54Ancient DNA (aDNA) sequencing has enabled unprecedented reconstruction of speciation, 55 migration, and admixture events for extinct taxa 1 . Outside the permafrost, however, irreversible 56 aDNA post-mortem degradation 2 has so far limited aDNA recovery within the ~0.5 million years (Ma) 57 time range 3 . Tandem mass spectrometry (MS)-based collagen type I (COL1) sequencing provides 58 direct access to older biomolecular information 4 , though with limited phylogenetic use. In the 59 absence of molecular evidence, the speciation of several Early and Middle Pleistocene extinct 60 species remain contentious. In this study, we address the phylogenetic relationships of the Eurasian 61 Pleistocene Rhinocerotidae 5-7 using ~1.77 million years (Ma) old dental enamel proteome sequences 62 of a Stephanorhinus specimen from the Dmanisi archaeological site in Georgia (South Caucasus) 8 . 63Molecular phylogenetic analyses place the Dmanisi Stephanorhinus as a sister group to the woolly 64 (Coelodonta antiquitatis) and Merck's rhinoceros (S. kirchbergensis) clade. We show that 65Coelodonta evolved from an early Stephanorhinus lineage and that this genus includes at least two 66 distinct evolutionary lines. As such, the genus Stephanorhinus is currently paraphyletic and its 67 systematic revision is therefore needed. We demonstrate that Early Pleistocene dental enamel 68 proteome sequencing overcomes the limits of ancient collagen-and aDNA-based phylogenetic 69 inference, and also provides additional information about the sex and taxonomic assignment of the 70 specimens analysed. Dental enamel, the hardest tissue in vertebrates, is highly abundant in the fossil 71 record. Our findings reveal that palaeoproteomic investigation of this material can push 72 biomolecular investigation further back into the Early Pleistocene. 73 74 75 samples 23 , is particularly high, in some cases close to 100%, in full agreement with the age of the 126 specimens investigated. (Fig. 2a). Other forms of non-enzymatic modifications are also present. 127 Tyrosine (Y) experienced mono-and di-oxidation while tryptophan (W) was extensively converted 128 into multiple oxidation products. (Fig. 5b). Oxidative degradation of histidine (H) and conversion of 129 arginine (R) leading to ornithine accumulation were also observed. These modifications are 130 absent, or much less frequent, in a medieval ovicaprine dental enamel control sample, further 131 confirming the authenticity of the sequences reconstructed. Similarly, unlike in the control, the 132 peptide length distribution in the Dmanisi dataset is dominated by short overlapping fragments, 133 generated by advanced, diagenetically-induced, terminal hydrolysis ( Fig. 5c and d). 134

This study presents the first phylogenetic analysis focused on the subfamily Aceratheriinae to date, with 392 characters (361 parsimony-informative characters) coded from 50 taxa at the species level. We added 80 newly defined and 33 revised characteristics to an existing matrix, covering features of the skull, teeth, and postcranial bones. Based on the results of ordered and unordered analyses, combined with a diagnosis in accordance with traditional morphological taxonomy, we revised the diagnosis of Aceratheriinae and reconstructed the phylogeny of Aceratheriinae. The tribe Teleoceratini, as well as the tribe Aceratheriini, was reclassified within Aceratheriinae; however, the traditionally established contents of each tribe were changed somewhat. Aceratheriinae underwent evolutionary adaptation several times during the early stages of its evolution, and several genera are herein reconstructed as early-diverging taxa, such as Floridaceras, Chilotheridium, and Plesiaceratherium. Turkanatherium and Protaceratherium are excluded from Aceratheriinae in this study. We suggest another two subclades of Aceratheriinae, containing Hoploaceratherium and Aprotodon, respectively. Aceratheriini and Teleoceratini are redefined as two highly specialized groups of Aceratheriinae.

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