Periodic Reporting for period 1 - MethylRIDE (MethylRIDE: Charting DNA methylation reprogramming of Ice Age horses in the face of global climate change and extinction) Reporting period: 2021-01-01 to 2022-12-31 Summary of the context and overall objectives of the project The Late Pleistocene period (i.e. the last Ice Age) is characterized by a succession of climatic events, notably the extended cold phase of the Last Glacial Maximum (LGM; ca. 22–18 kBP), and the subsequent rapid warming and cooling events that signify the transition from the Pleistocene (14–11 kBP) to the current inter-glacial Holocene period (11 kBP–now). The Late Pleistocene roughly coincides with the mass extinction of iconic megafaunal species. MethylRIDE especially focuses on the contribution of (epi)genetic mechanisms to megafaunal phenotypic plasticity and the role it plays in micro-evolutionary change in horses. In MethylRIDE, we exploit a highly novel experimental system by using more than 100 well-preserved equine bones and teeth dating back up to 50,000 years in North America, as well as modern blood samples from horses caretaken by Native Peoples, to record and compare the way in which the (epi)genome responded to the dramatic swings in climate and environment that occurred during this time period. MethylRIDE builds upon this remarkable paleontological material and record in the Americas, as well as an equivalent material panel in Siberia for comparison, and applies cutting-edge biotechnologies to measure (epi)genetic variation in ancient horse genomes, and determine the tempo and mode of change throughout this time period and until their extinction. We contrast these patterns with detailed climatic and paleoenvironmental records to identify relationships between (epi)genetic change and potential environmental stressors, and specifically monitor the extent to which American and Siberian horse population remained isolated or connected through Beringia as a consequence of global environmental change. MethylRIDE has direct relevance to research in a wide range of fields, including evolution, ecology, archaeology, domestication, animal breeding, anthropology, indigenous cultural preservation, extinction theory and conservation. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far A total of 44 ancient horse samples from northwestern Siberia showed sufficient DNA preservation levels for the characterization of 29 ancient (epi)genomes. A total of 64 ancient North American horse samples, 14 historical North American samples and 5 Ancient South American horse samples were screened for DNA preservation. Collectively, these samples proved sufficient for the successful characterization of 73 ancient (epi)genomes (including 9 historical), sex determination for 93 samples. Genome comparative data were also generated for American horses associated with the first Nations (Oglala Lakota, providing a total of 41 modern horse genomes in collaboration with Sacred Way Sanctuary), 3 historical horses pre-dating the Puebloan Revolt of 1680 CE, and 16 additional ones radiocarbon dated to the 18th and 19th centuries CE. The (epi)genome data generated represent considerably extend the almost non-existent genetic data for ancient horses in the Americas.The DNA libraries of 73 specimens were subjected to shotgun sequencing, while the remaining were subjected to DNA capture, together with 25 additional ones that provided experimental controls and validation. Direct processing of the underlying sequencing data through DamMet, as originally planned, failed detecting hyper-methylated regions, which required new statistical methodologies (Liu et al. 2023). Preliminary data analyses did not reveal the presence of genomic regions showing differential DNA methylation levels, neither between ancient and modern horses, or various categorizations of ancient American horses (e.g. temporal and geographic binning). This may indicate limited statistical power in our analyses, potentially due to the limited set of genomic regions targeted. Due to the cross-cultural emphasis of MethylRIDE and our attempt to develop a truly collaborative project with our Indigenous partners, the communication, dissemination and reporting schedule has required adjustment. YC serves as a co-author for the following papers: Early dispersal of domestic horses into the Great Plains and Northern Rockies (published and providing the basis for the cover page of Science on March 31, 2023), and DNA methylation-based profiling of archaeological horse remains for age-at-death and castration (published in iScience on February 1, 2023). Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Further publications leveraging the full extent of the data generated during the project are pending. Our collaboration with our Oglala Lakota partners has been ground-breaking as true exchange between scientific systems has been achieved. The members of our Traditional Indigenous Review Board (IRB) have made the decision to bring their unique scientific systems forward, and are calling for a new era of collaboration in science. As part of this, one additional academic article has been published, and 2 more have been conceptualized and drafted. The 1st paper credits MSCA support and is entitled ‘Standing for Unči Maka (Grandmother Earth) and all Life: An Introduction to Lakota Traditional Sciences, Principles and Protocols and the Birth of a New Era of Scientific Collaboration (published by the Maxwell Museum of Anthropology, UNM) on Feb 21, 2023. Much has been gained from this revolutionary cross-cultural collaboration, including a global call for integrating Indigenous knowledge and sciences as a response to the ongoing climate shift. The MSCA support in this initiative was truly essential and the additional articles are aimed at sharing our successful collaborative experience to empower similar research initiatives and help others to be able to successfully navigate both scientific systems and reach authentic collaboration.MethylRIDE was also able to create an opportunity that could potentially revolutionize the future of the field of ancient genomics. Applying Lakota scientific protocols regarding the extraction and screening of ancient DNA helped isolate high-quality DNA. This is worth further exploration, as it may extend the sensitivity of the techniques currently available. Finally, the project results can be potentially useful to those interested in conservation efforts, scientific collaboration, wild horse management, ecology, sustainability and rewilding efforts, as well as preservation of Indigenous culture as it relates to particular keystone species. YC and LO discuss findings for publication regarding dispersal of horses in North America. YC performing DNA extraction for MethylRIDE project. YC Drilling ancient North American horse bones for MethylRIDE project.
