Research paper

New single amino acid hydroxyproline radiocarbon dates for twoproblematic American Mastodon fossils from Alaska

Shweta Nalawade-Chavan a,*, Grant Zazula b, Fiona Brock a, John Southon c,Ross MacPhee d, Patrick Druckenmiller eaResearch Laboratory for Archaeology & History of Art, University of Oxford, Dyson Perrins Building, South Parks Road, Oxford OX1 3QY, UKb Yukon Palaeontology Program, Department of Tourism & Culture, Government of Yukon, P.O. Box 2703, Whitehorse, Yukon, Canada Y1A 2C6cKeck-CCAMS Group, Earth System Science Department, B321 Croul Hall, University of California, Irvine, Irvine, CA 92697-3100, USAdAmerican Museum of Natural History, 200 Central Park West, NY 10024, USAeUniversity of Alaska Museum, 907 Yukon Dr., Fairbanks, AK 99775, USA

a r t i c l e i n f o

Article history:Received 26 April 2013Received in revised form3 October 2013Accepted 16 October 2013Available online 25 October 2013

Keywords:American mastodonRadiocarbon datingBone collagenAmino acidsHydroxyprolineHPLC

a b s t r a c t

American mastodon (Mammut americanum) was amongst the widest ranging of Pleistocene megafaunalspecies, though their fossils are rare in Alaska and northwest Canada. Questions remain about theirextinction chronology at high latitudes because of the limited numbers of available radiocarbon dates.New radiocarbon dates for two American mastodon fossils were generated at two separate acceleratormass spectrometry laboratories using two different approaches, dating ultrafiltered ‘collagen’ vs. singleamino acid fractions. The bulk dates for these specimens are significantly younger than the single aminoacid (hydroxyproline) dates, which are in turn close to the background threshold for radiocarbon dating.On closer study we established that contamination from consolidants used in museum conservation wasnot removed thoroughly despite extensive physical and chemical cleaning procedures having beenapplied, and this led to the anomalous ultrafiltered ‘collagen’ results. The new hydroxyproline dates givesupport to older ages for American mastodons in the Arctic.

! 2013 Elsevier B.V. All rights reserved.

1. Introduction

Accurate radiocarbon dating of bones remains challenging de-cades after the introduction of acceleratormass spectrometry in thelate 1970s. Although different organic fractions can be extractedfrom archaeological and palaeontological bone using differentmethods for the purpose of radiocarbon dating, in most instances,the fraction dated is the organic fraction commonly described as‘collagen’, which consists predominantly of collagen as well asother proteins (hereafter referred to as collagen).

Despite the advancement of collagen-extraction proceduresover recent years, including the application of ultrafiltration,(Brown et al., 1988; Bronk Ramsey et al., 2004; Brock et al., 2007;Beaumont et al., 2010) the presence of contamination withinsamples remains a persistent issue. It is demonstrably difficult toachieve a contaminant-free sample that might be regarded as

representing the original material, unaltered by any natural orartificial processes over the years since it ceased interaction withthe biosphere. In routine radiocarbon pre-treatment methods thesamples are screened based on the C:N atomic ratios of purifiedcollagen, the collagen yield and carbon content, as well as d13C andd15N values prior to accelerator mass spectrometry (AMS) analysis(e.g. van Klinken, 1999). Fourier Transform Infrared Spectroscopy(FTIR) has also been used to screen bones and the products ofcollagen extraction, prior to dating, to identify the potential pres-ence of contaminants (e.g. Gianfrate et al., 2007; Yuan et al., 2007;France et al., 2011). However, whether these techniques areemployed independently or collectively, they are not always effi-cient in detecting trace levels of contamination that have managedto resist pre-treatment procedures. For example, an infinitely oldsample with just 1% modern carbon contamination will notanalytically produce an age greater than 38000 radiocarbon years(Bowman, 1995), yet the C:N atomic ratio for the contaminatedbone would remain within the acceptable range. Moreover, theaccuracy of these pre-treatment methods is generally assessed ontheir ability to precisely date a standard material of known radio-carbon activity. These standards are usually chosen because they

* Corresponding author. Tel.: !44 1865 285203; fax: !44 1865 265222.E-mail addresses: Shweta.Chavan@rlaha.ox.ac.uk, drshwetachavan@gmail.com

(S. Nalawade-Chavan).

