03.06.2025
In the past decade, ancient protein sequences have emerged as a valuable source of
data for deep-time phylogenetic inference1–4. Still, even though ancient proteins have
been reported from the Middle–Late Miocene5,6, the recovery of protein sequences
providing subordinal-level phylogenetic insights does not exceed 3.7 million years
ago (Pliocene)1. Here, we push this boundary back to 21–24 million years ago (Early
Miocene) by retrieving enamel protein sequences of a rhinocerotid (Epiaceratherium
sp.; CMNFV59632) from Canada’s High Arctic. We recover partial sequences of seven
enamel proteins and more than 1,000 peptide–spectrum matches, spanning at least
251 amino acids. Endogeneity is in line with thermal age estimates and is supported
by indicators of protein damage, including several spontaneous and irreversible
chemical modifications accumulated during prolonged diagenesis. Bayesian tip-dating
places the divergence time of CMNFV59632 in the Middle Eocene–Oligocene, coinciding
with a phase of high rhinocerotid diversification7. This analysis identifies a later
Oligocene divergence for Elasmotheriinae, weakening alternative models suggesting
a deep basal split between Elasmotheriinae and Rhinocerotinae8,9. The findings are
consistent with hypotheses on the origin of the enigmatic fauna of the Haughton
Crater, which, in spite of considerable endemism, has similarity to distant Eurasian
faunas10,11. Our findings demonstrate the potential of palaeoproteomics in obtaining
phylogenetic information from a specimen that is approximately ten times older than
any sample from which endogenous DNA has been obtained so far.
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