Periodic Reporting for period 1 - IBERHUNT (Palaeolithic ungulate hunting strategies in the eastern Iberian Peninsula through advanced proteomic profiling)
Okres sprawozdawczy: 2022-01-03 do 2024-01-02
Recent zooarchaeological analyses have suggested that Neanderthals were skilled hunters. Yet, there is still a vivid debate on whether Middle Palaeolithic human groups were also capable of more effective, and diverse, sophisticated food-processing behaviours, in particular through specialised hunting. Traditionally, archaeologists have relied on the osteological analysis of bone assemblages to determine prey mortality patterns, where species variability, age, and seasonality data are calculated. Although this approach has yielded a great deal of important information about the Pleistocene hominin hunting strategies, there is a considerable lack of sex-based prey data for most Pleistocene bone assemblages, key information that has direct implications for hominin planning depth, anticipatory abilities, mobility, and land use.
The IBERHUNT project will conduct advanced biomolecular analyses of ungulate dental assemblages and sediments from different rich-faunal Middle and Upper Palaeolithic sites on the eastern Iberian Peninsula. The anthropic occupation deposits range Marine Isotope Stages (MIS) 5 to 3, making them exemplary case studies for comparative-based research.
Our specific goals are to 1) determine the biological sex of ungulate remains by reading protein sequences through the analysis of tooth enamel amelogenin and 2) identify palaeoenvironmental biomarkers by the analysis of sediments’ compound-specific isotope ratios. This unique combination of state of the art techniques will provide contextualised and quantifiable high-resolution data on Pleistocene human foraging efficiency, enabling a re-evaluation of Neanderthal predatory economics through an optimal foraging perspective that includes climate adaptations. IBERHUNT results will broaden the discussion of evolutionary relationships between Neanderthals and modern humans and contribute to the development of new methods for archaeological research.
The IBERHUNT project will conduct advanced biomolecular analyses of ungulate dental assemblages and sediments from different rich-faunal Middle and Upper Palaeolithic sites on the eastern Iberian Peninsula. The anthropic occupation deposits range Marine Isotope Stages (MIS) 5 to 3, making them exemplary case studies for comparative-based research.
Our specific goals are to 1) determine the biological sex of ungulate remains by reading protein sequences through the analysis of tooth enamel amelogenin and 2) identify palaeoenvironmental biomarkers by the analysis of sediments’ compound-specific isotope ratios. This unique combination of state of the art techniques will provide contextualised and quantifiable high-resolution data on Pleistocene human foraging efficiency, enabling a re-evaluation of Neanderthal predatory economics through an optimal foraging perspective that includes climate adaptations. IBERHUNT results will broaden the discussion of evolutionary relationships between Neanderthals and modern humans and contribute to the development of new methods for archaeological research.
One of the key innovations in our project has been the development and application of novel proteomics methodological workflows. This has yielded significant findings, including a deeper understanding of the preservation, integrity, and recovery of the amelogenin protein in enamel samples from both burnt and non-burnt modern and archaeological contexts. Furthermore, we have made notable progress in the field of biological sex estimation, particularly in modern and archaeological samples from a diverse range of animal species.
Overview of the work performed per Work Package:
WP1: The PI has mainly focused on intensive hands-on training by Prof Parker and supervision by Prof. Eerkens at UC Davis, focusing on proteomic analytical and interpretation skills (i.e. powdered or cut enamel samples cleaning and pre-treatment and amelogenin extraction for tandem mass-spectrometry liquid injection). During the initial months, Dr. Égüez worked with a modern set of samples from Bos taurus, experimenting with protein thermoalteration to understand the preservation and quality recovery of amelogenin on dental enamel samples. Also, a modern amelogenin reference database for European male and female mammal species was built. This was an excellent way for the PI to learn the complete laboratory workflow before processing archaeological and unique samples that range from 400.000 years ago to 35.000 before the present. At the same time, the PI received training for peptide sequence searches. As amino acid variations are unique for each species, and there are no published amelogenin-specific databases, a great deal of time was devoted to recovering and matching amelogenin genes from AMELX and AMELY isoforms from annotated UniProt protein accession numbers to establish peptide sequences and variations that could be used in the project. Later, WP1 was completed with supervision and analysis monitoring for the remaining sampling sets and sites, followed by an interpretation phase and preparation of paper manuscripts. In total, the PI has processed and analysed 138 samples from 13 different animal species (Bison priscus, Bison bonasus, Bos taurus, Bos primigenius, Cervus elaphus, Capra sp., Capreolus capreolus, Equus ferus, Equus caballus, Ovis aries, Ursus arctos, Canis lupus familiaris, and Canis lupus) from modern referential collections and 6 Middle and Upper Palaeolithic archaeological sites (TD10 Gran Dolina, Spain; El Salt, Spain; Cova del Gegant, Spain; Les Cottés, France; Cueva Des-Cubierta, Spain and Peçtera Muierii, Romania).
