High‐throughput collagen fingerprinting of intact microfaunal remains; a low‐cost method for distinguishing between murine rodent bones

Abstract

RationaleMicrofaunal skeletal remains can be sensitive indicators of the contemporary ecosystem in which they are sampled and are often recovered in owl pellets in large numbers. Species identification of these remains can be obtained using a range of morphological criteria established for particular skeletal elements, but typically dominated by a reliance on cranial characters. However, this can induce biases under different environmental and taphonomic conditions. The aim of this research was to develop a high‐throughput method of objectively identifying rodent remains from archaeological deposits using collagen fingerprinting, most notably the identification of rats from other myomorph rodents as a means to identify disturbances in the archaeofauna through the presence of invasive taxa not contemporary with the archaeological deposits.MethodsCollagen was extracted from complete microfaunal skeletal remains in such a manner as to leave the bones morphologically intact (i.e., weaker concentration of acid than previously used over shorter length of time). Acid‐soluble collagen was then ultrafiltered into ammonium bicarbonate and digested with trypsin prior to dilution in the MALDI matrix and acquisition of peptide mass fingerprints using a matrix‐assisted laser desorption/ionisation time‐of‐flight (MALDI‐TOF) mass spectrometer.ResultsCollagen fingerprinting was able to distinguish between Rattus, Mus, Apodemus and Micromys at the genus level; at the species level, R. rattus and R. norvegicus could be separated whereas A. flavicollis and A. sylvaticus could not. A total of 12,317 archaeological microvertebrate samples were screened for myomorph signatures but none were found to be invasive rats (Rattus) or mice (Mus). Of the contemporary murine fauna, no harvest mice (Micromys) were identified and only 24 field mouse (Apodemus) discovered.ConclusionsAs a result, no evidence of recent bioturbation could be inferred from the faunal remains of these archaeological deposits. More importantly this work presents a method for high‐throughput screening of specific taxa and is the first application of collagen fingerprinting to microfaunal remains of archaeological specimens. © 2016 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd.

Direct dating of Neanderthal remains from the site of Vindija Cave and implications for the Middle to Upper Paleolithic transition

“…We used matrixassisted laser desorption/ionization mass spectrometry to determine the relative mass-to-charge (m/z) values of the tryptic digest peptides in each bone sample. By comparing these values with a library of known fauna, we identified bones to genus or species in some instance (27)(28)(29)(30)(31). We screened a total of 383 unidentified bone samples from Vindija Cave (levels G1, G3, and stratigraphic unit G1-G3).…”

Species identification and decay assessment of Late Pleistocene fragmentary vertebrate remains from Pin Hole Cave (Creswell Crags, UK) using collagen fingerprinting

“…Although we have emphasised how promising this relatively new technique of species identification is, the taxonomic resolution remains the key limitation when compared with aDNA (although it is considerably greater than can be achieved using the other molecular and nonmolecular methods discussed above). Even though the method is typically capable of obtaining genus-level information, with elephantids being a notable previously known exception ), and to the species level with some (Rybczynski et al 2013;Buckley et al 2016), there were further limitations identified in this study in some lineages. In particular these were within the carnivores, particularly the feliformes of the order Carnivora, where distinction was only observed at the level of subfamily (e.g.…”

“…Figs S3-S7). The ancient material consisted of screening the 12 317 fragments from Jenkinson's 1980s excavations (reposited in Creswell Crags Heritage Centre) for which peptide fingerprints were obtained in Buckley et al (2016) following a similar but less damaging method (i.e. demineralization with 0.3 M HCl over 4 h).…”

“…demineralization with 0.3 M HCl over 4 h). Although described in detail in Buckley et al (2016), the acid-solubilized collagen was ultrafiltered using 30 kDa molecular weight cut-off ultrafilters into 50 mM ammonium bicarbonate, digested with trypsin overnight at 37°C and subsequently diluted in alpha hydroxycinnamic acid matrix and spotted for analysis using a MALDI-ToF mass spectrometer, also collecting 2000 laser acquisitions for each. One example from each taxonomic group identified is presented in the Supporting Information (Figs S8-S12).…”

“…From the 12 317 fingerprints from Buckley et al (2016) initially screened for murine rodent remains, 782 could be matched to the larger mammalian taxa described above (Table S2). As expected, the most abundant taxon identified was reindeer (Rangifer), making up almost half of the identified fragments ( Fig.…”