- Email: [email protected]
Species identification of voles and lemmings from Late Pleistocene deposits in Pin Hole Cave (Creswell Crags, UK) using collagen fingerprinting
Quaternary International xxx (2018) 1e7
Contents lists available at ScienceDirect
Quaternary International journal homepage: www.elsevier.com/locate/quaint
Species identification of voles and lemmings from Late Pleistocene deposits in Pin Hole Cave (Creswell Crags, UK) using collagen fingerprinting Michael Buckley a, *, Muxin Gu b, Jeremy Herman c, Juho-Antti Junno d, Christiane Denys e, Andrew T. Chamberlain a a
School of Earth and Environmental Sciences, University of Manchester, UK School of Biological Sciences, University of Manchester, UK National Museums Scotland, UK d Archaeology, University of Oulu, PO Box 8000, 90014, Oulun yliopisto, Finland e Institut de Systematique, Evolution, Biodiversit e (ISYEB), UMR7205, Museum National d’Histoire naturelle, France b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 June 2017 Received in revised form 28 February 2018 Accepted 16 March 2018 Available online xxx
Microfaunal remains are commonly used as palaeoenvironmental proxies and have been proposed as a means to identify relative ages of Late Quaternary deposits through biostratigraphy (i.e., utilising ‘Mammal Assemblage Zones’). However, assemblages of faunal remains can include a diverse range of taxa which are often difficult to distinguish using morphological aspects of the surviving bones, particularly when diagnostic features are eroded or missing due to fragmentation. Here we investigate the application of a recently developed method of species identification by collagen fingerprinting to arvicoline rodents (voles and lemmings), the dominant mammalian taxonomic group present at the Late Pleistocene site of Pin Hole Cave, Creswell Crags U.K., which is currently the designated British type locality for the Pin Hole Mammal Assemblage Zone (Oxygen Isotope Stage 3) fauna. We also further explore the potential for studying collagen decay rate via deamidations, and their applicability across the species boundary, in terms of its use for relative ageing of remains. Our results demonstrate the ease with which some taxa can be objectively distinguished to genus (e.g. Microtus from Myodes and Lemmus from Dicrostonyx) and in some cases species (e.g., M. gregalis from M. oeconomus), but that the potential for relative ageing is complicated by a range of taphonomic factors. The results highlight the potential for this new technique in much larger-scale palaeoenvironmental studies investigating temporal changes in vertebrate biodiversity. © 2018 Published by Elsevier Ltd.
Keywords: Collagen fingerprinting ZooMS Microtinae Voles Lemmings Cave bones Microfaunal identification
1. Introduction The remains of animal bones in the fossil record have widely been used to study past environmental conditions (Behrensmeyer et al., 1979; Klein, 1988; Bryan, 1973), particularly during the Pleistocene with its substantially changing climate. This includes some attempts at linking faumal assemblage compositions with particular time periods for specific regions as a form of palaeobiostratigraphy (Ubilla, 2004; Lister, 1992; Schreve, 2001). Such an approach is considered more robust on isolated islands, with Britain being a particularly interesting example due to its repeated
* Corresponding author. E-mail address: [email protected] (M. Buckley).
connections to the continental mainland that periodically allowed for the introduction of new taxa. However, although ancient megafauna can be somewhat informative, it is the sub-fossil microfaunal remains that are considered a more ideal proxy for the longer term environmental conditions, particular because of the nature in which these species quickly colonise a new environment (i.e., with faster population turnover and growth (Mihok et al., 1985)) and their sensitivity to changing environmental conditions (Demirel et al., 2011). It is also easier to interpret the modes of natural accumulation, with the changing accumulation of megafaunal remains at a site being further complicated by developing human technology during the Late Pleistocene (Bar-Yosef and Kuhn, 1999). The cricetid rodents, including lemmings and voles
https://doi.org/10.1016/j.quaint.2018.03.015 1040-6182/© 2018 Published by Elsevier Ltd.
Please cite this article in press as: Buckley, M., et al., Species identification of voles and lemmings from Late Pleistocene deposits in Pin Hole Cave (Creswell Crags, UK) using collagen fingerprinting, Quaternary International (2018), https://doi.org/10.1016/j.quaint.2018.03.015
2
M. Buckley et al. / Quaternary International xxx (2018) 1e7
(arvicolines), are of particular interest in dating archaeological strata through analysis of their molar teeth (particularly for voles; (Maul et al., 1998)); the vole clock attempts to take advantage of the morphological change in teeth through the same lineage, but has been met with caution (Martin, 2014). The family Cricetidae consists of several subfamilies, but the most populous group in the Northern Hemisphere is the Arvicolinae (Ranta and Kaitala, 1997). Members of this group are typically distinguished by the morphology of their molar teeth, with various arrangements of the prismatic cusps relating to their more or less herbivorous diets (Luzi et al., 2016; Haddadian Shad et al., 2014). Microfaunal remains such as those of voles and lemmings are most commonly preserved through owl pellet accumulations and therefore do not typically suffer from the same types of fragmentation seen with larger vertebrate remains. As such, the teeth are usually considered ideal and targeted alone, although even morphological identification of these can be problematic due to the extent of their variability (McGuire, 2011). However, it should be noted that many birds of prey, including barn owls, remove the heads from their rodent prey prior to consuming the remainder of the carcass (e.g., Glue, 1967) yet few studies attempt to identify post-cranial rodent remains (e.g. Romaniuk et al., 2016). Newer and more sophisticated analytical techniques have been developed over the last few decades, involving either morphometrics to characterise morphological variation (McGuire, 2011) or DNA-based methods (Martínkov a et al., 2013), but these typically suffer from limitations in either time and costs associated with these approaches. Additionally, the chronological extent of DNA survival in the case of the latter is also much more limited, where the small sample sizes available make destructive sampling methods less routinely carried out (with generally lower success rates, e.g., <50% in the case of Lagerholm et al. (2014)). To address some of these limitations, recently there has been the development of a promising alternative approach using collagen fingerprinting for species identification (Buckley et al., 2009), which is amenable to high-throughput analysis of thousands of bone specimens in a relatively non-destructive manner (Buckley et al., 2016). Here we investigate the application of this recently developed methodology of species identification by collagen fingerprinting (also known as Zooarchaeology by Mass Spectrometry or ’ZooMS’) to rodents of the cricetid subfamily Arvicolinae (predominantly voles and lemmings), the most abundant taxon present at the Late Pleistocene site of Pin Hole Cave, Creswell Crags, (Derbyshire, UK; 53.262130 N/001.202414 W). It is a cave formed in Magnesian limestone that measures approximately 31 m long but only 1e2 m wide, with one small chamber on the eastern side around 17 m in. Not only was it the first cave excavated in the Creswell Crags gorge but it has been excavated several times, the last of which carried out in the 1980s recovered nearly 30,000 microvertebrate remains. This cave site is particularly important because it is currently the designated type locality for fauna belonging to the British Mammal Assemblage Zone corresponding to Marine Isotope Stage 3 (c. ~6030 Ka; (Currant and Jacobi, 2001; Currant and Jacobi, 2011; Van Meerbeeck et al., 2009)) but which continues sporadically into the Holocene and contains three phases of human occupation during the Late Pleistocene (Armstrong, 1932). We also further explore the potential for studying collagen decay rate for relative ageing via the measurement of glutamine deamidation and its applicability across the species boundary in a larger range of rodents than tested before. 2. Materials and methods The archaeological samples were loaned from the Creswell Crags Museum and Heritage Centre and collagen fingerprints
obtained following Buckley et al. (2016) which left most skeletal remains morphologically intact. In brief, complete bone fragments were submerged in a 0.3 M hydrochloric acid solution for 3 h, ultrafiltered with 30 kDa molecular weight cut-off filters, exchanged into 50 mM ammonium bicarbonate and digested overnight with sequencing grade trypsin. 2 mL samples were spotted onto stainless steel MALDI target plates whilst being mixed with 10 mg/mL alpha cyano hydroxycinnamic acid matrix in 50% acetonitrile/0.1% trifluoroacetic acid (TFA), allowed to dry and then analysed by a Bruker Ultraflex II Matrix Assiste Laser Desorption Ionization Time of Flight mass spectrometer (with up to 2000 laser acquisitions). Raw peaks were identified by the R package MALDIquant (Gibb and Strimmer, 2012). Further noise modelling was performed by fitting a normal function to the heights of all peaks within 100 to þ100 m/z of a particular peak and computing the probability of this peak for being noise. Peaks that have probability of being noise >1.0 -106 were discarded. Remaining signal peaks were calibrated to a set of reference peaks by fitting a linear model to the m/z versus error. Calibrated peaks for all samples were combined into a data matrix for further analyses. Bone specimens from modern reference material of all arvicoline taxa known from previous morphological studies of the British contemporary faunal record (MIS3 onwards) were sampled and analysed following a similar protocol albeit with a tenfold dilution in 0.1% TFA prior to mixing. The species examined were common vole (Microtus arvalis), field vole (M. agrestis), tundra vole (M. oeconomus) and water vole (Arvicola amphibius) from the National Museums Scotland (UK), narrow-headed vole (M. gregalis) and collared (Arctic) lemming (Dicrostonyx torquatus) from the Mammals collections of the Museum national of Natural History, Paris (France), bank vole (Myodes glareolus) from the University of Sheffield (UK) and both steppe lemming (Lagurus lagurus) and Norway lemming (Lemmus lemmus) from the University of Oulu (Finland). To confirm the identity of the proposed peptide biomarkers, additional peptide sequencing analyses were carried out using an LC-Orbitrap Elite mass spectrometer following Buckley et al. (2015) on the best representative samples of each reference taxon. 3. Results and discussion The study was largely separated into three main components, the first being to identify the taxonomic level at which species identifications could be made, the second was to apply this to the large Pin Hole Cave assemblage of thousands of microfaunal samples for identification, and then thirdly to test the measures of decay between taxa of different environmental conditions (and assumed time periods). 3.1. Taxonomic resolution amongst reference material The collagen fingerprints from the modern reference material revealed differences between all nine of the taxa investigated in this study (Table 1), including some from the same sub-genus Microtus (e.g., M. agrestis and M. arvalis; Fig. 1). It was particularly interesting to note that some of the most abundant peptide markers were not in line with known systematics (e.g., m/z 1443.7 in M. arvalis and M. gregalis, the latter of which belongs to a distinct sub-genus (Stenocranius), with the homologous marker at m/z 1459.7 for the other arvicolines, but this is as would be expected as the amenability to ionization is more directly related to biochemical properties than to similarity in molecular structure). However, there were some markers that clearly identify some taxonomic groups; we have previously pointed out that the marker at m/z 2695.4 (homologous to m/z 2705.4 in many other mammalian groups) is representative of most rodents, which holds true for
