung fossils might represent a mix of early Late Pleistocene and later specimens (terminal Pleistocene to Holocene) related to modern Pongo. The taxonomy and phylogeny of the complete Punung sample needs to be further investigated.

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	020Enamel20_2144.raw	Raw
	020Enamel80_2144.raw	Raw
	020Etch_2a_2132.raw	Raw
	020Etch_2at_2132.raw	Raw
	020Etch_2b_2132.raw	Raw

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Micro-destructive palaeoproteomic analysis of dental enamel from 23 Homo naledi specimens shows no evidence of conventional biological male markers

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