Early Pleistocene enamel proteome sequences from Dmanisi resolve Stephanorhinus phylogeny

Abstract

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

A comparison of proteomic, genomic, and osteological methods of archaeological sex estimation

“…The proteins are cleaved into peptides in situ, as part of enamel formation during tooth biogenesis 50 , 72 , 73 . In order to extract and analyze this peptide population, researchers need to demineralize the enamel and most use acid-based 33 – 36 , 45 , 46 , 55 , 58 , 74 approaches. There are two analytical options: a targeted approach focused on a limited number of specific amelogenin peptides 34 , 36 , 54 , or a shotgun proteomics approach that seeks to identify all proteins in the proteome and then selectively measure all amelogenin peptides bioinformatically after peptide spectral matching 35 , 45 , 46 , 54 , 55 .…”

Cretaceous dinosaur bone contains recent organic material and provides an environment conducive to microbial communities

“…However, recent discoveries of relatively intact macromolecular organic material in fossils and sub-fossils challenge this view. These include ancient DNA (Meyer et al, 2012; Orlando et al, 2013) and peptide (Buckley, 2015; Demarchi et al, 2016; Cappellini et al, 2018) sequences in sub-fossils, as well as ancient biomolecules such as sterols (Melendez et al, 2013), melanin (Vinther et al, 2008), amino acids (Curry et al, 1991), and porphyrins (Wiemann et al, 2018a). These findings show that organic remains can potentially persist for thousands to millions of years, depending on the biomolecules and environmental conditions.…”

DNA from resin-embedded organisms: Past, present and future

“…analysis of fragmentation or degradation patterns. Proteins are thought to be more stable than DNA [ 92 , 93 ] and seem to provide similar phylogenetic information [ 2 ]. Some experiments detected biomolecules such as chitin-protein complexes in insect cuticle from sub-fossil resin but failed to do so when Dominican amber samples were tested [ 88 ], while other more recent studies found amino acids from fossil feathers in amber from almost 100 million years BP [ 58 ].…”

“…Ideally, fossil—or ancient—DNA (aDNA) might serve as an alternative to morphological analysis and palaeoethology, which for centuries have served as the only tools available for scientists to determine the phylogenetic relationships between past organisms. Genomic sequences also provide insights into molecular evolutionary changes over time, clarify evolutionary relationships among taxa and yield information on mutualism rates [ 1 , 2 ].…”

“…Additionally, aDNA research has yielded promising results [e.g. 18 , 54 – 57 ]; positive studies have been reported for aDNA from beetles in museum collections and permafrost [ 43 , 49 ] and more recently, novel insights have been obtained into biomolecule preservation in amber samples [ 58 ] and other fossils [ 2 , 59 ], including chewed birch pitch [ 60 ].…”