Please cite this article in press as: Buckley, M., et al., Species identification of voles and lemmings from Late Pleistocene deposits in Pin Hole Cave (Creswell Crags, UK) using collagen fingerprinting, Quaternary International (2018), https://doi.org/10.1016/j.quaint.2018.03.015
M. Buckley et al. / Quaternary International xxx (2018) 1e7
3
Table 1 Peptide mass fingerprint markers from tryptic digestion of collagen from ten rodents labelled following position in collagen alpha chains (see (Buckley, 2016)); *homologous markers in all taxa not identified. Grey shading used to highlight homologous markers useful for taxonomic discrimination; aMarker previously published (Buckley et al., 2009, 2016); *incomplete set of markers that may not be homologous; *not confirmed by LC-Orbitrap analyses (see Supplementary Figures S2-7 for LC-MS/MS sequence spectra). Common name
Species
2t85
1t62a
2t26
2t60
*
Field vole
Microtus agrestis
1459.7
3417.5
Microtus oeconomus
2523.3
3297.5
Common vole
Microtus arvalis
Narrow-headed vole
Microtus gregalis
Bank vole
Myodes glareolus
Water vole
Arvicola amphibius
Steppe lemming
Lagurus lagurus
Norway lemming
Lemmus lemmus
1592.8/ 1608.8 1592.8/ 1608.8 1592.8/ 1608.8 1592.8/ 1608.8 1592.8/ 1608.8 1592.8/ 1608.8 1592.8/ 1608.8 1623.8
2523.3
Tundra vole
Collared lemming
Dicrostonyx torquatus Apodemus sp.
1594.8/ 1610.8 1592.8/ 1608.8
2523.3
Field mouse
1187.6/ 1203.6 1187.6/ 1203.6 1187.6/ 1203.6 1187.6/ 1203.6 1187.6/ 1203.6 1187.6/ 1203.6 1178.6/ 1194.6 1187.6/ 1203.6 1187.6/ 1203.6 1187.6/ 1203.6
1459.7 1443.7 1443.7 1459.7 1459.7 1459.7 1459.7 1459.7 1443.7
2537.3 2551.3 2551.3
3417.5
2551.3
3389.5
2551.3 2523.3
Fig. 1. MALDI ToF mass spectrometry peptide mass fingerprints from five arvicoline reference taxa: D. torquatus, M. gregalis, Lemmus, M. agrestis and M. arvalis; see Supplementary Figure S1 for reference spectra for the remaining four reference taxa.
these arvicolines (not shown in Table 1). One peptide marker that appears diagnostic of this group (particularly in combination with the latter) is at m/z 2129.1, found in all taxa here (not shown in Table 1; note that rabbits and hares also have the marker at m/z 2129.1, but with the m/z 2705.4 peak rather than m/z 2695.4). The marker at m/z 1592.8/1608.8 was found to represent the voles with the homologous marker at m/z 1594.8/1610.8 in the collared lemming and m/z 1623.8 in the Norway lemming (tribes Lemmini and Dicrostonychini respectively) whereby a substitution in the sequence belonging to the latter of one of the proline residues amenable to biological oxidation appears to affect the clear observation of the two oxidation forms seen in the others (Supplementary Figure S2).
The ability to distinguish between some of these taxa further advances the taxonomic resolution achievable in relation to suspected divergence times. Biochemical evidence supports morphological studies in an estimate for the origins of the Arvicolidae family at ~5 Ma (Chaline and Graf, 1988), with some earlier estimates even as far back as 8e9 Ma (Michaux et al., 2001) and subsequent diversification ~3e5 Ma (Conroy and Cook, 1999). Regardless of the estimates of this basal split between the voles and the lemmings, the ability to distinguish within Microtus is particularly interesting; Microtus is thought to have arisen between 0.5 and 2 Ma (Repenning, 1980; Chaline et al., 1999), but genome analysis indicates much higher rates of molecular evolution than observed in other mammals (Triant and DeWoody, 2006).
Please cite this article in press as: Buckley, M., et al., Species identification of voles and lemmings from Late Pleistocene deposits in Pin Hole Cave (Creswell Crags, UK) using collagen fingerprinting, Quaternary International (2018), https://doi.org/10.1016/j.quaint.2018.03.015
4
M. Buckley et al. / Quaternary International xxx (2018) 1e7
Collagen fingerprinting therefore provides a new tool for analysis of micromammal assemblages, allowing accurate identification of individual teeth amongst other skeletal remains from all arvicoline rodent genera, and most individual species, present in the Late Pleistocene and Holocene of the British Isles (Sutcliffe and Kowalski, 1976). Isolated teeth of arvicoline rodents constitute a large proportion of the potentially identifiable remains in microfaunal assemblages like these, but often cannot be specifically identified from their morphology alone (Sutcliffe and Kowalski, 1976). This is exemplified through examples such as at Beckford (Worcestershire, UK), where the presence of reported M. arvalis is incongruous with the otherwise Arctic tundra faunal assemblage but potentially derived from mis-identified M. gregalis isolated teeth. Similar mis-identifications have been known to occur between M. arvalis and M. agrestis (Sutcliffe and Kowalski, 1976). However, the importance of collagen fingerprinting as a palaeoecological tool stems from its ability to achieve this at low unit cost, which is currently a major drawback for the alternative DNAbased methods. Collagen fingerprinting is likely to be of similar use in many other sites from northern and western Europe, where assemblages are composed of the same, or similar suites of species. 3.2. Pin Hole Cave arvicoline identifications From an original >13,022 spectral fingerprints from the Pin Hole Cave assemblage (including specimens from the spoil and elsewhere in the cave than in our previous report, Buckley et al., 2016), we filtered out poor quality spectra resulting in 6,192 that we considered of good enough quality for likely species identification and contained the marker m/z 1105.6 diagnostic of most mammals (excludes cetaceans; Buckley et al., 2014). Of these, over at least 4,262 appeared to contain the rodent marker at m/z 2695 (Buckley et al., 2016) although 42 appear to derive from Apodemus (Buckley et al., 2016). Of the remainder, 2773 could be categorised as lemming (546 Lemmus; 2,227 Dicrostonyx) and 1,489 as vole (Fig. 2). More specifically, the majority (1,218) of the voles appear to derive from the narrow-headed vole (with the marker at approximately m/z 2552.3/2553.3; the increase from the marker at m/z 2551.3 in modern reference taxa is consistent with deamidation at one or two residues; Fig. 3). At least 78 could be identified as from common vole (the observed marker at m/z 2539.3 similarly increased above that of the modern reference) and 118 could be identified as
either M. agrestis or M. oeconomus; the majority appeared to contain the marker for the field vole, but caution should be noted in separating these two using the presented markers as their sequences could not be confirmed within this study; similarly, it is likely that of the 75 specimens of other voles (e.g., water or bank voles) the majority (at least 30) of those that retained the higher mass markers appeared reflective of bank vole (~30), with only a few (four) with markers for water vole, the much reduced number being consistent with the selection criteria in which only bone fragments small enough to fit into the microtitre plate well (~8 mm) were typically analysed. Contrarily to the ease at which some of the Microtus taxa could be separated, the separation of some taxa, e.g., M. agrestis from M. oeconomus or between Myodes and Arvicola was surprisingly more difficult to achieve given the absence of several potential markers observed in the modern reference material (not all listed) but these particular taxa only represented a small component of those present in this assemblage (i.e., both groups representing <5% of the assemblage). By far the dominant taxon in the assemblage appears to be the collared lemming (Dicrostonyx) with more than four times as many specimens identified as the next most abundant rodent, the Norway lemming. The presence of the lemmings D. torquatus and L. lemmus, the bank vole My. glareolus, the water vole A. amphibius, the tundra vole M. oeconomus and the narrow-headed vole M. gregalis are expected in the Late Pleistocene fauna of Pin Hole Cave; Dicrostonyx, Lemmus, and M. gregalis are known to characterise the Late Glacial period in Britain, with the other taxa present before and after the Late Glacial maximum although both lemmings have also been placed within the Windermere (Bølling-Allerød) interstadial in the later part of the Devensian glaciation (Yalden, 2010), around 13-11 Ka. Both lemmings at present have a circumpolar distribution, but it is the collared lemming that is known for being more adapted for colder environments, with fur that turns white in winter and complex winter claws (Yalden, 2010). The abundance of the narrow-headed vole, present today in the tundra region of northern Europe and Asia, is also of particular interest as it is thought to prefer wetter areas than the other Microtus species (De Jonge and Dienske, 1978) and Dicrostonyx (Yalden, 2010), but it is often found associated with the latter in fossil communities (Cordy, 1991). The occurrence of a small number of M. agrestis and Apodemus specimens identified is presumably indicative of the Windermere interstadial in this assemblage; fossil evidence indicates that their post-Weichselian
Fig. 2. Faunal composition of the vole and lemming remains from Pin Hole Cave, Creswell Crags, UK.
