Ancient bones — how old?

22 01 2021

Radiocarbon (14C) dating was developed by Nobel-Prize winning chemist Willard Libby, and has become the predominant method to build chronologies of ancient populations and species using the Quaternary fossil record. I have just published a research paper about 14C dating of fossil bone reviewing the four standard chemical pretreatments of bone collagen to avoid sample contamination and generate accurate fossil ages: gelatinization, ultrafiltration, XAD purification and hydroxyproline isolation. Hydroxyproline isolation is perceived as the most accurate pretreatment in a questionnaire survey completed by 132 experts from 25 countries, but remains costly, time-consuming and not widely available. I argue that (1) innovation is urgently required to develop affordable analytical chemistry to date low-mass samples of collagen amino acids, (2) those developments should be overseen by a certification agency, and (3) 14C users should be more conceptually involved in how (much) 14C chemistry determines dating accuracy. Across the board, scientific controversies like the timing of Quaternary extinctions need not be fuelled by inaccurate chronological data.


Megafauna bones from the Quaternary fossil record. Top: excavation of a partial skeleton of a short-faced kangaroo Procoptodon browneorum at Tight Entrance Cave (Western Australia) [1]: these bones are close to the limit of radiocarbon (14C) dating in a geological context 43000 to 49000 years old. Middle: metacarpal of the extinct horse Hippidion cf. devillei from Casa del Diablo (Peru) 14C dated at 11980 ± 100 years before present (BP) (CAMS-175039) following XAD purification of collagen gelatin [2]. Bottom: collection of skeletal remains of (mostly) red deer Cervus elaphus from El Cierro Cave (Spain) 14C dated at 15520 ± 75 years BP on ultrafiltered gelatin (OxA-27869 and OxA-27870 average) [3].


Scientists have widely been interested in the present and future state of biodiversity. Ecologists (the main audience of this blog) have also looked into the past with pioneering investigations addressing the composition of ancient forests and the origins of agriculture in layers of fossil pollen accumulated in lake sediments [4]. But it took us decades to see the fossil record as a useful tool (combining biological, geochemical and molecular techniques) to answer basic ecological questions. Some of those questions are critical for conserving today’s biodiversity [5, 6]: for example, when did human impacts on ecosystems become global or what extinct species have best tolerated past environmental change and what that means to modern species? [7].

The study of (pre)historic biological events relies one way or another on our ability to time when a certain animal, human, or plant occurred and what environmental conditions they experienced, and relies on concepts borrowed from archaeology (past human activity), palaeontology (fossils), palaeocology (species responses to past environments), and geochronology (age of fossils and/or their geological context). Among the range of chronological methods available to date biological and cultural samples [8], radiocarbon (14C) dating has become the most important for dating bones aged modern to late Quaternary (last ~ 50,000 years). Hereafter, ‘bone’ comprises antler, bone, ivory and teeth. 14C dating of bones is appealing at least for four reasons: 

  1. Conceptually simple (see video here): the more 14C found in a bone, the younger the bone. Counting 14C atoms by accelerator mass spectrometry (AMS, see the guts of an accelerator here) has outcompeted the original method of counting beta particles (measuring radioactivity, still used by some 14C laboratories) conceived by Willard Libby.
  2. Low-mass samples: we can date bone samples below 1 g, a crucial factor for a fragmented fossil record frequently represented by a single, incomplete bone per specimen (see video on how bones fossilise).
  3. Direct: if one is interested in timing when a species existed, directly dating a bone will often be more accurate than indirectly dating the surrounding soils and sediments, because fossils are frequently found outside the geological context of the death of the individual to which the bones belonged.
  4. Universal: more than 150 AMS facilities are doing 14C dating throughout the world, so the method is widely available wherever scientists do their work.

Most ages of fossil bone generated at modern AMS 14C laboratories are estimated from collagen (a fibrillary protein with three strands of amino acids twisted together like a rope). However, fossil collagen is not chemically inert but incorporates carbon from the environment (soil, sediment, rain, ground water) [9, 10]. AMS accelerators cannot differentiate 14C atoms belonging to a fossil bone from 14C atoms that bone has taken up from the environment. Imagine we are counting white beans in a plastic bag: we cannot tell whether different beans came from different plants, orchards, regions or countries; they are just beans as much as 14C atoms in a fossil bone are just 14C atoms. Contamination with foreign 14C can cause errors in the estimation of the geological age of a bone by tens to thousands of years, potentially distorting downstream inferences like what climatic conditions (measured through a palaeoclimate archive) align with a species’ extinction time (quantified from a time series of 14C dates). Therefore, prior to dating, collagen is subjected to four major purification steps — collectively known as ‘pretreatment’, which I summarise in the following panel.


increasing accuracy, complexity, cost & turnaround; decreasing datable mass/sample

pretreatment gelatinisation ultrafiltration XAD purification hydroxyproline
isolation
chemistry removal of mineral (apatite) in cold acid, then heating in slightly acidic water resulting in collagen gelatin gelatin filtered through a 30 kDalton (kDa) membrane gelatin broken into free amino acids in strong acid & passed through a non-polar XAD resin that binds to contaminants gelatin broken into free amino acids in acid & passed through a chromatographic column isolating hydroxyproline by its structure
dated fraction gelatin gelatin molecules > 30 kDa all 18 amino acids amino acid hydroxyproline
rationale apatite more prone to uptake carbon contaminants than collagen large molecules are collagen; small molecules are contaminants amino acids are free of contaminants; collagen is not hydroxyproline is specific to bone

Chemistry of collagen radiocarbon (14C) dating using accelerator mass spectrometry (AMS). The ultimate goal is to count the collagen-specific 14C atoms remaining in a bone sample (converted to gas or graphite) after removing the 14C atoms that the bone has incorporated from the environment since the individual (animal, human) died. What pretreatment to use balances (1) cost, (2) purification efficiency, (3) turnaround time, (4) geological provenance, (5) the true geological age of the fossil, (6) the size (mass) of the sample, and (7) how the bone was handled from sampling to storage. Examples of two extreme samples from low to high contamination (and collagen degradation) could be a Holocene-age bone sampled yesterday in southernmost Patagonia versus a Pleistocene-age bone from the banks of the river Guadalquivir curated for fifty years in a museum.


