Title: Sensitive detection of mycobacterial cell wall lipid biomarkers in Neanderthal skeletons from Subalyuk, Hungary

Authors: Oona Y-C. Lee^1,2, Houdini H.T. Wu^1,2,3, David E. Minnikin¹,Gurdyal S. Besra¹, Gareth Llewellyn⁴,Christopher M. Williams⁴, Heidi Yoko Jäger⁵, Frank Maixner⁵, Albert Zink⁵, Mihály Gasparik⁶, Ildikó Pap⁶ and György Pálfi⁷

Affiliations: ¹Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, United Kingdom B15 2TT; ²Coventry Road Medical Centre, Heartlands Hospital, Birmingham, United Kingdom B5 9SS; ³UK Health Security Agency, Public Health Birmingham laboratory, Heartlands Hospital, Birmingham, United Kingdom B5 9SS; ⁴National Mass Spectrometry Service Centre, School of Medicine, Grove Building, Swansea University, Swansea, UK; ⁵Institute for Mummies Studies, Eurac Research, Bolzano, Italy; ⁶Department of Anthropology, Hungarian Natural History Museum, Budapest, Hungary; ⁷Department of Biological Anthropology, University of Szeged, Szeged, Hungary

This paper is dedicated to the memory of Professor David Ernest Minnikin (1939–2021), who has established the use of mycobacterial cell wall lipid biomarkers in archaeological and TB evolution research.

Abstract: Mycobacterial cell wall lipid biomarkers analysis was carried out on the skeletal remains of two Neanderthal individuals, a 25- to a 35-year-old woman and a 3- to 4-year-old child that was discovered in Subalyuk Cave in North-Eastern Hungary (Bartucz, 1940; Pap et al., 1996) in 1932. Radiocarbon dating of the female remains revealed an age of 39,650 - 38,610 cal BP. Paleopathological studies of these Neanderthal remains revealed distinct evidence of skeletal infections. Alterations of the sacrum of the adult specimen suggest early-stage sacroiliitis, while several vertebral bodies indicate superficial osseous remodelling of infectious origin. The most characteristic lesions were observed on the endocranial surface of the child skull, reflecting a reaction of meningeal tissues. Paleoradiological analyses also confirmed the observed morphological lesions. Two specimens (vertebra and cranium samples) from the child Neanderthal revealed definitive signals for C₃₂-mycoserosate, a very characteristic component of the Mycobacterium tuberculosis complex (MTBC). A vertebra from the adult provided weak evidence for mycocerosate biomarkers. The correlation of distinctive skeletal lesions with characteristic amplified ancient DNA fragments and a proven lipid biomarker points to the presence of tuberculosis in these Neanderthals. In particular, the closely similar biomarker profiles, for two distinct juvenile cranial and vertebral bones, strengthen this diagnosis. These findings support the hypothesis that Tuberculosis would be one of the contributing factors to the relatively rapid demise of the genus Homo neanderthalis around 40,000 years ago.