Please cite this article in press as: Buckley, M., et al., Species identification of voles and lemmings from Late Pleistocene deposits in Pin Hole Cave (Creswell Crags, UK) using collagen fingerprinting, Quaternary International (2018), https://doi.org/10.1016/j.quaint.2018.03.015
M. Buckley et al. / Quaternary International xxx (2018) 1e7
5
Fig. 3. Two sections of example MALDI ToF mass spectrometry peptide mass fingerprints from six different arvicoline taxa from the Pin Hole assemblage.
arrival in Britain was during the Bølling-Allerød interstadial in the case of the field vole (Herman and Searle, 2011) and perhaps as late as the Holocene in the case of the wood mouse (Herman et al., 2017), although it was reported from Cat Hole, Glamorgan in the Bølling period along with Dicrostonyx, M. agrestis and My. glareolus and in the Allerød period with Lemmus, M. oeconomus and M. agrestis (Campbell, 1977). Overall, environmental inferences drawn from this study of microfauna is consistent with that observed from our previous analysis of the megafaunal remains (Buckley et al., 2017) in which we observed a dominance of fauna adapted for the severe conditions of the Late Glacial, with a dominance of reindeer, and substantial proportions of woolly rhinoceros and woolly mammoth. However, these microfaunal remains are likely to be more representative of the closer environment to the cave assemblage. 3.3. Glutamine deamidation across arvicoline taxa Given that the range of arvicoline rodents present at Pin Hole represent a range in climatic conditions over approximately 40,000 years, if relative decay measurements of collagen through peptide mass fingerprinting were indicative of relative age (Doorn et al., 2012) we would expect to see elevated decay in the colderadapted taxa from earlier in the Late Pleistocene (e.g., D. torquatus, M. oeconomus, M. gregalis) compared to the more warm-adapted taxa from the early Holocene (e.g., M. agrestis or My. glareolus). To explore this we measured the deamidation of the m/z 1105.6 peptide in a sample of rodents of different geological ages (albeit only based on known information about the taxa in this region). Although there were substantial differences between the levels of deamidation observed in the field mouse (sylvaticusA. ), a species that was restricted to the Holocene in this assemblage, and those of the Late Pleistocene voles and lemmings (Fig. 4), the direction of this difference contradicted what we would anticipate for their relative geological ages. This further highlights the potential unreliability of these decay markers as a means of relative ageing across the species boundary, when differences in accumulation and pre-taphonomic histories
are likely. Previously we postulated that the higher levels of decay observed in the field mouse compared with older Late Pleistocene carnivores, such as the arctic fox and hyaena, were the result of exposure to acidic conditions while passing through the digestive tract of a predator (most likely that of an owl; Buckley et al., 2017). However, this explanation is shown less plausible here given that the difference is between rodent taxa and noting that the collared lemming was expected to have a larger decay range than the other taxa, not only due to the greater number of specimens but also to the larger known temporal range (even if present throughout the range of the other taxa within the chronological period of this assemblage). Therefore, although the measurements of deamidation provide a means to rapidly identify more recent intrusions into an archaeological or palaeontological assemblage (Buckley et al., 2017), and may still prove valuable for the relative ageing of megafaunal remains of more comparable taphonomic histories, whether or not it could provide useful relative ageing information will depend on better understanding the accumulation and depositional processes affecting the rodent taxa being studied. One particular advantage with the study of microfaunal remains is that although there appears to be heterogeneity in the deamidation values across a single specimen (Simpson et al., 2016), as well as across archaeological sites (Doorn et al., 2012), here the evaluations are encompassing the entirety of the specimens giving a more appropriate average value. 4. Conclusions We report the further evaluation of a relatively new and underutilised methodology in collagen fingerprinting for species identification of ancient microfaunal remains. Specifically with the assemblage from Pin Hole Cave, we were able to go much further than studying the presence or absence of particular microfauna and look at the overall relative abundances of many of the different lemming and vole taxa. Here we found that the lemmings formed nearly two thirds of the material, dominated by the collared lemming, with the narrow-headed vole as dominant amongst the voles. Although we do show that they can yield species-level
Please cite this article in press as: Buckley, M., et al., Species identification of voles and lemmings from Late Pleistocene deposits in Pin Hole Cave (Creswell Crags, UK) using collagen fingerprinting, Quaternary International (2018), https://doi.org/10.1016/j.quaint.2018.03.015
6
M. Buckley et al. / Quaternary International xxx (2018) 1e7
Fig. 4. Box plots of decay measurements, as the ratio of the peak height of m/z 1106.6/1105.6, from Apodemus, M. arvalis, M. gregalis, Lemmus and Dicrostonyx.
information within Microtus, we further confirmed some of the cross-species issues with the use of decay measurements via deamidation as a tool for relative age estimation within an assemblage. Together these reveal two forms of latent data within geoarchaeological archives that contain faunal remains. The capabilities of the method has far-reaching implications for the reliable identification of material from similar sites throughout Europe, many of which will include morphologically undiagnostic teeth and other skeletal elements. Funding This work was supported by the Royal Society [UF120473]. for fellowship funding and the original studies as part of a fellowship funded by the Natural Environment Research Council [NE/H015132/ 1]. Acknowledgements We would like to thank the National Museums Scotland (Edinburgh, UK), University of Oulu (Oulu, Finland), Mammals collections of the Museum National d’Histoire naturelle (Paris, France), the University of Sheffield's Department of Archaeology (Sheffield, UK) and the Creswell Crags Heritage Centre (Derbyshire, UK) for access to samples. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.quaint.2018.03.015. References Armstrong, A.L., 1932. Excavations in the Pin Hole cave, Creswell Crags, Derbyshire. Proc. Prehist. Soc. East Anglia 6, 330e334. Bar-Yosef, O., Kuhn, S.L., 1999. The big deal about blades: laminar technologies and human evolution. Am. Anthropol. 101, 322e338. Behrensmeyer, A.K., Western, D., Boaz, D.E.D., 1979. New perspectives in vertebrate paleoecology from a recent bone assemblage. Paleobiology 5, 12e21. Bryan, A.L., 1973. Paleoenvironments and cultural diversity in late Pleistocene South America. Quat. Res. 3, 237e256. Buckley, M., 2016. Species identification of bovine, ovine and porcine type 1 collagen; comparing peptide mass fingerprinting and LC-based proteomics methods. Int. J. Mol. Sci. 17, 445. Buckley, M., Collins, M., Thomas-Oates, J., Wilson, J.C., 2009. Species identification by analysis of bone collagen using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 23, 3843e3854.