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Quaternary Geochronology

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1871-1014/$ e see front matter ! 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.quageo.2013.10.004

Quaternary Geochronology 20 (2014) 23e28

are clean (or in the case of bone, very easily cleaned to produceinfinite dates) and are used to assess the amount of contaminationadded in the laboratory. These standards are completely unsuitableto assess the ability of a certain treatment to remove a specificcontaminant. Also, many times the reliability of a given radiocarbondate is judged based on the archaeological or palaeontologicalcontext from which the sample is recovered, the demonstratedpurity of the material analysed, and the known accuracy and pre-cision of the analytical method employed. Generally, the apparentreliability of a radiocarbon date comes under scrutiny only when itseriously deviates from expectations, such as the inferred age of thecontext in which a sample was found. However, if the context isunknown or cannot be reconstructed, an erroneous date can easilybe accepted as the actual date of the material. A radiocarbon datingprocedure considered to be reliable based on the selection of onlycertain of the criteria mentioned above cannot guarantee its uni-versal suitability or accuracy for all samples.

Radiocarbon dating single amino acid, hydroxyproline has beendemonstrated to fulfill all the basic criteria to be deemed asmethodof choice for dating problematic bone materials (McCullagh et al.,2010; Marom et al., 2012). Here we show how hydroxyprolineradiocarbon dating can be used to resolve problems in defining theage of recalcitrant paleontological specimens.

American mastodons (Mammut americanum) were widespreadmembers of the Pleistocene megafauna in North America, rangingfrom the tropics of Central America in Honduras to near the Arcticcoast of Alaska (Kurtén and Anderson, 1980; Faunmap WorkingGroup, 1996). As tracked by radiocarbon dating, mastodons livingnear the Great Lakes region of the AmericanMidwestwere amongstthe latest surviving members of the Pleistocene megafauna, lastinguntil w10000 14C yr BP (Woodman and Athfield, 2009). However,questions remain regarding the chronology of this species at highlatitudes and their possible persistence in the cold, dry steppe-tundra environment that characterized glacial periods in Alaska(Guthrie, 1990). Many researchers have assumed that Americanmastodons only lived at high latitudes during Pleistocene intergla-cial periods when boreal forest habitats recolonized the arctic andsubarctic landscape (Harington, 1990). The radiocarbon dates re-ported in this paper suggest how lack of available samples for datingand a reliable radiocarbon chronology for mastodons in Alaska hasprevented a robust understanding of how this large mammalresponded to late Pleistocene environmental change, and the timingof their extinction on a continental scale.

American mastodon fossils have been collected in Alaska fornearly a century, though they are relatively rare in comparison toremains of the other extinct proboscideans that inhabited NorthAmerica in the latter part of the Pleistocene, Mammuthus pri-migenius and Mammuthus columbi (the woolly and Columbianmammoths, respectively). Harington (1978) estimates that lessthan 5% of proboscidean fossil remains in eastern Beringia repre-sent American mastodons (c.f. Guthrie, 1968).

Glues, varnishes, and other preservatives have been used sincethe earliest days of paleontology to protect fossils from damage ordeterioration. Such materials are usually carbon based, and mayaffect the radiocarbon date. Therefore, best practice is to collect asample for dating before the application of any reagents. However,a record of consolidant application is usually lacking for fossilsconserved in the early twentieth century.

We encountered this last problem whilst conducting a study ofhigh latitude American mastodon fossils. Among our specimenswere two samples that yielded ultrafiltered ‘collagen’ radiocarbondates from as late as the Last Glacial Maximum, and which weresignificantly younger than those recovered for other samples.Becausemastodon survival this late in northwestern North Americaseemed improbable, we questioned whether the presence of

undetected consolidants in the samples might have skewed ourradiocarbon dating results. We tested this idea using a novelapproach developed at the Oxford Radiocarbon Accelerator Unit(ORAU) that utilizes radiocarbon dating of a single amino acid,hydroxyproline. In brief, we found that the single amino acid dateswere close to background 14C levels, and thus are in better agree-ment with ages derived for other high latitude mastodon samples.

