Periodic Reporting for period 1 - PRISTINE (PRehistoric fISheries assisTing marIne baseliNEs)
Reporting period: 2022-11-01 to 2024-11-30
PRISTINE was born out of the need to understand the roots of the shifted baseline in Indo-Pacific fisheries, one of the most threatened marine regions. To achieve this, it was essential to expand the time frame beyond traditional fisheries records, which often rely on data from recent decades—insufficient for assessing the long-term impacts of fish exploitation. Given that the Indo-Pacific has a long history of marine resource exploitation dating back to at least the Neolithic, we targeted this period to establish a starting point for the baseline. This is why the project was named PRISTINE (PRehistoric fIsheries Assisting Marine Baselines).
We delved into ichthyoarchaeological data to learn about and identify the fish species that ancient Neolithic communities were fishing in different regions of the Indo-Pacific. However, detailed taxonomic identification of Indo-Pacific fish remains is challenging due to the region's high species diversity and the limitations of morphological analysis. These challenges hinder our understanding of prehistoric fisheries and their long-term impacts. To overcome this, we relied on cutting-edge paleoproteomic techniques to obtain accurate species identification of prehistoric fish remains through the following objectives: 1) develop a collagen reference database of representative modern Indo-Pacific fishes, 2) obtain taxonomic identifications of Neolithic fish remains, and 3) conduct a long-term evaluation of Indo-Pacific fisheries.
We delved into ichthyoarchaeological data to learn about and identify the fish species that ancient Neolithic communities were fishing in different regions of the Indo-Pacific. However, detailed taxonomic identification of Indo-Pacific fish remains is challenging due to the region's high species diversity and the limitations of morphological analysis. These challenges hinder our understanding of prehistoric fisheries and their long-term impacts. To overcome this, we relied on cutting-edge paleoproteomic techniques to obtain accurate species identification of prehistoric fish remains through the following objectives: 1) develop a collagen reference database of representative modern Indo-Pacific fishes, 2) obtain taxonomic identifications of Neolithic fish remains, and 3) conduct a long-term evaluation of Indo-Pacific fisheries.
Successful collagen extraction was performed on modern fish samples. In total, 72 fish species were analyzed using MALDI-TOF (ZooMS) to obtain collagen type I spectra, which were visualized to meet established quality standards, and theoretical biomarkers were assigned. Twenty-five selected species were further sequenced using LC-MS/MS (proteomics) to confirm the identified biomarkers. For this, project focused on the families Serranidae, Scaridae, Carangidae, Carcharhinidae, and Lethrinidae, as these are the most abundant in the archaeological record, and several of their species are currently threatened. A reference collagen database was compiled and it is currently in preparation for online publication.
Ninety-eight fish tissue samples were subjected to DNA extraction. Five complete mitogenomes were sequenced using Illumina technology and assembled to obtain the full mitogenome. COI barcoding was extracted, amplified, and sequenced using Sanger technology. All sequences were carefully curated before submission to BOLD.
Archaeological fish remains were measured and photographed before successful collagen extraction, ensuring all morphological features were documented. Identification of archaeological fish remains was carried out via ZooMS, both genus and species levels were achieved. At the species level, we were able to identify closely related species within the genera Epinephelus and Scarus. The collagen spectra of the archaeological material were verified for quality, confirming that the method was effective.
Ninety-eight fish tissue samples were subjected to DNA extraction. Five complete mitogenomes were sequenced using Illumina technology and assembled to obtain the full mitogenome. COI barcoding was extracted, amplified, and sequenced using Sanger technology. All sequences were carefully curated before submission to BOLD.
Archaeological fish remains were measured and photographed before successful collagen extraction, ensuring all morphological features were documented. Identification of archaeological fish remains was carried out via ZooMS, both genus and species levels were achieved. At the species level, we were able to identify closely related species within the genera Epinephelus and Scarus. The collagen spectra of the archaeological material were verified for quality, confirming that the method was effective.
Some of PRISTINE’s results exceeded expectations at both biomolecular levels: proteomic and genomic.
Regarding proteomics, the project successfully sequenced collagen from fish species whose amino acid sequences were previously unrecorded in protein databases. This achievement represents a significant advancement beyond the current state of the art, as it expands the molecular reference data available for fish species identification and contributes to the growing field proteomics and its application in ichthyoarchaeoogy. Additionally, using MALDI-TOF (ZooMS), we were able to identify closely related species in the archaeological record, such as those from the genera Epinephelus and Scarus, both of which are currently threatened in the region.
Furthermore, the project generated substantial genomic data from reference fish in the western Indo-Pacific, including five complete mitogenomes—an outcome that was not initially anticipated—as well as 64 COI sequences. These genetic sequences are particularly valuable, as they come from a region where such genomic information is often scarce or unavailable. By making these datasets accessible through BOLD, PRISTINE will facilitate further research in fish taxonomy, biodiversity assessments, and conservation efforts. The open availability of this data will not only enhance scientific knowledge in the Indo-Pacific region but also support global initiatives in fisheries management and marine biodiversity conservation.
Regarding proteomics, the project successfully sequenced collagen from fish species whose amino acid sequences were previously unrecorded in protein databases. This achievement represents a significant advancement beyond the current state of the art, as it expands the molecular reference data available for fish species identification and contributes to the growing field proteomics and its application in ichthyoarchaeoogy. Additionally, using MALDI-TOF (ZooMS), we were able to identify closely related species in the archaeological record, such as those from the genera Epinephelus and Scarus, both of which are currently threatened in the region.
Furthermore, the project generated substantial genomic data from reference fish in the western Indo-Pacific, including five complete mitogenomes—an outcome that was not initially anticipated—as well as 64 COI sequences. These genetic sequences are particularly valuable, as they come from a region where such genomic information is often scarce or unavailable. By making these datasets accessible through BOLD, PRISTINE will facilitate further research in fish taxonomy, biodiversity assessments, and conservation efforts. The open availability of this data will not only enhance scientific knowledge in the Indo-Pacific region but also support global initiatives in fisheries management and marine biodiversity conservation.