lsdo ttir, A., 2014. Species Buckley, M., Fraser, S., Herman, J., Melton, N., Mulville, J., Pa identification of archaeological marine mammals using collagen fingerprinting. J. Archaeol. Sci. 41, 631e641. ~ a, R.A., Lawless, C., Tambusso, P.S., Varela, L., Carlini, A.A., Buckley, M., Farin Powell, J.E., Martinez, J.G., 2015. Collagen sequence analysis of the extinct giant ground sloths Lestodon and Megatherium. PLoS One 10 e0144793. Buckley, M., Gu, M., Shameer, S., Patel, S., Chamberlain, A.T., 2016. High-throughput collagen fingerprinting of intact microfaunal remains; a low-cost method for distinguishing between murine rodent bones. Rapid Commun. Mass Spectrom. 30, 805e812. Buckley, M., Harvey, V.L., Chamberlain, A.T., 2017. Species identification and decay assessment of Late Pleistocene fragmentary vertebrate remains from Pin Hole Cave (Creswell Crags, UK) using collagen fingerprinting. Boreas 46, 402e411. Campbell, J.B., 1977. The Upper Palaeolithic of Britain: a Study of Man and Nature in the Late Ice Age. Oxford University Press. Chaline, J., Brunet-Lecomte, P., Montuire, S., Viriot, L., Courant, F., 1999. Anatomy of the arvicoline radiation (Rodentia): palaeogeographical, palaeoecological history and evolutionary data. Ann. Zool. Fenn. 36, 239e267. Chaline, J., Graf, J.-D., 1988. Phylogeny of the Arvicolidae (Rodentia): biochemical and paleontological evidence. J. Mammal. 69, 22e33. Conroy, C.J., Cook, J.A., 1999. MtDNA evidence for repeated pulses of speciation within arvicoline and murid rodents. J. Mammalian Evol. 6, 221e245. Cordy, J.-M., 1991. Palaeoecology of the late glacial and early postglacial of Belgium and neighbouring areas. In: Barton, N., Roberts, A.J., Roe, D.A. (Eds.), CBA Research Report. Council for British Archaeology, vol. 77, pp. 40e47. Currant, A., Jacobi, R., 2001. A formal mammalian biostratigraphy for the Late Pleistocene of Britain. Quat. Sci. Rev. 20, 1707e1716. Currant, A.P., Jacobi, R., 2011. The Mammal Faunas of the British Late Pleistocene. In: Ashton, N., Lewi, S.G., Stringer, C. (Eds.), In, The Ancient Human Occupation of Britain, Developments in Quaternary Sciences, vol. 14, pp. 165e180. De Jonge, G., Dienske, H., 1978. Habitat and interspecific displacement of small mammals in The Netherlands. Neth. J. Zoology 29, 177e214. Demirel, A., Andrews, P., Yalçınkaya, I., Ersoy, A., 2011. The taphonomy and palaeoenvironmental implications of the small mammals from Karain Cave, Turkey. J. Archaeol. Sci. 38, 3048e3059. Doorn, N.L., Wilson, J., Hollund, H., Soressi, M., Collins, M.J., 2012. Site-specific deamidation of glutamine: a new marker of bone collagen deterioration. Rapid Commun. Mass Spectrom. 26, 2319e2327. Gibb, S., Strimmer, K., 2012. MALDIquant: a versatile R package for the analysis of mass spectrometry data. Bioinformatics 28, 2270e2271. Glue, D.E., 1967. Prey taken by the Barn Owl in England and Wales. Bird Study 14, 169e183. Haddadian Shad, H., Darvish, J., Mohammadian, T., Mahmoudi, A., Alaie Kakhki, N., Ghanbarifardi, M., Molavi, F., Barani-Beiranvand, H., 2014. Preliminary study of rodents using pellets of predatory birds in Iran. Iran. J. Animal Biosyst. 10. hannesdo ttir, F., Jones, E.P., McDevitt, A.D., Michaux, J.R., White, T.A., Herman, J.S., Jo jcik, J.M., Searle, J.B., 2017. Post-glacial colonization of Europe by the wood Wo mouse, Apodemus sylvaticus: evidence of a northern refugium and dispersal with humans. Biol. J. Linn. Soc. 120, 313e332. Herman, J.S., Searle, J.B., 2011. Post-glacial partitioning of mitochondrial genetic variation in the field vole. Proc. R. Soc. B 278, 3601e3607. Klein, R.G., 1988. The archaeological significance of animal bones from Acheulean sites in southern Africa. Afr. Archaeol. Rev. 6, 3e25. Lagerholm, V.K., Sandoval-Castellanos, E., Ehrich, D., Abramson, N.I., , M., Angerbjo €rn, A., Stewart, J.R., Nadachowski, A., Kalthoff, D.C., Germonpre n, L., 2014. On the origin of the Norwegian lemming. Mol. Ecol. 23, Dale 2060e2071. Lister, A., 1992. Mammalian fossils and Quaternary biostratigraphy. Quat. Sci. Rev. 11,
Please cite this article in press as: Buckley, M., et al., Species identification of voles and lemmings from Late Pleistocene deposits in Pin Hole Cave (Creswell Crags, UK) using collagen fingerprinting, Quaternary International (2018), https://doi.org/10.1016/j.quaint.2018.03.015
M. Buckley et al. / Quaternary International xxx (2018) 1e7 329e344. pez-García, J.M., Blasco, R., Rivals, F., Rosell, J., 2016. Variations in Microtus Luzi, E., Lo arvalis and Microtus agrestis (Arvicolinae, rodentia) dental Morphologies in an archaeological context: the case of teixoneres cave (late Pleistocene, northeastern Iberia). J. Mammalian Evol. 24, 1e9. Martin, R.A., 2014. A critique of vole clocks. Quat. Sci. Rev. 94, 1e6. , N., Barnett, R., Cucchi, T., Struchen, R., Pascal, M., Pascal, M., Martínkova Fischer, M.C., Higham, T., Brace, S., Ho, S.Y., 2013. Divergent evolutionary processes associated with colonization of offshore islands. Mol. Ecol. 22, 5205e5220. Maul, L., Masini, F., Abbazzi, L., Turner, A., 1998. Geochronometric application of evolutionary trends in the dentition of fossil Arvicolidae. Meded. Ned. Inst. Toegepaste Geowetenschappen TNO 60, 565e572. McGuire, J.L., 2011. Identifying California Microtus species using geometric morphometrics documents Quaternary geographic range contractions. J. Mammal. 92, 1383e1394. Michaux, J., Reyes, A., Catzeflis, F., 2001. Evolutionary history of the most speciose mammals: molecular phylogeny of muroid rodents. Mol. Biol. Evol. 18, 2017e2031. Mihok, S., Turner, B.N., Iverson, S.L., 1985. The characterization of vole population dynamics. Ecol. Monogr. 55, 399e420. Ranta, E., Kaitala, V., 1997. Travelling waves in vole population dynamics. Nature 390, 456.
7
Repenning, C.A., 1980. Faunal exchanges between Siberia and North America. Can. J. Anthropol. 1, 37e44. Romaniuk, A.A., Shepherd, A.N., Clarke, D.V., Sheridan, A.J., Fraser, S., Bartosiewicz, L., Herman, J.S., 2016. Rodents: food or pests in neolithic Orkney. R. Soc. Open Sci. 3, 160514. Schreve, D.C., 2001. Differentiation of the British late Middle Pleistocene interglacials: the evidence from mammalian biostratigraphy. Quat. Sci. Rev. 20, 1693e1705. Simpson, J.P., Penkman, K.E.H., Demarchi, B., Koon, H., Collins, M.J., Thomas-Oates, J., Shapiro, B., Stark, M., Wilson, J., 2016. The effects of demineralisation and sampling point variability on the measurement of glutamine deamidation in type I collagen extracted from bone. J. Archaeol. Sci. 69, 29e38. Sutcliffe, A.J., Kowalski, K., 1976. Pleistocene rodents of the British Isles. Br. Mus. Nat. Hist. A 27, 33e147. Triant, D.A., DeWoody, J.A., 2006. Accelerated molecular evolution in Microtus (Rodentia) as assessed via complete mitochondrial genome sequences. Genetica 128, 95e108. Ubilla, M., 2004. Mammalian biostratigraphy of Pleistocene fluvial deposits in Northern Uruguay, South America. Proc. Geologists' Assoc. 115, 347e357. Van Meerbeeck, C., Renssen, H., Roche, D., 2009. How did marine Isotope stage 3 and last glacial maximum climates differ?eperspectives from equilibrium simulations. Clim. Past 5, 33e51. Yalden, D., 2010. The History of British Mammals. A&C Black, London.