2. Samples

Specimen 1, UAMES 7663 from the University of AlaskaMuseumEarth Sciences Collection is an American mastodon cranium (Fig. 1)recovered at a placer gold mine at Livengood, Alaska, approxi-mately 80 km (50 mi) north of Fairbanks, in 1941 and donated tothe University of Alaska Museum of the North in the early 1980s.Exposed surfaces of this cranium are coveredwith a thick coating ofan unknown consolidant. Specimen 2, AMNH 103277 is registeredin the American Museum of Natural History, vertebrate paleon-tology catalog as a mandible recovered at Little Eldorado Creek,near Fairbanks, Alaska, in 1940, but no further information isavailable. We are of course aware that these samples may alsocontain carbon-containing contaminants other than those derivedfrom consolidants, but FTIR analysis (see below) indicates that thelatter were certainly present and most probably the major, if notthe exclusive, source of error. Sample AMNH 103277 showed clearsigns of the presence of surface consolidants. Presence of con-solidants was also suspected on sample UAMES 7663 although notvisible to the naked eye.

3. Methods

3.1. UCIAMS [University of California Irvine accelerator massspectrometer] ultrafiltered ‘collagen’ extraction method

After removal of the bone surface with a Dremel" grinding tool,w150 mg of bone was crushed to mm-sized chips. Since the pres-ence of consolidants was known or suspected, the crushed bonesampleswere sonicated in acetone, methanol andwater (for 30mineach) before decalcification. The 2nd specimen (the mandible)produced lots of flocculent material and so the solvent wash

Fig. 1. Mammut americanum cranium, UAMES 7663, at the University of AlaskaMuseum.

S. Nalawade-Chavan et al. / Quaternary Geochronology 20 (2014) 23e2824

sequence was repeated. Bone standards and blanks run with thisbatch were subjected to the same treatment.

Procedures used in the preparation of the ultrafiltered(>30 kDa) gelatin are detailed in Beaumont et al. (2010). Briefly, thecrushed bone was decalcified for 24 h in 0.5 N HCl, washed in Milli-Q" water and gelatinized overnight at 60 "C in 5 ml of 0.01 N HCl.The gelatin solution was transferred to pre-cleaned 30 kDa MWCOCentriprep" ultrafilters, using ultrafine pipets (Samco" 233) toavoid pipetting solids. The gelatin was centrifuged twice to reducethe volume to<1 ml, diluted to 3 ml with Milli-Q"water to reducethe residual salt content of the >30 kDa fraction, and then centri-fuged twice more. The >30 kDa gelatin was then pipetted intoheavy-walled 13mm culture tubes, frozenwith liquid nitrogen, anddried overnight in a vacuum centrifuge. The combustion andgraphitization protocols used, and the operation of the UCIAMSinstrument, are described in Beverly et al. (2010).

These samples were part of a larger batch of mastodon bonesthat were all thought to have been treated with consolidants. As acheck, two other samples from the batch were run with andwithout the solvent/sonication treatment, and the solvent-treatedsamples gave ages several thousand years older than the un-treated, indicating that younger material (presumably consolidant)was indeed being removed. Note that the older ages could not bedue to solvent residues, because almost half the carbon in theultrafiltered gelatin would have had to come from the solvents,which would have produced highly anomalous C:N atomic ratiosfor the treated samples.

