Periodic Reporting for period 1 - IsoTroph (Advanced techniques in isotopic biogeochemistry for the estimation of fossil mammal trophic position)
Période du rapport: 2022-09-01 au 2024-08-31
A deep understanding of present and past food web dynamics is crucial for comprehension of the evolution of ecosystems and sustainable management of natural and anthropogenic environments. In this field, isotope paleoecology holds a vast potential for investigating organism-environment interactions in a diachronic view.
Following the fundamental principle that every organism reflects the stable isotope composition of its diet, the isotope analysis equalized the possibilities to study the ecology of contemporary and fossil animals. One of the most common uses of stable isotopes in paleoecology is estimating an animal's trophic position (TP). TP estimation is possible due to the metabolic fractionation of isotopes, followed by the progressive predominance of heavier isotopes over light ones through the trophic chain. For bone collagen, the nitrogen isotopic composition in collagen is widely used as a proxy of TP due to distinct fractionation of 15N and 14N isotopes along the trophic chain, resulting in higher 15N concentrations in consumers in relation to their food. The 15N to 14N ratio is denoted as δ15N, i.e. in relation to an internationally defined standard, atmospheric nitrogen. Higher δ15N values can be interpreted as a higher trophic position of the specimen. This heavy isotope enrichment between a consumer and its diet can be quantified as a trophic discrimination factor (TDF). This parameter is used in numerical modeling to track detailed trophic relationships of individuals and between taxa. More detailed TP estimations are possible when individual TDFs of particular amino acids of collagen are compared.
The IsoTroph project aimed at bringing methodological improvements to the single amino acid isotopic analysis of terrestrial mammals' bone collagen. In particular, the scientific objective of the project was focused on the δ15N glutamate-phenylalanine (Glx-Phe) trophic discrimination factor (TDFGlx-Phe) in bone collagen of terrestrial carnivorans. This quantitative parameter is crucial in reconstructions of TP, but its numerical value has never been directly measured in mammal collagen. The specific research questions to investigate in the project were:
1. Is the 7.6‰ value, usually adopted in paleoecological studies, an exact approximation of the collagen-to-collagen TDFGlx-Phe in mammals?
2. Is the collagen-to-collagen TDFGlx-Phe constant between different mammal consumers?
The conclusions are that the TDFGlx-Phe=7.6‰ is not valid for mammalian bone collagen, but the actual value is around the range of 4.5-5.5‰ and seems constant among terrestrial carnivorans.
Following the fundamental principle that every organism reflects the stable isotope composition of its diet, the isotope analysis equalized the possibilities to study the ecology of contemporary and fossil animals. One of the most common uses of stable isotopes in paleoecology is estimating an animal's trophic position (TP). TP estimation is possible due to the metabolic fractionation of isotopes, followed by the progressive predominance of heavier isotopes over light ones through the trophic chain. For bone collagen, the nitrogen isotopic composition in collagen is widely used as a proxy of TP due to distinct fractionation of 15N and 14N isotopes along the trophic chain, resulting in higher 15N concentrations in consumers in relation to their food. The 15N to 14N ratio is denoted as δ15N, i.e. in relation to an internationally defined standard, atmospheric nitrogen. Higher δ15N values can be interpreted as a higher trophic position of the specimen. This heavy isotope enrichment between a consumer and its diet can be quantified as a trophic discrimination factor (TDF). This parameter is used in numerical modeling to track detailed trophic relationships of individuals and between taxa. More detailed TP estimations are possible when individual TDFs of particular amino acids of collagen are compared.
The IsoTroph project aimed at bringing methodological improvements to the single amino acid isotopic analysis of terrestrial mammals' bone collagen. In particular, the scientific objective of the project was focused on the δ15N glutamate-phenylalanine (Glx-Phe) trophic discrimination factor (TDFGlx-Phe) in bone collagen of terrestrial carnivorans. This quantitative parameter is crucial in reconstructions of TP, but its numerical value has never been directly measured in mammal collagen. The specific research questions to investigate in the project were:
1. Is the 7.6‰ value, usually adopted in paleoecological studies, an exact approximation of the collagen-to-collagen TDFGlx-Phe in mammals?
2. Is the collagen-to-collagen TDFGlx-Phe constant between different mammal consumers?
The conclusions are that the TDFGlx-Phe=7.6‰ is not valid for mammalian bone collagen, but the actual value is around the range of 4.5-5.5‰ and seems constant among terrestrial carnivorans.
To achieve the project goals, mean δ15N values of Glx and Phe in bones of selected carnivores and their prey were measured, and the TDFGlx-Phe was calculated between the carnivore’s average and its prey’s average values (the latter based on the proportion of prey species biomass in diet). The research material of the project were animal bone collections from Poland: modern wolf and European lynx from Bialowieża forest; modern red fox from Potok-Serderki mine; and Late Pleistocene cave hyena from Perspektywiczna Cave.
The first part of the project was focused on estimation of proportion of particular prey in the diet of studied carnivores. The task was completed based on: 1) data from previously published literature (for modern carnivores); and 2) determined as a part of the project activity, based on combined taphonomic and proteomic (Zooarchaeology by Mass Spectrometry, ZooMS) analyses (in case of cave hyena).
The second stage of the project included laboratory works: collagen extraction, collagen hydrolysis and amino acid derivatization, isotope ratio mass spectrometry measurements of bulk bone collagen and gas chromatography-combustion-isotope ratio mass spectrometry measurements of collagen individual amino acids. The results of spectrometric analyses were used in the the final stage of the project to calculate the TDFGlx-Phe. These stages constituted the training of the fellow in single amino acid paleoecology.