Please cite this article in press as: Buckley, M., et al., Species identification of voles and lemmings from Late Pleistocene deposits in Pin Hole Cave (Creswell Crags, UK) using collagen fingerprinting, Quaternary International (2018), https://doi.org/10.1016/j.quaint.2018.03.015
Contents lists available at ScienceDirect
Quaternary International journal homepage: www.elsevier.com/locate/quaint
Species identification of voles and lemmings from Late Pleistocene deposits in Pin Hole Cave (Creswell Crags, UK) using collagen fingerprinting Michael Buckley a, *, Muxin Gu b, Jeremy Herman c, Juho-Antti Junno d, Christiane Denys e, Andrew T. Chamberlain a a
School of Earth and Environmental Sciences, University of Manchester, UK School of Biological Sciences, University of Manchester, UK National Museums Scotland, UK d Archaeology, University of Oulu, PO Box 8000, 90014, Oulun yliopisto, Finland e Institut de Systematique, Evolution, Biodiversit e (ISYEB), UMR7205, Museum National d’Histoire naturelle, France b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 June 2017 Received in revised form 28 February 2018 Accepted 16 March 2018 Available online xxx
Microfaunal remains are commonly used as palaeoenvironmental proxies and have been proposed as a means to identify relative ages of Late Quaternary deposits through biostratigraphy (i.e., utilising ‘Mammal Assemblage Zones’). However, assemblages of faunal remains can include a diverse range of taxa which are often difficult to distinguish using morphological aspects of the surviving bones, particularly when diagnostic features are eroded or missing due to fragmentation. Here we investigate the application of a recently developed method of species identification by collagen fingerprinting to arvicoline rodents (voles and lemmings), the dominant mammalian taxonomic group present at the Late Pleistocene site of Pin Hole Cave, Creswell Crags U.K., which is currently the designated British type locality for the Pin Hole Mammal Assemblage Zone (Oxygen Isotope Stage 3) fauna. We also further explore the potential for studying collagen decay rate via deamidations, and their applicability across the species boundary, in terms of its use for relative ageing of remains. Our results demonstrate the ease with which some taxa can be objectively distinguished to genus (e.g. Microtus from Myodes and Lemmus from Dicrostonyx) and in some cases species (e.g., M. gregalis from M. oeconomus), but that the potential for relative ageing is complicated by a range of taphonomic factors. The results highlight the potential for this new technique in much larger-scale palaeoenvironmental studies investigating temporal changes in vertebrate biodiversity. © 2018 Published by Elsevier Ltd.
Keywords: Collagen fingerprinting ZooMS Microtinae Voles Lemmings Cave bones Microfaunal identification
1. Introduction The remains of animal bones in the fossil record have widely been used to study past environmental conditions (Behrensmeyer et al., 1979; Klein, 1988; Bryan, 1973), particularly during the Pleistocene with its substantially changing climate. This includes some attempts at linking faumal assemblage compositions with particular time periods for specific regions as a form of palaeobiostratigraphy (Ubilla, 2004; Lister, 1992; Schreve, 2001). Such an approach is considered more robust on isolated islands, with Britain being a particularly interesting example due to its repeated
* Corresponding author. E-mail address: [email protected] (M. Buckley).
connections to the continental mainland that periodically allowed for the introduction of new taxa. However, although ancient megafauna can be somewhat informative, it is the sub-fossil microfaunal remains that are considered a more ideal proxy for the longer term environmental conditions, particular because of the nature in which these species quickly colonise a new environment (i.e., with faster population turnover and growth (Mihok et al., 1985)) and their sensitivity to changing environmental conditions (Demirel et al., 2011). It is also easier to interpret the modes of natural accumulation, with the changing accumulation of megafaunal remains at a site being further complicated by developing human technology during the Late Pleistocene (Bar-Yosef and Kuhn, 1999). The cricetid rodents, including lemmings and voles
https://doi.org/10.1016/j.quaint.2018.03.015 1040-6182/© 2018 Published by Elsevier Ltd.
Please cite this article in press as: Buckley, M., et al., Species identification of voles and lemmings from Late Pleistocene deposits in Pin Hole Cave (Creswell Crags, UK) using collagen fingerprinting, Quaternary International (2018), https://doi.org/10.1016/j.quaint.2018.03.015
2
M. Buckley et al. / Quaternary International xxx (2018) 1e7
(arvicolines), are of particular interest in dating archaeological strata through analysis of their molar teeth (particularly for voles; (Maul et al., 1998)); the vole clock attempts to take advantage of the morphological change in teeth through the same lineage, but has been met with caution (Martin, 2014). The family Cricetidae consists of several subfamilies, but the most populous group in the Northern Hemisphere is the Arvicolinae (Ranta and Kaitala, 1997). Members of this group are typically distinguished by the morphology of their molar teeth, with various arrangements of the prismatic cusps relating to their more or less herbivorous diets (Luzi et al., 2016; Haddadian Shad et al., 2014). Microfaunal remains such as those of voles and lemmings are most commonly preserved through owl pellet accumulations and therefore do not typically suffer from the same types of fragmentation seen with larger vertebrate remains. As such, the teeth are usually considered ideal and targeted alone, although even morphological identification of these can be problematic due to the extent of their variability (McGuire, 2011). However, it should be noted that many birds of prey, including barn owls, remove the heads from their rodent prey prior to consuming the remainder of the carcass (e.g., Glue, 1967) yet few studies attempt to identify post-cranial rodent remains (e.g. Romaniuk et al., 2016). Newer and more sophisticated analytical techniques have been developed over the last few decades, involving either morphometrics to characterise morphological variation (McGuire, 2011) or DNA-based methods (Martínkov a et al., 2013), but these typically suffer from limitations in either time and costs associated with these approaches. Additionally, the chronological extent of DNA survival in the case of the latter is also much more limited, where the small sample sizes available make destructive sampling methods less routinely carried out (with generally lower success rates, e.g., <50% in the case of Lagerholm et al. (2014)). To address some of these limitations, recently there has been the development of a promising alternative approach using collagen fingerprinting for species identification (Buckley et al., 2009), which is amenable to high-throughput analysis of thousands of bone specimens in a relatively non-destructive manner (Buckley et al., 2016). Here we investigate the application of this recently developed methodology of species identification by collagen fingerprinting (also known as Zooarchaeology by Mass Spectrometry or ’ZooMS’) to rodents of the cricetid subfamily Arvicolinae (predominantly voles and lemmings), the most abundant taxon present at the Late Pleistocene site of Pin Hole Cave, Creswell Crags, (Derbyshire, UK; 53.262130 N/001.202414 W). It is a cave formed in Magnesian limestone that measures approximately 31 m long but only 1e2 m wide, with one small chamber on the eastern side around 17 m in. Not only was it the first cave excavated in the Creswell Crags gorge but it has been excavated several times, the last of which carried out in the 1980s recovered nearly 30,000 microvertebrate remains. This cave site is particularly important because it is currently the designated type locality for fauna belonging to the British Mammal Assemblage Zone corresponding to Marine Isotope Stage 3 (c. ~6030 Ka; (Currant and Jacobi, 2001; Currant and Jacobi, 2011; Van Meerbeeck et al., 2009)) but which continues sporadically into the Holocene and contains three phases of human occupation during the Late Pleistocene (Armstrong, 1932). We also further explore the potential for studying collagen decay rate for relative ageing via the measurement of glutamine deamidation and its applicability across the species boundary in a larger range of rodents than tested before. 2. Materials and methods The archaeological samples were loaned from the Creswell Crags Museum and Heritage Centre and collagen fingerprints
obtained following Buckley et al. (2016) which left most skeletal remains morphologically intact. In brief, complete bone fragments were submerged in a 0.3 M hydrochloric acid solution for 3 h, ultrafiltered with 30 kDa molecular weight cut-off filters, exchanged into 50 mM ammonium bicarbonate and digested overnight with sequencing grade trypsin. 2 mL samples were spotted onto stainless steel MALDI target plates whilst being mixed with 10 mg/mL alpha cyano hydroxycinnamic acid matrix in 50% acetonitrile/0.1% trifluoroacetic acid (TFA), allowed to dry and then analysed by a Bruker Ultraflex II Matrix Assiste Laser Desorption Ionization Time of Flight mass spectrometer (with up to 2000 laser acquisitions). Raw peaks were identified by the R package MALDIquant (Gibb and Strimmer, 2012). Further noise modelling was performed by fitting a normal function to the heights of all peaks within 100 to þ100 m/z of a particular peak and computing the probability of this peak for being noise. Peaks that have probability of being noise >1.0 -106 were discarded. Remaining signal peaks were calibrated to a set of reference peaks by fitting a linear model to the m/z versus error. Calibrated peaks for all samples were combined into a data matrix for further analyses. Bone specimens from modern reference material of all arvicoline taxa known from previous morphological studies of the British contemporary faunal record (MIS3 onwards) were sampled and analysed following a similar protocol albeit with a tenfold dilution in 0.1% TFA prior to mixing. The species examined were common vole (Microtus arvalis), field vole (M. agrestis), tundra vole (M. oeconomus) and water vole (Arvicola amphibius) from the National Museums Scotland (UK), narrow-headed vole (M. gregalis) and collared (Arctic) lemming (Dicrostonyx torquatus) from the Mammals collections of the Museum national of Natural History, Paris (France), bank vole (Myodes glareolus) from the University of Sheffield (UK) and both steppe lemming (Lagurus lagurus) and Norway lemming (Lemmus lemmus) from the University of Oulu (Finland). To confirm the identity of the proposed peptide biomarkers, additional peptide sequencing analyses were carried out using an LC-Orbitrap Elite mass spectrometer following Buckley et al. (2015) on the best representative samples of each reference taxon. 3. Results and discussion The study was largely separated into three main components, the first being to identify the taxonomic level at which species identifications could be made, the second was to apply this to the large Pin Hole Cave assemblage of thousands of microfaunal samples for identification, and then thirdly to test the measures of decay between taxa of different environmental conditions (and assumed time periods). 3.1. Taxonomic resolution amongst reference material The collagen fingerprints from the modern reference material revealed differences between all nine of the taxa investigated in this study (Table 1), including some from the same sub-genus Microtus (e.g., M. agrestis and M. arvalis; Fig. 1). It was particularly interesting to note that some of the most abundant peptide markers were not in line with known systematics (e.g., m/z 1443.7 in M. arvalis and M. gregalis, the latter of which belongs to a distinct sub-genus (Stenocranius), with the homologous marker at m/z 1459.7 for the other arvicolines, but this is as would be expected as the amenability to ionization is more directly related to biochemical properties than to similarity in molecular structure). However, there were some markers that clearly identify some taxonomic groups; we have previously pointed out that the marker at m/z 2695.4 (homologous to m/z 2705.4 in many other mammalian groups) is representative of most rodents, which holds true for