3.2. ORAU [Oxford Radiocarbon Accelerator Unit] ultrafiltered‘collagen’ extraction method

The surfaces of both samples were carefully cleaned by airabrasion using aluminium oxide powder, prior to drilling 600e700 mg of bone powder from each using a tungsten carbide drill bitat low speed. The samples were then subjected to sequential sol-vent washes with acetone (45 "C), methanol (45 "C), and chloro-form (room temperature) for a minimum of 1 h each to remove anyremaining consolidant. Sample AMNH 103277 underwent twoseparate acetone washes because the first rinse was observed toremove some material from the bone specimen. The samples werethen left to air dry thoroughly before undergoing routine collagen-extraction procedures. This method, described in detail by Brocket al. (2010), involves sequential washes with HCl (0.5 M, 3e4rinses overw18 h), NaOH (0.1M, 30min), and HCl (0.5M,15min) atroom temperature, with thorough rinses with ultrapure Milli-Q"water after each stage. The crude collagen was then gelatinized inpH3 solution at 75 "C for 20 h and the resultant solution filteredwith a cleaned Ezee"-filter (Elkay, UK). The filtrate was transferredto a pre-cleaned Vivaspin 15" 30 kDa MWCO ultrafilter (Sartorius)and centrifuged until 0.5e1.0 mL of the>30 kDa fraction remained.This fraction was then removed from the ultrafilter with ultrapurewater before being freeze-dried.

The final collagen products were converted to CO2 and graphi-tised prior to AMS dating as described by Brock et al. (2010). Duringthe conversion to CO2, the C:N atomic weight ratio and carbon andnitrogen stable isotopic values of the collagen were measured.Background corrections were applied to the radiocarbon dates asdescribed by Wood et al. (2010).

3.3. ORAU [Oxford Radiocarbon Accelerator Unit] Hydroxyprolinemethod

The second approach at ORAU was to separate the single aminoacid hydroxyproline from hydrolysed bone collagen using mixed-mode preparative chromatography. Bone samples were

sandblasted to clean before any chemical pre-treatment. For sam-ple AMNH 103277 (Mandible) ultrafiltered collagen excess wasused. Due to insufficient amount of ultrafiltered collagen excess,sample UAMES 7663 (Cranium) was subjected to sequential solventwashes with acetone (45 "C), methanol (45 "C), and chloroform(room temperature) for a minimum of 1 h each to remove anyremaining consolidant. The sample was then left to air dry thor-oughly before undergoing collagen-extraction proceduresdescribed in O’Connell and Hedges (1999) and O’Connell et al.(2001), involves 3e4 rinses of 0.5 M HCl over w18 h followed bythorough rinses with ultrapure Milli-Q" water. The crude collagenwas then gelatinized in pH3 solution at 70 "C for 24 h and theresultant solution was freeze-dried. Ultrafiltration was notemployed as chromatographic separation alone has been demon-strated to be more effective in removal of contamination (Maromet al., 2012). Bone collagen was hydrolysed prior to chromato-graphic separation by treating with excess of 6 M hydrochloric acidin a nitrogen atmosphere at 105 "C for 24 h. Hydrochloric acid wasremoved using a vacuum evaporator and the washed residue wasthen reconstituted in pH3 water. This hydrolysate solution wasfiltered through 0.2m PTFE syringe filters and thenwas injected ontothe Primesep A HPLC column.

The mixed mode chromatographic method has been improvedfrom Marom et al. (2012) by replacing the 0.3% ortho-phosphoricacid mobile phase with Milli-Q"water. As non-acidity of themobile phase can lead to dissolution of atmospheric CO2, themobile phase was sparged with N2 and amino acid fractions werecollected in test tubes containing a drop of concentrated hydro-chloric acid so as to ensure a pH of 2 in the fractions collected(Nalawade-Chavan et al., 2013). The hydroxyproline (Hyp) frac-tions collected were evaporated to dryness using a vacuumevaporator and reconstituted in pH3 water so as to facilitate theiraddition onto Chromosorb" in tin capsules on the day of com-bustion. ORAU’s routine procedures were followed for combustion,graphitization and AMS dating of the hydroxyproline (Brock et al.,2010). Hydroxyproline fractions, using the same procedures wereseparated from a laboratory background age standard, an Alaskanbison bone thought to be 60,000 to 70,000 years old (Brock et al.,2007).

3.4. Fourier transform infrared spectroscopy (FTIR)

Infrared spectra of whole bone powder and collagen extract atORAU were obtained using a Varian Excalibur series FTIR with aSpecac Golden Gate ATR. Data were measured and manipulatedusing Digilab Resolutions Pro 4.0 software. Each sample was runfor 64 scans, and each spectrum subject to backgroundsubtraction.

