Periodic Reporting for period 1 - BACKWARD (Overcoming the frontiers of biomolecular studies on human history and adaptation using palaeoproteomics)
Période du rapport: 2022-01-01 au 2023-06-30
The family tree of humans, once so simple is now a tangled shrub, as more and more fossils add layer upon layer of complexity to what is arguably the most fundamental of questions: where did we come from? Although the adoption of transformative experimental methods has repeatedly propelled the advancement of palaeoanthropological research, genetics can be arguably considered the single approach that, in the last decades, provided the most impressive results. Specifically, high-throughput ancient DNA (aDNA) sequencing has been central to dramatically advance our understanding of human evolution, migration, and admixture after the divergence of modern humans from archaic ones in the Middle Pleistocene. However, no aDNA data have so far been recovered from hominid remains older than ~0.4 million years (Ma), due to the irreversible chemical degradation of ancient DNA.
In stark contrast, ancient proteins represent a more durable source of genetic information, reported to survive up to ~2 Ma, in hominid dental enamel, even from sub-tropical regions. Their sequences can be reliably used to confidently reconstruct the evolutionary relationships between extant and extinct species, or to determine the biological species and the sex of the organisms the fossil teeth originate from. Ultimately, mass spectrometry (MS)-based ancient protein sequencing, i.e. palaeoproteomics, already convincingly demonstrated its transformative value, currently being the only approach enabling molecular-based evolutionary reconstructions for species that went extinct millions of years ago. BACKWARD addresses major unsettled debates about African and Asian extinct hominid phylogeny, by developing and deploying a new generation of palaeoproteomic workflows, relying on the most advanced mass spectrometry and bioinformatic solutions currently available. In particular, BACKWARD aims to define the phylogenetic position of a set of extinct hominids, dated up to ~2 million years and including (i) Homo naledi, Homo erectus, Paranthropus robustus, Australopithecus africanus and Australopithecus sediba, from South Africa, and (ii) Homo erectus, Meganthropus palaojavanicus, Sivapithecus spp., and Lufengpithecus spp. from Eurasia. BACKWARD also aims to screen large sets of morphologically non-informative isolated fossil fragments of bones and teeth, to identify the species and sex of the organism from which they originated. Deciphering the evolutionary history of our own species, has always represented an inspirational target for the broad audience globally. BACKWARD’s discoveries will be shared internationally with the largest possible audience using the web and social media, public outreach events focused on different target categories based both in the EU and in the extra-EU countries from which the fossil specimens originate.
In stark contrast, ancient proteins represent a more durable source of genetic information, reported to survive up to ~2 Ma, in hominid dental enamel, even from sub-tropical regions. Their sequences can be reliably used to confidently reconstruct the evolutionary relationships between extant and extinct species, or to determine the biological species and the sex of the organisms the fossil teeth originate from. Ultimately, mass spectrometry (MS)-based ancient protein sequencing, i.e. palaeoproteomics, already convincingly demonstrated its transformative value, currently being the only approach enabling molecular-based evolutionary reconstructions for species that went extinct millions of years ago. BACKWARD addresses major unsettled debates about African and Asian extinct hominid phylogeny, by developing and deploying a new generation of palaeoproteomic workflows, relying on the most advanced mass spectrometry and bioinformatic solutions currently available. In particular, BACKWARD aims to define the phylogenetic position of a set of extinct hominids, dated up to ~2 million years and including (i) Homo naledi, Homo erectus, Paranthropus robustus, Australopithecus africanus and Australopithecus sediba, from South Africa, and (ii) Homo erectus, Meganthropus palaojavanicus, Sivapithecus spp., and Lufengpithecus spp. from Eurasia. BACKWARD also aims to screen large sets of morphologically non-informative isolated fossil fragments of bones and teeth, to identify the species and sex of the organism from which they originated. Deciphering the evolutionary history of our own species, has always represented an inspirational target for the broad audience globally. BACKWARD’s discoveries will be shared internationally with the largest possible audience using the web and social media, public outreach events focused on different target categories based both in the EU and in the extra-EU countries from which the fossil specimens originate.
