Middle Pleistocene protein sequences from the rhinoceros genus and the phylogeny of extant and extinct Middle/Late Pleistocene Rhinocerotidae.

Background: Ancient protein sequences are increasingly used to elucidate the phylogenetic relationships between extinct and extant mammalian taxa. Here, we apply these recent developments to Middle Pleistocene bone specimens of the rhinoceros genus . No biomolecular sequence data is currently available for this genus, leaving phylogenetic hypotheses on its evolutionary relationships to extant and extinct rhinoceroses untested. Furthermore, recent phylogenies based on Rhinocerotidae (partial or complete) mitochondrial DNA sequences differ in the placement of the Sumatran rhinoceros (). Therefore, studies utilising ancient protein sequences from Middle Pleistocene contexts have the potential to provide further insights into the phylogenetic relationships between extant and extinct species, including and .

Methods: ZooMS screening (zooarchaeology by mass spectrometry) was performed on several Late and Middle Pleistocene specimens from the genus , subsequently followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to obtain ancient protein sequences from a Middle Pleistocene specimen. We performed parallel analysis on a Late Pleistocene woolly rhinoceros specimen and extant species of rhinoceroses, resulting in the availability of protein sequence data for five extant species and two extinct genera. Phylogenetic analysis additionally included all extant Perissodactyla genera (, ), and was conducted using Bayesian (MrBayes) and maximum-likelihood (RAxML) methods.

Results: Various ancient proteins were identified in both the Middle and Late Pleistocene rhinoceros samples. Protein degradation and proteome complexity are consistent with an endogenous origin of the identified proteins. Phylogenetic analysis of informative proteins resolved the Perissodactyla phylogeny in agreement with previous studies in regards to the placement of the families Equidae, Tapiridae, and Rhinocerotidae. is shown to be most closely related to the genera and . The protein sequence data further places the Sumatran rhino in a clade together with the genus , opposed to forming a clade with the black and white rhinoceros species.

Discussion: The first biomolecular dataset available for places this genus together with the extinct genus and the extant genus . This is in agreement with morphological studies, although we are unable to resolve the order of divergence between these genera based on the protein sequences available. Our data supports the placement of the genus in a clade together with extant species. Finally, the availability of protein sequence data for both extinct European rhinoceros genera allows future investigations into their geographic distribution and extinction chronologies.