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The sequencing of ancient DNA has enabled the reconstruction of speciation, migration and admixture events for extinct taxa1. However, the irreversible post-mortem degradation2of ancient DNA has so far limited its recovery—outside permafrost areas—to specimens that are not older than approximately 0.5 million years (Myr)3. By contrast, tandem mass spectrometry has enabled the sequencing of approximately 1.5-Myr-old collagen type I4, and suggested the presence of protein residues in fossils of the Cretaceous period5—although with limited phylogenetic use6. In the absence of molecular evidence, the speciation of several extinct species of the Early and Middle Pleistocene epoch remains contentious. Here we address the phylogenetic relationships of the Eurasian Rhinocerotidae of the Pleistocene epoch7–9, using the proteome of dental enamel from a Stephanorhinustooth that is approximately 1.77-Myr old, recovered from the archaeological site of Dmanisi (South Caucasus, Georgia)10. Molecular phylogenetic analyses place this Stephanorhinusas a sister group to the clade formed by the woolly rhinoceros (Coelodonta antiquitatis) and Merck’s rhinoceros (Stephanorhinus kirchbergensis). We show that Coelodontaevolved from an early Stephanorhinuslineage, and that this latter genus includes at least two distinct evolutionary lines. The genus Stephanorhinusis therefore currently paraphyletic, and its systematic revision is needed. We demonstrate that sequencing the proteome of Early Pleistocene dental enamel overcomes the limitations of phylogenetic inference based on ancient collagen or DNA. Our approach also provides additional information about the sex and taxonomic assignment of other specimens from Dmanisi. Our findings reveal that proteomic investigation of ancient dental enamel—which is the hardest tissue in vertebrates11, and is highly abundant in the fossil record—can push the reconstruction of molecular evolution further back into the Early Pleistocene epoch, beyond the currently known limits of ancient DNA preservation.

Understanding how animal populations have evolved in response to palaeoenvironmental conditions is essential for predicting the impact of future environmental change on current biodiversity. Analyses of ancient DNA provide a unique opportunity to track population responses to prehistoric environments. We explored the effects of palaeoenvironmental change on the colonial tuco-tuco (Ctenomys sociabilis), a highly endemic species of Patagonian rodent that is currently listed as threatened by the IUCN. By combining surveys of modern genetic variation from throughout this species' current geographic range with analyses of DNA samples from fossil material dating back to 10000ybp, we demonstrate a striking decline in genetic diversity that is concordant with environmental events in the study region. Our results highlight the importance of non-anthropogenic factors in loss of diversity, including reductions in smaller mammals such as rodents.

Summary: We present Serial SimCoal, a program that models population genetic data from multiple time points, as with ancient DNA data. An extension of SIMCOAL, it also allows simultaneous modeling of complex demographic histories, and migration between multiple populations. Further, we incorporate a statistical package to calculate relevant summary statistics, which, for the first time allows users to investigate the statistical power provided by, conduct hypothesis-testing with, and explore sample size limitations of ancient DNA data. Availability: Source code and Windows/Mac executables at http://www.stanford.edu/group/hadlylab/ssc.html contact: senka@stanford.edu