Please cite this article in press as: Buckley, M., et al., Species identification of voles and lemmings from Late Pleistocene deposits in Pin Hole Cave (Creswell Crags, UK) using collagen fingerprinting, Quaternary International (2018), https://doi.org/10.1016/j.quaint.2018.03.015
M. Buckley et al. / Quaternary International xxx (2018) 1e7
3
Table 1 Peptide mass fingerprint markers from tryptic digestion of collagen from ten rodents labelled following position in collagen alpha chains (see (Buckley, 2016)); *homologous markers in all taxa not identified. Grey shading used to highlight homologous markers useful for taxonomic discrimination; aMarker previously published (Buckley et al., 2009, 2016); *incomplete set of markers that may not be homologous; *not confirmed by LC-Orbitrap analyses (see Supplementary Figures S2-7 for LC-MS/MS sequence spectra). Common name
Species
2t85
1t62a
2t26
2t60
*
Field vole
Microtus agrestis
1459.7
3417.5
Microtus oeconomus
2523.3
3297.5
Common vole
Microtus arvalis
Narrow-headed vole
Microtus gregalis
Bank vole
Myodes glareolus
Water vole
Arvicola amphibius
Steppe lemming
Lagurus lagurus
Norway lemming
Lemmus lemmus
1592.8/ 1608.8 1592.8/ 1608.8 1592.8/ 1608.8 1592.8/ 1608.8 1592.8/ 1608.8 1592.8/ 1608.8 1592.8/ 1608.8 1623.8
2523.3
Tundra vole
Collared lemming
Dicrostonyx torquatus Apodemus sp.
1594.8/ 1610.8 1592.8/ 1608.8
2523.3
Field mouse
1187.6/ 1203.6 1187.6/ 1203.6 1187.6/ 1203.6 1187.6/ 1203.6 1187.6/ 1203.6 1187.6/ 1203.6 1178.6/ 1194.6 1187.6/ 1203.6 1187.6/ 1203.6 1187.6/ 1203.6
1459.7 1443.7 1443.7 1459.7 1459.7 1459.7 1459.7 1459.7 1443.7
2537.3 2551.3 2551.3
3417.5
2551.3
3389.5
2551.3 2523.3
Fig. 1. MALDI ToF mass spectrometry peptide mass fingerprints from five arvicoline reference taxa: D. torquatus, M. gregalis, Lemmus, M. agrestis and M. arvalis; see Supplementary Figure S1 for reference spectra for the remaining four reference taxa.
these arvicolines (not shown in Table 1). One peptide marker that appears diagnostic of this group (particularly in combination with the latter) is at m/z 2129.1, found in all taxa here (not shown in Table 1; note that rabbits and hares also have the marker at m/z 2129.1, but with the m/z 2705.4 peak rather than m/z 2695.4). The marker at m/z 1592.8/1608.8 was found to represent the voles with the homologous marker at m/z 1594.8/1610.8 in the collared lemming and m/z 1623.8 in the Norway lemming (tribes Lemmini and Dicrostonychini respectively) whereby a substitution in the sequence belonging to the latter of one of the proline residues amenable to biological oxidation appears to affect the clear observation of the two oxidation forms seen in the others (Supplementary Figure S2).
The ability to distinguish between some of these taxa further advances the taxonomic resolution achievable in relation to suspected divergence times. Biochemical evidence supports morphological studies in an estimate for the origins of the Arvicolidae family at ~5 Ma (Chaline and Graf, 1988), with some earlier estimates even as far back as 8e9 Ma (Michaux et al., 2001) and subsequent diversification ~3e5 Ma (Conroy and Cook, 1999). Regardless of the estimates of this basal split between the voles and the lemmings, the ability to distinguish within Microtus is particularly interesting; Microtus is thought to have arisen between 0.5 and 2 Ma (Repenning, 1980; Chaline et al., 1999), but genome analysis indicates much higher rates of molecular evolution than observed in other mammals (Triant and DeWoody, 2006).
Please cite this article in press as: Buckley, M., et al., Species identification of voles and lemmings from Late Pleistocene deposits in Pin Hole Cave (Creswell Crags, UK) using collagen fingerprinting, Quaternary International (2018), https://doi.org/10.1016/j.quaint.2018.03.015
4
M. Buckley et al. / Quaternary International xxx (2018) 1e7
Collagen fingerprinting therefore provides a new tool for analysis of micromammal assemblages, allowing accurate identification of individual teeth amongst other skeletal remains from all arvicoline rodent genera, and most individual species, present in the Late Pleistocene and Holocene of the British Isles (Sutcliffe and Kowalski, 1976). Isolated teeth of arvicoline rodents constitute a large proportion of the potentially identifiable remains in microfaunal assemblages like these, but often cannot be specifically identified from their morphology alone (Sutcliffe and Kowalski, 1976). This is exemplified through examples such as at Beckford (Worcestershire, UK), where the presence of reported M. arvalis is incongruous with the otherwise Arctic tundra faunal assemblage but potentially derived from mis-identified M. gregalis isolated teeth. Similar mis-identifications have been known to occur between M. arvalis and M. agrestis (Sutcliffe and Kowalski, 1976). However, the importance of collagen fingerprinting as a palaeoecological tool stems from its ability to achieve this at low unit cost, which is currently a major drawback for the alternative DNAbased methods. Collagen fingerprinting is likely to be of similar use in many other sites from northern and western Europe, where assemblages are composed of the same, or similar suites of species. 3.2. Pin Hole Cave arvicoline identifications From an original >13,022 spectral fingerprints from the Pin Hole Cave assemblage (including specimens from the spoil and elsewhere in the cave than in our previous report, Buckley et al., 2016), we filtered out poor quality spectra resulting in 6,192 that we considered of good enough quality for likely species identification and contained the marker m/z 1105.6 diagnostic of most mammals (excludes cetaceans; Buckley et al., 2014). Of these, over at least 4,262 appeared to contain the rodent marker at m/z 2695 (Buckley et al., 2016) although 42 appear to derive from Apodemus (Buckley et al., 2016). Of the remainder, 2773 could be categorised as lemming (546 Lemmus; 2,227 Dicrostonyx) and 1,489 as vole (Fig. 2). More specifically, the majority (1,218) of the voles appear to derive from the narrow-headed vole (with the marker at approximately m/z 2552.3/2553.3; the increase from the marker at m/z 2551.3 in modern reference taxa is consistent with deamidation at one or two residues; Fig. 3). At least 78 could be identified as from common vole (the observed marker at m/z 2539.3 similarly increased above that of the modern reference) and 118 could be identified as
either M. agrestis or M. oeconomus; the majority appeared to contain the marker for the field vole, but caution should be noted in separating these two using the presented markers as their sequences could not be confirmed within this study; similarly, it is likely that of the 75 specimens of other voles (e.g., water or bank voles) the majority (at least 30) of those that retained the higher mass markers appeared reflective of bank vole (~30), with only a few (four) with markers for water vole, the much reduced number being consistent with the selection criteria in which only bone fragments small enough to fit into the microtitre plate well (~8 mm) were typically analysed. Contrarily to the ease at which some of the Microtus taxa could be separated, the separation of some taxa, e.g., M. agrestis from M. oeconomus or between Myodes and Arvicola was surprisingly more difficult to achieve given the absence of several potential markers observed in the modern reference material (not all listed) but these particular taxa only represented a small component of those present in this assemblage (i.e., both groups representing <5% of the assemblage). By far the dominant taxon in the assemblage appears to be the collared lemming (Dicrostonyx) with more than four times as many specimens identified as the next most abundant rodent, the Norway lemming. The presence of the lemmings D. torquatus and L. lemmus, the bank vole My. glareolus, the water vole A. amphibius, the tundra vole M. oeconomus and the narrow-headed vole M. gregalis are expected in the Late Pleistocene fauna of Pin Hole Cave; Dicrostonyx, Lemmus, and M. gregalis are known to characterise the Late Glacial period in Britain, with the other taxa present before and after the Late Glacial maximum although both lemmings have also been placed within the Windermere (Bølling-Allerød) interstadial in the later part of the Devensian glaciation (Yalden, 2010), around 13-11 Ka. Both lemmings at present have a circumpolar distribution, but it is the collared lemming that is known for being more adapted for colder environments, with fur that turns white in winter and complex winter claws (Yalden, 2010). The abundance of the narrow-headed vole, present today in the tundra region of northern Europe and Asia, is also of particular interest as it is thought to prefer wetter areas than the other Microtus species (De Jonge and Dienske, 1978) and Dicrostonyx (Yalden, 2010), but it is often found associated with the latter in fossil communities (Cordy, 1991). The occurrence of a small number of M. agrestis and Apodemus specimens identified is presumably indicative of the Windermere interstadial in this assemblage; fossil evidence indicates that their post-Weichselian
Fig. 2. Faunal composition of the vole and lemming remains from Pin Hole Cave, Creswell Crags, UK.
