Periodic Reporting for period 1 - PALEOS (seda DNA: proxy for mapping presence of domesticated Horse mitochondrial haplogroups in the Pyrenees)
Reporting period: 2021-08-23 to 2023-08-22
The process of animal domestication brought about dramatic changes in the daily lives and economies of early human ancestors. Understanding the earliest stages of animal domestication has, however, remained challenging due to the lack of reliable, well-preserved archaeological records. Fossil preservation along with scarcity of fossils yielding analysable DNA add to the problem. Hence there is a need of technique that does not rely on the availability of reliable fossils. Sedimentary ancient DNA (sedaDNA) uses the DNA extracted from sediments deposited in different environmental settings for paleoecosystem reconstructions. These different environmental settings include marine sediments, riverine sediments, lacustrine sediments, cave sediments, paleosols (ancient soils), peat-bogs, thrombolites and many more. We wanted to design the tool that combines the detailed analytical approach used in fossil based paleogenomics to sedimentary DNA hence leading to holistic analysis of history domesticated animals while circumventing the fossil preservation and scarcity problem. Using different set of samples, we address the following objectives in the PALEOS: 1) To make a collection of probes to track horse domestication in any environmental sample, 2) To find if any pattern of preferential mitochondrial (maternal), and potentially Y-chromosomal (paternal), haplogroups existed in the use of horses for transport, 3) To expand the collection of probes to include other domesticated animals of major economic importance, namely: donkeys, sheep, goats, pigs, cattle, dogs and pigs, 4) To assess the suitability of our methodology to characterize the DNA content of coprolites.
For PALEOS, we focused on mitochondrial DNA sequences of different domesticated animals and Y chromosomal sequences of horses. Although, these sequences are identical in individuals of the same species, there are a few differences distributed across the sequence. These differences, known as single nucleotide polymorphisms (SNPs) work as informative indicators of the specific groups called haplogroups. These haplogroups show temporal phylogeographic distribution of maternal and paternal lineage of any species. This makes it important to conclusively know to which haplogroup an individual from whom the ancient DNA is obtained, belongs. Here, we successfully designed RNA-probes for the most economically relevant domesticated animals, namely: horses, donkeys, pigs, cattle, goats, sheep, dogs and chicken. These probes target SNPs of different haplogroups of these animals. We tested the efficiency of these probes on different samples which included ancient DNA extracts obtained from diverse horse fossils. Such positive controls established the sensitivity and specificity of our in-solution DNA capture system to increase coverage by magnitudes beyond 1,000x, relative to shotgun sequencing. We further demonstrated that these probes provide sufficient power to detect the presence of different domesticated animals when applied on the DNA extracted from soil belonging to the local farm harboring different domestic animals. Our system can, thus, be used in routine for a genetic monitoring of domesticated animals present in any given environment, in the absence of invasive animal sampling; it is, thus, respectful of the animal welfare. After successful testing of probes for accuracy and efficiency, we applied this tool on ancient sediments and coprolites (~40,000 years old). Sediments were obtained from the paleolithic rock shelter Abrigo de la Malia located in the Iberian Peninsula (the region of interest), which showed early human occupation and the presence of several other mammals as well as birds. Our tool detected the presence of DNA originating from sheep in sediments deposited during past few decades when cave was being used by the farmers as a storage facility for their equipment and livestock. The mitochondrial sequences grouped with haplogroup B which is the haplogroup for domesticated sheep with ubiquitous distribution in the Mediterranean and middle Europe. This demonstrated the capacity of our methodology to detect the presence of domestic animals in the region. Applying this methodology to coprolites obtained from the cave Cassenade, France, we detected the presence of one horse maternal lineage in one coprolite, alongside those of local cave hyenas showing that the cave hyena fed on a horse before defecating in the cave. Overall, our experiments successfully demonstrate that the tool developed by PALEOS can be applied on wide variety of environmental samples. These results were presented in two conferences: The Animal Farm Meeting, Toulouse 11-13th June, 2022 and 10th International Council of Archaeozoology- Archaeozoology, Genetics, Proteomics & Morphometrics (ICAZ-AGPM) meeting, Munich. Furthermore, two manuscripts are exclusively dedicated to describe the design and the results of these probes.
PALEOS for the first time established the suitability of sediments and environmental samples to investigate animal domestication. This greatly reduces the dependency of the scientists on the fossils. Furthermore, the design of PALEOS is so robust that it not only detects the correct haplogroup but does so with 1000X more coverage even in case of samples where shotgun gives 1.5x coverage. This high sensitivity adds to the success of PALEOS. Extraction of DNA from fossils often requires the destruction of fossils and such fossils then become non-usable for other morphological studies. But, sediment collection or collection of any environmental sample, is a non-invasive method and when used with tool provided by PALEOS gives comparable results. Hence, it is the first step towards preferentially using non-invasive and non-destructive methods of ancient DNA studies wherever fossils are indispensable. During PALEOS, Dr. Kuldeep developed collaboration with Dr. Yvette Running Horse Collin, another MSCA fellow, who worked on combining the western science with Oglala Lakota traditional science from USA at CAGT, Toulouse. We discussed on how the tool developed by PALEOS can be used extensively in US on indigenous sites to discover the ecosystem maintenance practices followed by Oglala Lakota people since ancient times. Yvette is currently Executive Director and Principal Science Officer for “Taku Škaŋ Škaŋ Wasakliyapi: Global Institute for Traditional Sciences (GIFTS)”, the first ever institute for traditional Indigenous sciences. Using probes developed by PALEOS, we are planning to investigate the traditional oyster farming and forest maintenance practices followed by Oglala Lakota people. This collaboration further shows the far-reaching applicability of tools developed by PALEOS.
Besides ancient DNA, applying this tool to modern soil also demonstrated its potential as a surveillance technique for detecting the presence of domestic animals in various environmental settings. The technique can be expanded or reduced according to the objectives of the individual project. Its adaptability, allowing customization according to project objectives, renders it both highly efficient and exceptionally flexible. The results of PALEOS can be useful for those potentially interested in ancient DNA research, paleoecology, sediment DNA research, as well as for those involved in environmental monitoring of certain species of interest using environmental samples without the use of animal tissues.
Besides ancient DNA, applying this tool to modern soil also demonstrated its potential as a surveillance technique for detecting the presence of domestic animals in various environmental settings. The technique can be expanded or reduced according to the objectives of the individual project. Its adaptability, allowing customization according to project objectives, renders it both highly efficient and exceptionally flexible. The results of PALEOS can be useful for those potentially interested in ancient DNA research, paleoecology, sediment DNA research, as well as for those involved in environmental monitoring of certain species of interest using environmental samples without the use of animal tissues.