4. Results

Table 1 illustrates the ultrafiltered ‘collagen’ and amino acidhydroxyproline AMS dates obtained for these mastodon specimensat UCIAMS and ORAU. Note that the C:N ratios for ultrafiltered‘collagen’ of the treated bone are within the acceptable range of3.1e3.5 (van Klinken, 1999) but slightly higher than the 3.2e3.3values normally observed for bone collagen, possibly indicatinginadequate removal of consolidants. However, C:N ratios of thehydroxyproline fractions were very close to the theoretical C:Nvalue of 5.0 for hydroxyproline. Acid hydrolysis of the collagen thatwas performed to break the protein into individual amino acidsprior to HPLC separation seems to be the key step that may effec-tively break contaminants from the collagen that are then removedby chromatographic separation.

S. Nalawade-Chavan et al. / Quaternary Geochronology 20 (2014) 23e28 25

4.1. Correction algorithm for Hydroxyproline AMS dates

Thehydroxyprolinedates inTable 1 are correctedonly for routineprocedures such pre-treatment chemistry, combustion, and graph-itization, thus necessitating a correction algorithm for the carboncontamination derived from the chromatographic separation step.

The amount of total extraneous carbon added during HPLCseparation was estimated using an elemental analyzer (Carlo ErbaNA, 2000) linked to a Continuous Flow Isotope Ratio Mass Spec-trometer (CF IRMS Sercon 20e20). For this, 500 ml of mobile phasewas collected from a blank injection after passing through an HPLCcolumn and was found to be 0.022 mg/ml. The amount of carbonadded, alone by mobile phase was estimated in similar way butwithout using the HPLC column and was found to be 0.006 mg/ml.The extraneous carbon from mobile phase was believed to bemodern, as it originated from dissolved atmospheric CO2 and TOCof MilliQ" water. The difference between these two values,0.016 mg/ml, corresponds to the amount of carbon resulting fromcolumn bleed alone which is believed to be radiocarbon dead beingoriginated from hydrocarbon based fossil material. The dead andmodern carbon contamination in each hydroxyproline fractionwascalculated based on the volume of mobile phase collected, whichwas 120 ml for all the hydroxyproline dates reported.

For an uncontaminated sample:

Radiocarbon age;T # $8033 ln%F14C&

For a sample contaminated by extraneous old and moderncarbon:

Corrected radiocarbon age;TC # $8033 ln%F14C$ Cmod&=b

where,

F14C: Measured activityCmod: Modern contamination (mg)/Total carbon (mg)b # Ctotal%mg&$Modern C contamination%mg&$Dead C contamination%mg&

Ctotal%mg&

A combined error term, EComb, was calculated taking the pro-cedural error for old and modern carbon estimation into account.

where,

Ctotal # Graphite size (mg)Edead # Procedural error in estimation of dead carbon contam-ination (for n # 5)Emod # Procedural error in estimation of modern carboncontamination (for n # 5)EF14C # '1s error in determination of C14 activity

Table 2 illustrates the hydroxyproline radiocarbon dates cor-rected for the addition of the extraneous carbon added duringchromatographic separation. This correction algorithm was alsoapplied to the laboratory background age bone standard, an Alas-kan bison.

When the F14C is less than 2 sigma, which occurs when theradiocarbon error is >4000 14C years, radiocarbon dates are pre-sented as greater than ages (van der Plicht and Hogg, 2006), aspresented for few of the Alaskan Bison measurements.

4.2. Transform Infra-Red (FTIR) spectroscopy

Fourier Transform Infra-Red (FTIR) spectroscopy was used toidentify the presence of consolidants that may have been appliedduring curation and storage. FTIR spectra of mastodon bone spec-imens and extracted collagen (Specimen 1 and 2) were comparedwith those of a pig rib from the wreck of the Mary Rose (AD 1545),an ORAU in-house standard known not to have been subjected toconservation treatment (Bronk Ramsey et al., 2004). Only FTIRspectra of highly consolidated mastodon bone specimen, AMNH103227 are discussed in this paper.

The comparison of FTIR spectra of mastodon bone specimenwith that of Mary Rose Pig bone clearly suggested presence ofconsolidants (Fig. 2).