Please cite this article in press as: Buckley, M., et al., Species identification of voles and lemmings from Late Pleistocene deposits in Pin Hole Cave (Creswell Crags, UK) using collagen fingerprinting, Quaternary International (2018), https://doi.org/10.1016/j.quaint.2018.03.015
M. Buckley et al. / Quaternary International xxx (2018) 1e7
5
Fig. 3. Two sections of example MALDI ToF mass spectrometry peptide mass fingerprints from six different arvicoline taxa from the Pin Hole assemblage.
arrival in Britain was during the Bølling-Allerød interstadial in the case of the field vole (Herman and Searle, 2011) and perhaps as late as the Holocene in the case of the wood mouse (Herman et al., 2017), although it was reported from Cat Hole, Glamorgan in the Bølling period along with Dicrostonyx, M. agrestis and My. glareolus and in the Allerød period with Lemmus, M. oeconomus and M. agrestis (Campbell, 1977). Overall, environmental inferences drawn from this study of microfauna is consistent with that observed from our previous analysis of the megafaunal remains (Buckley et al., 2017) in which we observed a dominance of fauna adapted for the severe conditions of the Late Glacial, with a dominance of reindeer, and substantial proportions of woolly rhinoceros and woolly mammoth. However, these microfaunal remains are likely to be more representative of the closer environment to the cave assemblage. 3.3. Glutamine deamidation across arvicoline taxa Given that the range of arvicoline rodents present at Pin Hole represent a range in climatic conditions over approximately 40,000 years, if relative decay measurements of collagen through peptide mass fingerprinting were indicative of relative age (Doorn et al., 2012) we would expect to see elevated decay in the colderadapted taxa from earlier in the Late Pleistocene (e.g., D. torquatus, M. oeconomus, M. gregalis) compared to the more warm-adapted taxa from the early Holocene (e.g., M. agrestis or My. glareolus). To explore this we measured the deamidation of the m/z 1105.6 peptide in a sample of rodents of different geological ages (albeit only based on known information about the taxa in this region). Although there were substantial differences between the levels of deamidation observed in the field mouse (sylvaticusA. ), a species that was restricted to the Holocene in this assemblage, and those of the Late Pleistocene voles and lemmings (Fig. 4), the direction of this difference contradicted what we would anticipate for their relative geological ages. This further highlights the potential unreliability of these decay markers as a means of relative ageing across the species boundary, when differences in accumulation and pre-taphonomic histories
are likely. Previously we postulated that the higher levels of decay observed in the field mouse compared with older Late Pleistocene carnivores, such as the arctic fox and hyaena, were the result of exposure to acidic conditions while passing through the digestive tract of a predator (most likely that of an owl; Buckley et al., 2017). However, this explanation is shown less plausible here given that the difference is between rodent taxa and noting that the collared lemming was expected to have a larger decay range than the other taxa, not only due to the greater number of specimens but also to the larger known temporal range (even if present throughout the range of the other taxa within the chronological period of this assemblage). Therefore, although the measurements of deamidation provide a means to rapidly identify more recent intrusions into an archaeological or palaeontological assemblage (Buckley et al., 2017), and may still prove valuable for the relative ageing of megafaunal remains of more comparable taphonomic histories, whether or not it could provide useful relative ageing information will depend on better understanding the accumulation and depositional processes affecting the rodent taxa being studied. One particular advantage with the study of microfaunal remains is that although there appears to be heterogeneity in the deamidation values across a single specimen (Simpson et al., 2016), as well as across archaeological sites (Doorn et al., 2012), here the evaluations are encompassing the entirety of the specimens giving a more appropriate average value. 4. Conclusions We report the further evaluation of a relatively new and underutilised methodology in collagen fingerprinting for species identification of ancient microfaunal remains. Specifically with the assemblage from Pin Hole Cave, we were able to go much further than studying the presence or absence of particular microfauna and look at the overall relative abundances of many of the different lemming and vole taxa. Here we found that the lemmings formed nearly two thirds of the material, dominated by the collared lemming, with the narrow-headed vole as dominant amongst the voles. Although we do show that they can yield species-level
Please cite this article in press as: Buckley, M., et al., Species identification of voles and lemmings from Late Pleistocene deposits in Pin Hole Cave (Creswell Crags, UK) using collagen fingerprinting, Quaternary International (2018), https://doi.org/10.1016/j.quaint.2018.03.015
6
M. Buckley et al. / Quaternary International xxx (2018) 1e7
Fig. 4. Box plots of decay measurements, as the ratio of the peak height of m/z 1106.6/1105.6, from Apodemus, M. arvalis, M. gregalis, Lemmus and Dicrostonyx.
information within Microtus, we further confirmed some of the cross-species issues with the use of decay measurements via deamidation as a tool for relative age estimation within an assemblage. Together these reveal two forms of latent data within geoarchaeological archives that contain faunal remains. The capabilities of the method has far-reaching implications for the reliable identification of material from similar sites throughout Europe, many of which will include morphologically undiagnostic teeth and other skeletal elements. Funding This work was supported by the Royal Society [UF120473]. for fellowship funding and the original studies as part of a fellowship funded by the Natural Environment Research Council [NE/H015132/ 1]. Acknowledgements We would like to thank the National Museums Scotland (Edinburgh, UK), University of Oulu (Oulu, Finland), Mammals collections of the Museum National d’Histoire naturelle (Paris, France), the University of Sheffield's Department of Archaeology (Sheffield, UK) and the Creswell Crags Heritage Centre (Derbyshire, UK) for access to samples. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.quaint.2018.03.015. References Armstrong, A.L., 1932. Excavations in the Pin Hole cave, Creswell Crags, Derbyshire. Proc. Prehist. Soc. East Anglia 6, 330e334. Bar-Yosef, O., Kuhn, S.L., 1999. The big deal about blades: laminar technologies and human evolution. Am. Anthropol. 101, 322e338. Behrensmeyer, A.K., Western, D., Boaz, D.E.D., 1979. New perspectives in vertebrate paleoecology from a recent bone assemblage. Paleobiology 5, 12e21. Bryan, A.L., 1973. Paleoenvironments and cultural diversity in late Pleistocene South America. Quat. Res. 3, 237e256. Buckley, M., 2016. Species identification of bovine, ovine and porcine type 1 collagen; comparing peptide mass fingerprinting and LC-based proteomics methods. Int. J. Mol. Sci. 17, 445. Buckley, M., Collins, M., Thomas-Oates, J., Wilson, J.C., 2009. Species identification by analysis of bone collagen using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 23, 3843e3854.
