Project description:Groupers (Epinephelidae) are ecologically, commercially, and culturally important predatory fishes throughout their global distribution range in tropical, subtropical and occasionally temperate regions. They are key species for modern and ancient fisheries in the Mediterranean which have been heavily overfished in the past century leading to smaller catch sizes, lower CPUE, and decreased biomass. There are four species of grouper native to the Mediterranean within the Epinephelus genus.The abundance and distribution of grouper species prior to the 20th century in the Mediterranean remains poorly known. Using peptide mass fingerprinting, also known as Zooarchaeology by Mass Spectrometry (ZooMS), we investigated if ZooMS is a viable method for identifying intra-genus grouper bones to species level. Due to the lack of publicly available genomic sequences and for validation of ZooMS markers, we reconstructed collagen type I amino acid sequences using LC-MS/MS for four Epinephelus spp. Adequate variation between collagen sequences enabled the production of the best supported phylogenetic tree for Mediterranean Epinephelus spp. to date. We identified 23 previously undescribed ZooMS biomarkers capable of distinguishing groupers to the species level. Our novel biomarkers were applied to a case study of 23 grouper/comber fish bones from the Middle to Late Holocene archaeological site of Kinet Höyük, located along the coast of Iskenderun Bay, Turkey. ZooMS markers enabled species level identification of 19 bones with 18 identified as Epinephelus aeneus and 1 identified as Epinephelus marginatus. Combining ZooMS identifications with catch size reconstructions has revealed that E. aeneus is capable of growing ca. 30 cm larger than previously reported. This abundance and dominance of E. aeneus locally at Kinet Höyük is consistent with E. aeneus being the most prevalent grouper species in Iskenderun Bay today, testifying to several millennia of this species local population persistence despite fishing pressure, habitat degradation, and climatic changes.