Ancient bone remains are widely utilized when investigating vertebrate biodiversity of past animal populations but are often so highly fragmented that the majority of specimens cannot be identified to any meaningful taxonomic level. Recently, high‐throughput methods for objective species identification using collagen peptide mass fingerprinting have been created to overcome this with the added indication that they could also offer a means of relative ageing through decay measurement. Here we explore both species identification and decay measurements for the Pin Hole Cave ‘microfaunal’ assemblage, the site that has been designated as a representative for Marine Oxygen Isotope Stage 3 in Britain in terms of its suite of mammalian fauna. We explore the technique's potential to corroborate the faunal diversity established previously by macroscopic studies and evaluate the decay measurements across the species boundary. The results support that the analysis of fragmentary remains by collagen fingerprinting can yield a more diverse set of fauna, and offer additional information relating to taphonomy, than the analysis of morphologically intact bones on their own. However, although useful for identifying likely contaminations of an assemblage, there was an unexpected decrease in the decay measurements observed for some megafauna compared with much younger microfauna, indicating that other factors need to be carefully monitored before it could be used as a relative ageing technique in Quaternary deposits.

Collagen is the dominant organic component of bone and is intimately locked within the hydroxyapatite structure of this ubiquitous biomaterial that dominates archaeological and palaeontological assemblages. Radiocarbon analysis of extracted collagen is one of the most common approaches to dating bone from late Pleistocene or Holocene deposits, but dating is relatively expensive compared to other biochemical techniques. Numerous analytical methods have previously been investigated for the purpose of screening out samples that are unlikely to yield reliable dates including histological analysis, UV-stimulated fluorescence and, most commonly, the measurement of percentage nitrogen (%N) and ratio of carbon to nitrogen (C:N). Here we propose the use of collagen fingerprinting (also known as Zooarchaeology by Mass Spectrometry, or ZooMS, when applied to species identification) as an alternative screening method for radiocarbon dating, due to its ability to provide information on collagen presence and quality, alongside species identification. The method was tested on a series of sub-fossil bone specimens from cave systems on Cayman Brac (Cayman Islands), chosen due to the observable range in diagenetic alteration, and in particular, the extent of mineralisation. Six 14C dates, of 18 initial attempts, were obtained from remains of extinct hutia, Capromys sp. (Rodentia; Capromyidae), recovered from five distinct caves on Cayman Brac, and ranging from 393 ± 25 to 1588 ± 26 radiocarbon years before present (yr BP). All of the bone samples that yielded radiocarbon dates generated excellent collagen fingerprints, and conversely those that gave poor fingerprints also failed dating. Additionally, two successfully fingerprinted bone samples were screened out from a set of 81. Both subsequently generated 14C dates, demonstrating successful utilisation of ZooMS as an alternative screening mechanism to identify bone samples that are suitable for 14C analysis.

Morphological identification of ancient bone is often problematic due to heavy fragmentation that generally influences zooarchaeological assemblages. Fish bones are more taphonomically sensitive than those of other vertebrates as they are typically smaller and less biomineralised. Thus, taxonomic identification based on the preservation of morphological features is often extremely limited and can reduce or eliminate the usefulness of an assemblage for inferring taxon information. Currently, one of the most time--and cost--efficient methods of achieving faunal identity from ancient bone is by the collagen fingerprinting technique known as ZooMS (Zooarchaeology by Mass Spectrometry). ZooMS harnesses the potential of preserved collagen, which is the most dominant and time-stable protein in bone. In this research, ZooMS is applied to ancient Baltic region fish assemblages that are between 500 and 6000 years old in order to define species identity and construct assemblage compositions. Alongside inferences into environmental and biological shifts from the Neolithic era to present day in the Baltic region, we demonstrate for the first time the ability to distinguish between recently diverged members of the Salmo (salmon) and Scophthalmus (turbot) genera. ZooMS analysis highlights 7% of the collagen-containing assemblage as having been morphologically identified incorrectly and has facilitated taxonomic refinement of a further 28% of samples, including some of the morphologically indeterminate bone fragments. This research emphasises the great potential of ZooMS in identifying ichthyoarchaeological bone remains to species--level, and provides a case for the use of collagen fingerprinting in contributing to baseline fisheries and ecological data to inform modern management.

Advancements in molecular science are continually improving our knowledge of marine turtle biology and evolution. However, there are still considerable gaps in our understanding, such as past marine turtle distributions, which can benefit from advanced zooarchaeological analyses. Here, we apply collagen fingerprinting to 130 archaeological marine turtle bone samples up to approximately 2500 years old from the Caribbean and Florida's Gulf Coast for faunal identification, finding the vast majority of samples (88%) to contain preserved collagen despite deposition in the tropics. All samples can be identified to species-level with the exception of the Kemp's ridley (Lepidochelys kempii) and olive ridley (L. olivacea) turtles, which can be separated to genus level, having diverged from one another only approximately 5 Ma. Additionally, we identify a single homologous peptide that allows the separation of archaeological green turtle samples, Chelonia spp., into two distinct groups, which potentially signifies a difference in genetic stock. The majority of the archaeological samples are identified as green turtle (Chelonia spp.; 63%), with hawksbill (Eretmochelys imbricata; 17%) and ridley turtles (Lepidochelys spp.; 3%) making up smaller proportions of the assemblage. There were no molecular identifications of the loggerhead turtle (Caretta caretta) in the assemblage despite 9% of the samples being morphologically identified as such, highlighting the difficulties in relying on morphological identifications alone in archaeological remains. Finally, we present the first marine turtle molecular phylogeny using collagen (I) amino acid sequences and find our analyses match recent phylogenies based on nuclear and mitochondrial DNA. Our results highlight the advantage of using collagen fingerprinting to supplement morphological analyses of turtle bones and support the usefulness of this technique for assessing their past distributions across the Caribbean and Florida's Gulf Coast, especially in these tropical environments where DNA preservation may be poor.

Billfish from the families Xiphiidae (swordfish) and Istiophoridae (marlins and sailfish) are large, often pelagic fishes that are highly migratory. Although some billfish have been the target of global commercial and sport fisheries for decades, prehistoric billfish foraging is relatively rare, but includes systematic swordfish (Xiphias gladius) and/or striped marlin (Kajikia audax) exploitation in the Santa Barbara Channel region of California, the Gulf of Maine, and the northern coast of Chile. While whole vertebrae, rostra, and other elements can often be identified to species, fragments of these, or other non-diagnostic elements such as fin ray spines, as well as modified bones, are difficult to determine to species-level beyond general identification as billfish or "large fish." We performed collagen fingerprinting on modern (n = 17) and archaeological (n = 30) billfish and large tuna (Scombridae) remains from museum collections and Chumash archaeological sites in California's Santa Barbara Channel region to test this method for determining the species of fragmentary remains. These data demonstrate that collagen fingerprinting can distinguish between the families Istiophoridae, Xiphiidae, and Scombridae, although distinguishing between species within Istiophoridae needs additional research. All but one of our archaeological specimens are from swordfish, with just one striped marlin, suggesting that the Chumash were likely encountering or targeting swordfish more frequently than other billfish species. Our study demonstrates that collagen fingerprinting is an important technique for documenting ancient billfish and other fisheries around the world.