lsdo ttir, A., 2014. Species Buckley, M., Fraser, S., Herman, J., Melton, N., Mulville, J., Pa identification of archaeological marine mammals using collagen fingerprinting. J. Archaeol. Sci. 41, 631e641. ~ a, R.A., Lawless, C., Tambusso, P.S., Varela, L., Carlini, A.A., Buckley, M., Farin Powell, J.E., Martinez, J.G., 2015. Collagen sequence analysis of the extinct giant ground sloths Lestodon and Megatherium. PLoS One 10 e0144793. Buckley, M., Gu, M., Shameer, S., Patel, S., Chamberlain, A.T., 2016. High-throughput collagen fingerprinting of intact microfaunal remains; a low-cost method for distinguishing between murine rodent bones. Rapid Commun. Mass Spectrom. 30, 805e812. Buckley, M., Harvey, V.L., Chamberlain, A.T., 2017. Species identification and decay assessment of Late Pleistocene fragmentary vertebrate remains from Pin Hole Cave (Creswell Crags, UK) using collagen fingerprinting. Boreas 46, 402e411. Campbell, J.B., 1977. The Upper Palaeolithic of Britain: a Study of Man and Nature in the Late Ice Age. Oxford University Press. Chaline, J., Brunet-Lecomte, P., Montuire, S., Viriot, L., Courant, F., 1999. Anatomy of the arvicoline radiation (Rodentia): palaeogeographical, palaeoecological history and evolutionary data. Ann. Zool. Fenn. 36, 239e267. Chaline, J., Graf, J.-D., 1988. Phylogeny of the Arvicolidae (Rodentia): biochemical and paleontological evidence. J. Mammal. 69, 22e33. Conroy, C.J., Cook, J.A., 1999. MtDNA evidence for repeated pulses of speciation within arvicoline and murid rodents. J. Mammalian Evol. 6, 221e245. Cordy, J.-M., 1991. Palaeoecology of the late glacial and early postglacial of Belgium and neighbouring areas. In: Barton, N., Roberts, A.J., Roe, D.A. (Eds.), CBA Research Report. Council for British Archaeology, vol. 77, pp. 40e47. Currant, A., Jacobi, R., 2001. A formal mammalian biostratigraphy for the Late Pleistocene of Britain. Quat. Sci. Rev. 20, 1707e1716. Currant, A.P., Jacobi, R., 2011. The Mammal Faunas of the British Late Pleistocene. In: Ashton, N., Lewi, S.G., Stringer, C. (Eds.), In, The Ancient Human Occupation of Britain, Developments in Quaternary Sciences, vol. 14, pp. 165e180. De Jonge, G., Dienske, H., 1978. Habitat and interspecific displacement of small mammals in The Netherlands. Neth. J. Zoology 29, 177e214. Demirel, A., Andrews, P., Yalçınkaya, I., Ersoy, A., 2011. The taphonomy and palaeoenvironmental implications of the small mammals from Karain Cave, Turkey. J. Archaeol. Sci. 38, 3048e3059. Doorn, N.L., Wilson, J., Hollund, H., Soressi, M., Collins, M.J., 2012. Site-specific deamidation of glutamine: a new marker of bone collagen deterioration. Rapid Commun. Mass Spectrom. 26, 2319e2327. Gibb, S., Strimmer, K., 2012. MALDIquant: a versatile R package for the analysis of mass spectrometry data. Bioinformatics 28, 2270e2271. Glue, D.E., 1967. Prey taken by the Barn Owl in England and Wales. Bird Study 14, 169e183. Haddadian Shad, H., Darvish, J., Mohammadian, T., Mahmoudi, A., Alaie Kakhki, N., Ghanbarifardi, M., Molavi, F., Barani-Beiranvand, H., 2014. Preliminary study of rodents using pellets of predatory birds in Iran. Iran. J. Animal Biosyst. 10. hannesdo ttir, F., Jones, E.P., McDevitt, A.D., Michaux, J.R., White, T.A., Herman, J.S., Jo jcik, J.M., Searle, J.B., 2017. Post-glacial colonization of Europe by the wood Wo mouse, Apodemus sylvaticus: evidence of a northern refugium and dispersal with humans. Biol. J. Linn. Soc. 120, 313e332. Herman, J.S., Searle, J.B., 2011. Post-glacial partitioning of mitochondrial genetic variation in the field vole. Proc. R. Soc. B 278, 3601e3607. Klein, R.G., 1988. The archaeological significance of animal bones from Acheulean sites in southern Africa. Afr. Archaeol. Rev. 6, 3e25. Lagerholm, V.K., Sandoval-Castellanos, E., Ehrich, D., Abramson, N.I., , M., Angerbjo €rn, A., Stewart, J.R., Nadachowski, A., Kalthoff, D.C., Germonpre n, L., 2014. On the origin of the Norwegian lemming. Mol. Ecol. 23, Dale 2060e2071. Lister, A., 1992. Mammalian fossils and Quaternary biostratigraphy. Quat. Sci. Rev. 11,
Please cite this article in press as: Buckley, M., et al., Species identification of voles and lemmings from Late Pleistocene deposits in Pin Hole Cave (Creswell Crags, UK) using collagen fingerprinting, Quaternary International (2018), https://doi.org/10.1016/j.quaint.2018.03.015
M. Buckley et al. / Quaternary International xxx (2018) 1e7 329e344. pez-García, J.M., Blasco, R., Rivals, F., Rosell, J., 2016. Variations in Microtus Luzi, E., Lo arvalis and Microtus agrestis (Arvicolinae, rodentia) dental Morphologies in an archaeological context: the case of teixoneres cave (late Pleistocene, northeastern Iberia). J. Mammalian Evol. 24, 1e9. Martin, R.A., 2014. A critique of vole clocks. Quat. Sci. Rev. 94, 1e6. , N., Barnett, R., Cucchi, T., Struchen, R., Pascal, M., Pascal, M., Martínkova Fischer, M.C., Higham, T., Brace, S., Ho, S.Y., 2013. Divergent evolutionary processes associated with colonization of offshore islands. Mol. Ecol. 22, 5205e5220. Maul, L., Masini, F., Abbazzi, L., Turner, A., 1998. Geochronometric application of evolutionary trends in the dentition of fossil Arvicolidae. Meded. Ned. Inst. Toegepaste Geowetenschappen TNO 60, 565e572. McGuire, J.L., 2011. Identifying California Microtus species using geometric morphometrics documents Quaternary geographic range contractions. J. Mammal. 92, 1383e1394. Michaux, J., Reyes, A., Catzeflis, F., 2001. Evolutionary history of the most speciose mammals: molecular phylogeny of muroid rodents. Mol. Biol. Evol. 18, 2017e2031. Mihok, S., Turner, B.N., Iverson, S.L., 1985. The characterization of vole population dynamics. Ecol. Monogr. 55, 399e420. Ranta, E., Kaitala, V., 1997. Travelling waves in vole population dynamics. Nature 390, 456.
7
Repenning, C.A., 1980. Faunal exchanges between Siberia and North America. Can. J. Anthropol. 1, 37e44. Romaniuk, A.A., Shepherd, A.N., Clarke, D.V., Sheridan, A.J., Fraser, S., Bartosiewicz, L., Herman, J.S., 2016. Rodents: food or pests in neolithic Orkney. R. Soc. Open Sci. 3, 160514. Schreve, D.C., 2001. Differentiation of the British late Middle Pleistocene interglacials: the evidence from mammalian biostratigraphy. Quat. Sci. Rev. 20, 1693e1705. Simpson, J.P., Penkman, K.E.H., Demarchi, B., Koon, H., Collins, M.J., Thomas-Oates, J., Shapiro, B., Stark, M., Wilson, J., 2016. The effects of demineralisation and sampling point variability on the measurement of glutamine deamidation in type I collagen extracted from bone. J. Archaeol. Sci. 69, 29e38. Sutcliffe, A.J., Kowalski, K., 1976. Pleistocene rodents of the British Isles. Br. Mus. Nat. Hist. A 27, 33e147. Triant, D.A., DeWoody, J.A., 2006. Accelerated molecular evolution in Microtus (Rodentia) as assessed via complete mitochondrial genome sequences. Genetica 128, 95e108. Ubilla, M., 2004. Mammalian biostratigraphy of Pleistocene fluvial deposits in Northern Uruguay, South America. Proc. Geologists' Assoc. 115, 347e357. Van Meerbeeck, C., Renssen, H., Roche, D., 2009. How did marine Isotope stage 3 and last glacial maximum climates differ?eperspectives from equilibrium simulations. Clim. Past 5, 33e51. Yalden, D., 2010. The History of British Mammals. A&C Black, London.
Please cite this article in press as: Buckley, M., et al., Species identification of voles and lemmings from Late Pleistocene deposits in Pin Hole Cave (Creswell Crags, UK) using collagen fingerprinting, Quaternary International (2018), https://doi.org/10.1016/j.quaint.2018.03.015