Bone IR absorbance spectra below wavenumbers w1200 cm$1

primarily reflect vibrational modes of phosphate and carbonategroups (Figueirado et al., 2012) and are not diagnostic of organicconstituents. At higher wavenumbers, several features of the FTIRspectrum from the conserved bone in Fig. 2 are consistent withreference spectra (FTIR spectrum database, University of Tartu,Estonia, Institute of Chemistry, http://tera.chem.ut.ee/IR_spectra)from a variety of natural oils e castor, linseed, tung, safflower, etc.Similarities include strong absorbance peaks at w2850 and2950 cm$1 relative to the unconserved pig bone, reduced absor-bance in the 3300e3500 cm$1 and 1500 cm$1 regions, and thepresence of additional absorbance bands near 1380, 1460 and1700 cm$1. The peaks in Fig. 2 near 2950 and 2850 cm$1, andespecially at 1700e1750 cm$1, are broader than those in thereference oil spectra, and none of the oils listed show the increasedabsorbance near 1250 cm$1 that is present in the mastodon bonespectrum. However, natural resins do exhibit broader peaks in thegroup near 2900 cm$1, plus absorbance peaks near 1250 and1740 cm$1, with the latter peak possibly contributing to the rela-tively broad 1700e1750 cm$1 feature. In addition, at least someresins (e.g., rosin, Manila copal) also show relatively low absor-bance in the 3000e3500 cm$1 region. Hence, if the conservationmaterial applied was a mixture containing at least one natural oil

Ecomb # (%F14C$ Cmod&=b)

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!" EF14C%F14C$ Cmod&

#2!"EmodCtotal

#2"1b$

1%F14C$ Cmod&

#2!"

Edeadb* Ctotal

#2s

Table 1Radiocarbon dates for mastodon specimens.

Specimen#(element)

Componentdated

14C BP Lab# Collagenyield (%)

C:N

UAMES 7663Craniuma

Ultrafiltered‘collagen’

33,090 ' 470 UCIAMS 88768 1.8 3.3

Ultrafiltered‘collagen’

20,440 ' 130 OxA-25401 3.7 3.4

Hydroxyproline 48,200 ' 2600 OxA-X-2492-15 13.1 5.1AMNH 103277

MandibleUltrafiltered‘collagen’

33,810 ' 460 UCIAMS 88772 3.9 3.5

Ultrafiltered‘collagen’

29,610 ' 340 OxA-25402 5.9 3.4

Hydroxyproline 47,100 ' 2500 OxA-X-2490-48 5.9b 4.7

Note: All radiocarbon dates are background-corrected for pre-treatment chemistry,combustion, and graphitization. Refer to Table 2 for hydroxyproline radiocarbondates corrected for HPLC background.

a Thick coating of varnish.b Ultrafiltered collagen excess used.

S. Nalawade-Chavan et al. / Quaternary Geochronology 20 (2014) 23e2826

and one natural resin, this could explain many of the observedfeatures of the FTIR spectrum, together with the anomalouslyyoung radiocarbon ages.

The FTIR spectra of the mastodon collagen were comparable tothat of Mary Rose pig bone collagen (Fig. 3). Note that the peak at2350 cm$1 in both the collagen and bone spectra is due to CO2, andthe absorption at this wavenumber depends critically on vacuumconditions within the FTIR spectrometer.

5. Discussion

The difference in the ultrafiltered ‘collagen’ radiocarbon datesfrom UCIAMS and Oxford for the same sample probably reflects theamount of contaminant in the samples sent to these two

laboratories and how well the respective techniques removed it.Oxford may well have had more contaminated samples as UCIAMSgot the samples first and the least contaminated-looking materialwould presumably have been chosen.

The comparison of FTIR spectra of the collagen from bothmastodon specimens with that of Mary Rose Pig bone collagenwould suggest that the consolidant had been removed, whichclearly was not the case as indicated by the higher C:N ratios andyounger radiocarbon dates. By contrast, the hydroxyproline de-terminations demonstrate that the ultrafiltered ‘collagen’ dates aresignificant underestimates due to inadequate removal contami-nants that are predominantly modern carbon in age. Both hy-droxyproline dates obtained for the mastodon specimens arestatistically indistinguishable from hydroxyproline dates for thew60,000e70,000 BP Alaskan bison bone standard (Brock et al.,2007). The two mastodon specimens, effectively radiocarbon-dead, gave similar hydroxyproline dates, 51,300 ' 3900 and48,800 ' 3000 BP, very close to the method background. Datingsamples this close to background is not only extremely challengingdue to extreme sensitivity to modern contaminants, but also sub-ject to statistical fluctuations. For example, if a sample of infiniteage is dated 20 times, it should produce 19 greater than ages, andone finite date. Therefore, it is extremely likely that the singleamino acid dates for the mastodon samples are minimum ages, andthat the samples date beyond the limit of the radiocarbon method.

