Periodic Reporting for period 2 - IBERHUNT (Palaeolithic ungulate hunting strategies in the eastern Iberian Peninsula through advanced proteomic profiling)
Reporting period: 2024-01-03 to 2025-01-02
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 conducted advanced biomolecular analyses of ungulate dental assemblages and sediments from different rich-faunal Middle and Upper Palaeolithic sites on the eastern Iberian Peninsula.
Our specific goals were 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. In conclusion, IBERHUNT results broadened the discussion of evolutionary relationships between Neanderthals and modern humans and contributed to the development of new methods for archaeological research. The results were successfully achieved through the implementation of: 1) proteomic analytical and interpretation skills; 2) Recovering and matching amelogenin genes from AMELX and AMELY isoforms from annotated UniProt protein accession numbers to establish peptide sequences and variations; 3) Collection, interpretation, and organisation of mass chromatograms from nano-liquid tandem mass-spectrometry; 4) Extraction and analysis of palaeoenvironmental biomarkers; and 5) Multivariate statistics training in R software programming.
The IBERHUNT project conducted advanced biomolecular analyses of ungulate dental assemblages and sediments from different rich-faunal Middle and Upper Palaeolithic sites on the eastern Iberian Peninsula.
Our specific goals were 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. In conclusion, IBERHUNT results broadened the discussion of evolutionary relationships between Neanderthals and modern humans and contributed to the development of new methods for archaeological research. The results were successfully achieved through the implementation of: 1) proteomic analytical and interpretation skills; 2) Recovering and matching amelogenin genes from AMELX and AMELY isoforms from annotated UniProt protein accession numbers to establish peptide sequences and variations; 3) Collection, interpretation, and organisation of mass chromatograms from nano-liquid tandem mass-spectrometry; 4) Extraction and analysis of palaeoenvironmental biomarkers; and 5) Multivariate statistics training in R software programming.
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, and made notable progress in the field of biological sex estimation in modern and archaeological samples from a diverse range of animal species.
Overview of the work performed per Work Package:
WP1: Was focused on intensive hands-on training by Prof Parker and supervision by Prof. Eerkens at UC Davis, focusing on proteomic analytical and interpretation skills. During the initial months, a modern amelogenin reference database for European male and female mammal species was built, processing modern and archaeological samples ranging from 400.000 to 35.000 BP. 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. 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 4 Middle and Upper Palaeolithic archaeological sites (TD10 Gran Dolina, Spain; El Salt, Spain; Les Cottés, France and Peçtera Muierii, Romania).
WP2: Upon return to AMBI Lab, Dr. Égüez worked under Dr Mallol supervision on training for analysing palaeoenvironmental biomarkers. Sediment lipid extraction was performed successfully, and deuterium and carbon compound-specific isotope analysis are underway. Additionally, we included the analysis of tooth dentine for oxygen and carbon stable isotopes at UC Davis under supervision of Prof. Eerkens, complementing ecological and paleoenvironmental data while waiting for the sediments isotopes analysis.
WP3: This period was focused on developing computational skills in R programming, coupling it with critical 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. TD10 Gran Dolina, El Salt, Les Cottés, and Peçtera Muierii sites' proteomic pilot study successfully compared proteomic data on different species (Bison priscus, Bos primigenius, Equus ferus, and Canis lupus).
Main results achieved so far are:
3 peer-review articles in preparation
3 oral communications in international conferences:
1) Faunal sex estimation through amelogenin peptides analysis from the TD10.2 bison bone bed at Gran Dolina (Atapuerca, Spain). Natalia Égüez et al., XX UISPP World Congress (UISPP) (5-9 September 2023, Romania)
2) Deciphering Neanderthal Subsistence Approaches in El Salt's Stratigraphic Unit Xa (Alacant, Spain): Amelogenin Analysis of Equine Dental Samples. Natalia Égüez et al., 4th Virtual Conference for Women Archaeologists and Paleontologists (6-8 March 2024, online)
3) Amelogenin analysis of faunal dental samples in El Salt’s stratigraphic unit xa (alacant, spain): deciphering neanderthal subsistence approaches. Natalia Égüez et al.,30th EAA Annual Meeting (28-31 August 2024, Italy)
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: Was focused on intensive hands-on training by Prof Parker and supervision by Prof. Eerkens at UC Davis, focusing on proteomic analytical and interpretation skills. During the initial months, a modern amelogenin reference database for European male and female mammal species was built, processing modern and archaeological samples ranging from 400.000 to 35.000 BP. 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. 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 4 Middle and Upper Palaeolithic archaeological sites (TD10 Gran Dolina, Spain; El Salt, Spain; Les Cottés, France and Peçtera Muierii, Romania).
WP2: Upon return to AMBI Lab, Dr. Égüez worked under Dr Mallol supervision on training for analysing palaeoenvironmental biomarkers. Sediment lipid extraction was performed successfully, and deuterium and carbon compound-specific isotope analysis are underway. Additionally, we included the analysis of tooth dentine for oxygen and carbon stable isotopes at UC Davis under supervision of Prof. Eerkens, complementing ecological and paleoenvironmental data while waiting for the sediments isotopes analysis.
WP3: This period was focused on developing computational skills in R programming, coupling it with critical 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. TD10 Gran Dolina, El Salt, Les Cottés, and Peçtera Muierii sites' proteomic pilot study successfully compared proteomic data on different species (Bison priscus, Bos primigenius, Equus ferus, and Canis lupus).
Main results achieved so far are:
3 peer-review articles in preparation
3 oral communications in international conferences:
1) Faunal sex estimation through amelogenin peptides analysis from the TD10.2 bison bone bed at Gran Dolina (Atapuerca, Spain). Natalia Égüez et al., XX UISPP World Congress (UISPP) (5-9 September 2023, Romania)
2) Deciphering Neanderthal Subsistence Approaches in El Salt's Stratigraphic Unit Xa (Alacant, Spain): Amelogenin Analysis of Equine Dental Samples. Natalia Égüez et al., 4th Virtual Conference for Women Archaeologists and Paleontologists (6-8 March 2024, online)
3) Amelogenin analysis of faunal dental samples in El Salt’s stratigraphic unit xa (alacant, spain): deciphering neanderthal subsistence approaches. Natalia Égüez et al.,30th EAA Annual Meeting (28-31 August 2024, Italy)
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, as this is the first time this method has been successfully applied in Middle Palaeolithic European contexts. 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.