So far, the palaeoproteomic research activity has been focusing mostly on the investigation of dental enamel proteins from four dental enamel specimens of the Pleistocene hominin species Paranthropus robustus from the site of Swartkransin South Africa. The results obtained so far have been publicly shared as a pre-print document in the bioRxiv repository (doi: 10.1101/2023.07.03.547326). For this species, both phylogenetic relationships to other taxa and the nature of intraspecific variation are still disputed. The identification of AMELY-specific peptides and semi-quantitative MS data analysis enabled us to determine the biological sex of all the specimens. Our combined molecular and morphometric data also provide compelling evidence of a significant degree of variation within southern African Paranthropus, as previously suggested based on morphology alone. Finally, the molecular data also confirm the taxonomic placement of Paranthropus within the hominin clade. This study demonstrates the feasibility of recovering informative Early Pleistocene hominin enamel proteins from Africa. Crucially, it also shows how the analysis of these proteins can contribute to understanding whether hominin morphological variation is due to sexual dimorphism or to taxonomic differences. We anticipate that this approach can be widely applied to geologically-comparable sites within South Africa, and possibly more broadly across the continent.
Another research line led to the publication of a new workflow designed to achieve reliable species identification of a large sample set of bone fragments at a sustainable costs. Species determination based on genetic evidence is an indispensable tool in archaeology, forensics, ecology, and food authentication. Most available analytical approaches involve compromises with regard to the number of detectable species, high cost due to low throughput, or a labor-intensive manual process. The work published on Nature Communications (PMID: 35513387) describes “Species by Proteome INvestigation” (SPIN), a shotgun proteomics workflow for analyzing archaeological bone capable of querying over 150 mammalian species by liquid chromatography-tandem mass spectrometry (LC-MS/MS). While this initial study is focused on modern and archaeological mammalian bone, SPIN will be open and expandable to other biological tissues and taxa.
Preliminary palaeoproteomic data have also been generated to characterize the palaeodiversity of the genus Pongo, by analyzing 14 dental specimens of Late Pleistocene Pongo from Vietnam, Laos, Malaysia and Thailand. The preliminary results have been presented in a poster at the 12th annual meeting of the European Society for the Study of Human Evolution, which took place in Tübingen (Germany) between 22th and 24th September 2022. Finally multiple methodological works are currently under preparation to better describe and improve most of the steps of the palaeoproteomics workflow.
Another research line led to the publication of a new workflow designed to achieve reliable species identification of a large sample set of bone fragments at a sustainable costs. Species determination based on genetic evidence is an indispensable tool in archaeology, forensics, ecology, and food authentication. Most available analytical approaches involve compromises with regard to the number of detectable species, high cost due to low throughput, or a labor-intensive manual process. The work published on Nature Communications (PMID: 35513387) describes “Species by Proteome INvestigation” (SPIN), a shotgun proteomics workflow for analyzing archaeological bone capable of querying over 150 mammalian species by liquid chromatography-tandem mass spectrometry (LC-MS/MS). While this initial study is focused on modern and archaeological mammalian bone, SPIN will be open and expandable to other biological tissues and taxa.
Preliminary palaeoproteomic data have also been generated to characterize the palaeodiversity of the genus Pongo, by analyzing 14 dental specimens of Late Pleistocene Pongo from Vietnam, Laos, Malaysia and Thailand. The preliminary results have been presented in a poster at the 12th annual meeting of the European Society for the Study of Human Evolution, which took place in Tübingen (Germany) between 22th and 24th September 2022. Finally multiple methodological works are currently under preparation to better describe and improve most of the steps of the palaeoproteomics workflow.
BACKWARD already started delivering on some of its objectives. Specifically, the recovery of the ancient protein sequences from the P. robustus specimens currently represent the oldest genetic data from a human relative. Even though these results have been publicly shared only in a non-peer-reviewed preprint, this attracted the attention of the scientific press (doi: 10.1038/d41586-023-02242-z). Similarly the publication of the SPIN article described the first high-throughput and high-resolution workflow for cost sustainable species identification of bone fragments.