Collagen fingerprinting for the species identification of archaeological amphibian remains

Abstract

Amphibians are well known as being one of the main groups of animals today most threatened by environmental changes but they are also some of the least well understood of all terrestrial vertebrates. This gap in knowledge is much greater as we look further back into the relatively recent past, despite representing an invaluable resources in archaeological and palaeontological assemblages that are more indicative of palaeoclimate conditions than most other vertebrate taxa. This in part stems from their remains being typically much less studied, partly due to the less common forms of expertise required for identifications based on skeletal morphology – the most frequently observed tissue that remains in ancient assemblages. Here we apply a method of biomolecular species identification by collagen peptide mass fingerprinting to the British Late Pleistocene assemblage of Pin Hole Cave (Creswell Crags, UK) as well as a range of relevant extant taxa for comparison. Our results demonstrate the ability to separate at the species level with all modern taxa investigated, allowing for the identification of these archaeological remains to the amphibian taxa known to exist in Late Pleistocene Britain. Analyses of the Pin Hole assemblage found a dominance of the two species previously known from the site (common frogs and toads: Rana temporaria and Bufo bufo, respectively) and also a small number of the rarer natterjack toad (Epidalea calamita) not previously identified in the Creswell Crags region but known from other sites in the UK; additionally, one specimen appeared to yield the fingerprint of the moor frog (R. arvalis), now extinct in the UK. As such, collagen fingerprinting is here shown to widen the known palaeobiodiversity of taxa, and highlights the further potential to enhance our understanding of climate change in the past.

A primer for ZooMS applications in archaeology

“…From its earliest days, ZooMS was occasionally applied to nonmammals, but it is only recently that the method has been developed in earnest for birds ( 8 ), fish ( 21 , 22 , 92 ), amphibians ( 93 ), and reptiles ( 12 , 20 , 93 ). Fish have been shown to have the most collagen sequence variants ( 94 , 95 ) due to the relaxed functional constraints of their lower body temperature and buoyancy, as well as the presence of a distinct third ɑ-chain (COL1ɑ3) in most fishes ( 21 , 96 ).…”

Species identification of Australian marsupials using collagen fingerprinting

“…Peptide marker COL1A1 508–519 (P1) is often characterized as highly conserved with a peak at m/z 1105 for most terrestrial mammals [ 65 ] and at m/z 1079 for cetaceans [ 45 ]. For marsupials, this marker is present at m/z 1162, and for T. aculeatus at m/z 1120.…”

Leveraging palaeoproteomics to address conservation and restoration agendas

“…Faunal assemblages from cave deposits are accordingly a useful resource for assessing and reconstructing past geographical species ranges ( Frick et al., 2020 ). ZooMS analyses of Late Pleistocene microfauna remains from Pin Hole Cave, England, revealed the geographical presence of several now extirpated species, including horseshoe bat ( Buckley and Herman, 2019 ) and moor frog ( Buckley and Cheylan, 2020 ). Palaeoproteomic identification of eggshell remains from El Miron Cave, Spain, revealed that it once lay within the geographical range of the bearded vulture ( Demarchi et al., 2019 ).…”

“…The majority of reference markers developed for ZooMS to date belongs to medium- to large-sized Eurasian mammals. Although recent years have seen the expansion of the reference library to include a larger variety of ecologically interesting taxa such as micromammals ( Buckley and Herman, 2019 ; Harvey et al., 2019a ; Buckley et al., 2020 ), fish ( Richter et al., 2011 , 2020 ; Harvey et al., 2018 ), amphibians ( Buckley and Cheylan, 2020 ), and reptiles ( Harvey et al., 2019b ), significant development of databases is still required. The lack of reference sequences at genomic or transcriptomic level for many species of interest further contributes to the paucity of available reference datasets ( Sakalauskaite et al., 2020 ).…”