The results revealed that the prey collagen to carnivore collagen TDFGlx-Phe values are similar between the carnivores representing variable ecologies (hypercarnivores and omnivores), and stay between 4.5‰ and 5.5‰. This result confirmed the hypothesis that the TDFGlx-Phe differs from the literature's theoretical value used so far.
The project results were presented at nine scientific conferences, covering the scientific fields of isotope ecology and paleoecology, paleontology, archaeology, and molecular bioarchaeology. One peer-reviewed paper has been published, one has been accepted for publication, and two more are being prepared.
The first part of the project was focused on estimation of proportion of particular prey in the diet of studied carnivores. The task was completed based on: 1) data from previously published literature (for modern carnivores); and 2) determined as a part of the project activity, based on combined taphonomic and proteomic (Zooarchaeology by Mass Spectrometry, ZooMS) analyses (in case of cave hyena).
The second stage of the project included laboratory works: collagen extraction, collagen hydrolysis and amino acid derivatization, isotope ratio mass spectrometry measurements of bulk bone collagen and gas chromatography-combustion-isotope ratio mass spectrometry measurements of collagen individual amino acids. The results of spectrometric analyses were used in the the final stage of the project to calculate the TDFGlx-Phe. These stages constituted the training of the fellow in single amino acid paleoecology.
The results revealed that the prey collagen to carnivore collagen TDFGlx-Phe values are similar between the carnivores representing variable ecologies (hypercarnivores and omnivores), and stay between 4.5‰ and 5.5‰. This result confirmed the hypothesis that the TDFGlx-Phe differs from the literature's theoretical value used so far.
The project results were presented at nine scientific conferences, covering the scientific fields of isotope ecology and paleoecology, paleontology, archaeology, and molecular bioarchaeology. One peer-reviewed paper has been published, one has been accepted for publication, and two more are being prepared.
The project's direct aim and results were to enhance the methodology of using stable isotopes in ecology and paleoecology. Therefore, its primary relevance is scientific and potential users of the project results are expected within the academia, in particular among ecologists, paleoecologists, paleontologists, archaeologists, and animal conservation researchers.
The project's most important scientific result is establishing a parameter (the δ15N TDFGlx-Phe) that is needed in ecological modeling and that was calculated for the first time for bone collagen of terrestrial carnivorous mammals. The results improved the methodology of the TP calculation and questioned the validity of the method used so far, particularly in collagen-based studies. The new model of TP estimation is anchored at the herbivore baseline, which is the mathematic representation of herbivores δ15NGlx-δ15NPhe relationship in a form of linear regression. This allows to overcome the limitations of the isotopic values in past vegetation, which was needed in the previous method but was unknown and only assumed. The new data established in the course of the IsoTroph calls for revisiting the published studies and will significantly impact the interpretation of the paleodiet of ancient hominids.
The new approach of ZooMS analysis of digested bone fragments was the first ever applied for a cave hyena taphocenosis. The results revealed differences between diet estimation using classical taxonomical identifications based on bone morphology and the actual taxa eaten by hyenas. The new model regards the biomass of each species and points to the woolly rhino and mammoth as the main prey of the cave hyena. The results highlighted the usefulness of ZooMS analyses in the diet reconstruction of fossil assemblages and raised the call for action for more detailed studies combining morphology-based and ZooMS-based taxonomy identification for better understanding of the taphonomic formation processes of bone accumulations related to hunting and scavenging behavior of bone collectors.
Additionally, the project delivered unique information on collagen-to-collagen prey-predator trophic discrimination factor (bulk collagen Δ13C, bulk collagen Δ15N) in cave hyena. The results revealed that the Δ15N for hyena is similar to those previously known for other predators, but the Δ13C value is higher. This difference is diet-specific and relates to hyena's ability to digest bones. This observation has important implications for future ecological and paleoecological studies of other bone-eating carnivores, such as wolverine or striped hyena.
The project's most important scientific result is establishing a parameter (the δ15N TDFGlx-Phe) that is needed in ecological modeling and that was calculated for the first time for bone collagen of terrestrial carnivorous mammals. The results improved the methodology of the TP calculation and questioned the validity of the method used so far, particularly in collagen-based studies. The new model of TP estimation is anchored at the herbivore baseline, which is the mathematic representation of herbivores δ15NGlx-δ15NPhe relationship in a form of linear regression. This allows to overcome the limitations of the isotopic values in past vegetation, which was needed in the previous method but was unknown and only assumed. The new data established in the course of the IsoTroph calls for revisiting the published studies and will significantly impact the interpretation of the paleodiet of ancient hominids.
The new approach of ZooMS analysis of digested bone fragments was the first ever applied for a cave hyena taphocenosis. The results revealed differences between diet estimation using classical taxonomical identifications based on bone morphology and the actual taxa eaten by hyenas. The new model regards the biomass of each species and points to the woolly rhino and mammoth as the main prey of the cave hyena. The results highlighted the usefulness of ZooMS analyses in the diet reconstruction of fossil assemblages and raised the call for action for more detailed studies combining morphology-based and ZooMS-based taxonomy identification for better understanding of the taphonomic formation processes of bone accumulations related to hunting and scavenging behavior of bone collectors.
Additionally, the project delivered unique information on collagen-to-collagen prey-predator trophic discrimination factor (bulk collagen Δ13C, bulk collagen Δ15N) in cave hyena. The results revealed that the Δ15N for hyena is similar to those previously known for other predators, but the Δ13C value is higher. This difference is diet-specific and relates to hyena's ability to digest bones. This observation has important implications for future ecological and paleoecological studies of other bone-eating carnivores, such as wolverine or striped hyena.