The hydroxyproline determinations are corrected for extra-neous carbon added during preparative HPLC and are reported asgreater than ages. Dating samples close to the background limit ofradiocarbon dating is in any case very challenging, so it would besensible to view the hydroxyproline dates reported here as mini-mum ages, with the likelihood high that they are probably older.

6. Conclusions

This investigation highlights the importance of pretreatmentchemistry in reliable dating. The single amino acid (hydroxypro-line) radiocarbon dates for the two mastodon specimens understudy are close to 14C background levels, while bulk dates forcollagen from the same samples were much younger. Only thesingle amino acid approach is sufficiently reliable to date very oldsamples where even minuscule levels of modern contaminationcan distort radiocarbon dates significantly. Our results alsodemonstrate the efficacy of this technique for radiocarbon datingmuseum specimens that have been treated with glues and con-solidants for conservation purposes.

The radiocarbon dates generated in this particular case alsoclearly establish the reliability of the single amino acid approach ascompared to ultrafiltered ‘collagen’ radiocarbon dating. This in turnindicates that rigorous physical cleaning combined with singleamino acid dating should be looked upon as the method of choicefor dating archaeological and palaeontological samples that arepossibly or definitely consolidant-contaminated, especially in in-stances in which accurate dates are crucial but information oncontexts of specimen recovery is poor or unavailable.

Table 2Hydroxyproline AMS dates after correcting for the addition of extraneous carbon during chromatographic separation.

Sample ID 14C age BP('1s error)

F14C ' Graphitesize (mg)

Corrected F14C ' Corrected 14

C age BP ('1s error)Greater than age

Alaskan bison Hyp 1 48,000 ' 2800 0.00254 ' 0.00089 1.17 0.00193 ' 0.00089 50,200 ' 3700 e

Alaskan bison Hyp 2 47,700 ' 3900 0.00265 ' 0.00129 0.81 0.00177 ' 0.00129 50,900 ' 5900 >43,700Alaskan bison Hyp 3 47,800 ' 3700 0.00263 ' 0.00120 0.87 0.00181 ' 0.00120 50,700 ' 5300 >43,900Alaskan bison Hyp 4 46,000 ' 3500 0.00323 ' 0.00143 0.74 0.00227 ' 0.00144 48,900 ' 5100 >42,300UAMES 7663 Hyp 48,200 ' 2600 0.00249 ' 0.00081 0.88 0.00168 ' 0.00081 51,300 ' 3900 e

AMNH 103277 Hyp 47,100 ' 2500 0.00283 ' 0.00087 1.37 0.00231 ' 0.00087 48,800 ' 3000 e

Fig. 2. FTIR spectra of mastodon bone compared with FTIR spectra of ORAU in-housestandard bone.

Fig. 3. FTIR spectra of mastodon collagen compared with FTIR spectra of ORAU in-house standard collagen.

S. Nalawade-Chavan et al. / Quaternary Geochronology 20 (2014) 23e28 27

Accurate radiocarbon dating is essential to determine thechronology for the extinction of North Americas Pleistocenemegafauna, including the American mastodon. Further ongoingradiocarbon dating of fossil specimens from Alaska and adjacentNorthwest Canada will further resolve the timing of mastodoninhabitation and extirpation for this species in the high latitudes.

Acknowledgments

This work was funded by NERC grant NE/H/004491/1. C John-ston and A Crossley are thanked for permission to use, and assis-tance with, FTIR at the Department of Materials, University ofOxford & Begbroke Nano (supported by the DTI MNT CapitalProgramme).

Editorial handling by: R. Grun

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