WP3: The PI focused on developing computational skills in R programming, coupling it with key notions of empirical modelling within a human evolutionary framework. Multivariate statistics were used to analyse collective properties and tackle hypotheses testing over large sets of multi-taxa and multi-site sex-based patterns. El Salt site proteomic pilot study was successfully carried out by comparing proteomic data on three different species (Equus ferus, Capra sp., and Cervus elaphus).
The main results achieved so far for this research are as follows:
- 3 peer-review articles in preparation
- 4 oral communications in international conferences
-1 poster communication in national conference
- 5 talks as invited speaker in international venues
- 2 talks as invited speaker in national venues
- 1 dissemination activity funded by the European Commission MacaroNight 2023
Overview of the work performed per Work Package:
WP1: The PI has mainly focused on intensive hands-on training by Prof Parker and supervision by Prof. Eerkens at UC Davis, focusing on proteomic analytical and interpretation skills (i.e. powdered or cut enamel samples cleaning and pre-treatment and amelogenin extraction for tandem mass-spectrometry liquid injection). During the initial months, Dr. Égüez worked with a modern set of samples from Bos taurus, experimenting with protein thermoalteration to understand the preservation and quality recovery of amelogenin on dental enamel samples. Also, a modern amelogenin reference database for European male and female mammal species was built. This was an excellent way for the PI to learn the complete laboratory workflow before processing archaeological and unique samples that range from 400.000 years ago to 35.000 before the present. At the same time, the PI received training for peptide sequence searches. As amino acid variations are unique for each species, and there are no published amelogenin-specific databases, a great deal of time was devoted to recovering and matching amelogenin genes from AMELX and AMELY isoforms from annotated UniProt protein accession numbers to establish peptide sequences and variations that could be used in the project. Later, WP1 was completed with supervision and analysis monitoring for the remaining sampling sets and sites, followed by an interpretation phase and preparation of paper manuscripts. In total, the PI has processed and analysed 138 samples from 13 different animal species (Bison priscus, Bison bonasus, Bos taurus, Bos primigenius, Cervus elaphus, Capra sp., Capreolus capreolus, Equus ferus, Equus caballus, Ovis aries, Ursus arctos, Canis lupus familiaris, and Canis lupus) from modern referential collections and 6 Middle and Upper Palaeolithic archaeological sites (TD10 Gran Dolina, Spain; El Salt, Spain; Cova del Gegant, Spain; Les Cottés, France; Cueva Des-Cubierta, Spain and Peçtera Muierii, Romania).
WP3: The PI focused on developing computational skills in R programming, coupling it with key notions of empirical modelling within a human evolutionary framework. Multivariate statistics were used to analyse collective properties and tackle hypotheses testing over large sets of multi-taxa and multi-site sex-based patterns. El Salt site proteomic pilot study was successfully carried out by comparing proteomic data on three different species (Equus ferus, Capra sp., and Cervus elaphus).
The main results achieved so far for this research are as follows:
- 3 peer-review articles in preparation
- 4 oral communications in international conferences
-1 poster communication in national conference
- 5 talks as invited speaker in international venues
- 2 talks as invited speaker in national venues
- 1 dissemination activity funded by the European Commission MacaroNight 2023
The results obtained constitute an important new data addition to the proteomic research community, as amelogenin animal sequences are scarce and often poorly annotated. Our results successfully contribute to IBERHUNT's objective of providing quantifiable data to investigate Pleistocene human foraging efficiency and hunting strategies for determined species and ecosystem management. This represents a step forward for the archaeology of human behavior, particularly for understanding the Neandertal hunting skills in the Iberian Peninsula and Southern Europe. Moreover, the research has demonstrated the potential of amelogenin and other enamel proteins, such as enamelin, to contribute to phylogenetic studies of extinct mammals, allowing for detailed knowledge of the evolutionary relationships between species that can inform studies in ecology and conservation.
Methodologically, the review and improvement of sample cleaning pre-treatment, protein extraction, and sample storage constitute a new corpus of information that can be used for stakeholders and cultural heritage organisations, such as museums, thus contributing to future actions toward better preservation of biological samples.
Methodologically, the review and improvement of sample cleaning pre-treatment, protein extraction, and sample storage constitute a new corpus of information that can be used for stakeholders and cultural heritage organisations, such as museums, thus contributing to future actions toward better preservation of biological samples.