Robert Longin
Gelatinization is the minimum, if not final, pretreatment offered by the vast majority of laboratories.
It mimics an ancient culinary trick: slow simmering of bone in hot water dissolves collagen and produces a nutritious bone broth (gelatin). 
Adapted for 14C dating by Robert Longin as part of his PhD thesis at the University of Lyon (France) and published in Nature in 1971 [11].
Erle Nelson
Ultrafiltration is the most popular pretreatment of collagen gelatin these days. 
Developed in the 1980s by Erle Nelson at Simon Fraser University (Canada) [12], used by several AMS labs in North America since the 1990s, and introduced to Europe through Mike Richards’ PhD and first postdoc at Oxford University.
The Oxford Radiocarbon Accelerator Unit (ORAU) adopted ultrafiltration in June 2000, and other European labs followed at least 6 years later.
Thomas Stafford, Jr.
XAD purification uses non-polar resins that specifically bind to contaminants after the collagen gelatine has been hydrolysed to free amino acids. 
Developed by Tom Stafford in the 1980s [13, 15], it features in the menu of 14C services offered by several North American AMS laboratories.
The method has surprisingly not reached European facilities.
Richard (Dizzy) Gillespie
Dating hydroxyproline was hinted at in the 1960s [16] but Dizzy Gillespie was the first author to publish 14C dates using hydroxyproline isolation in 1984 [14].
This pretreatment uses liquid chromatography to isolate individual amino acids (hydrolysed from collagen) by their structure.
Hydroxyproline is an imino (or secondary) amino acid that animals produce in vivo (metabolically, not from the diet).

Authors making key collagen-chemistry developments for AMS radiocarbon (14C) dating. Gelatinisation alone and ultrafiltration will be safe for dating bone from cold climates and depositional environments with weak soil formation like caves but for the wrong reason; that is, absence or low occurrence of the greatest single source of 14C contamination: humic/fulvic acids. These compounds originate from the modification of plant matter, and form chemical bonds (cross-links) with collagen that are not broken by gelatinisation or ultrafiltration. Humates act on a collagen fibril like the tanning of a strip of leather, once the tanning touches the leather at one point, it spreads over the whole strip. This so-called Maillard reaction (honouring Louis Maillard) takes place daily in everyone’s lives as it is what gives a loaf of bread or your favourite biscuit its brown colour.


Questionnaire survey

I have spent > 3 years collating 14C dates from the scientific literature almost daily [e.g., 17]. Throughout, I have discovered that bone pretreatment is often not reported by authors publishing 14C dates. Finding this information becomes a detective task that I will discuss in a future post. My focus here is that (i) if different 14C laboratories offer different pretreatments, and (ii) if different pretreatments (sometimes the same pretreatment run by different laboratories) can produce different 14C ages of the same bone, and (iii) given that there are no universal guidelines about what pretreatment should be best for what sample, then neglecting the chemistry of how a 14C date has been generated eludes a critical criterion to rank the quality of 14C data. We currently lack a comprehensive assessment and quantification of the magnitude of this problem in published research.

With that concern in mind, I conceived my study. I asked 132 researchers, who use, generate and/or publish 14C dates of human and megafauna bones, to answer 12 questions about their preferences and concerns about the four most widespread pretreatments of collagen leading to AMS 14C dating (gelatinisation optionally followed by ultrafiltration, XAD purification or hydroxyproline isolation). I wanted to know the degree of consensus among top researchers in their fields (particularly archaeologists, palaeontologists and geochronologists), working at world-class research sites and/or AMS laboratories worldwide (25 countries). The research paper has been published open-access here, including main text (pdf), questionnaire design (html) + respondents’ raw responses (xlsx)+ supplementary figures and tables (pdf). A talk I gave in February 2020 in the Flinders University Ecology and Evolution Seminar Series about this work is available here.

The questionnaire survey revealed that, for dating bone suspected to be severely contaminated with foreign carbon, hydroxyproline isolation is the preferred chemical option (39% of the respondents), followed by ultrafiltration (23%), XAD purification (9%), and gelatinisation alone (<5%). 23% of the audience was unsure of the reliability of what pretreatment to rank first. Before submitting bone samples to a 14C laboratory, both contamination and the international prestige of 14C laboratories are the top-ranked criteria respondents ponder to choose pretreatment. And, one in every two respondents who submit bone samples for dating to a 14C laboratory do not choose pretreatment themselves but ask or let the 14C laboratory select pretreatment for them.


Products of bone chemical pretreatment for radiocarbon (14C) dating. Top: Rib fragment of a Mexican horse Equus conversidens from Wally’s Beach (Canada) before pretreatment. Bottom-left: collagen extracted with an alkali (potassium hydroxide, KOH) after decalcifying the bone, followed by acidification and freeze-drying (3 days of work in total), giving an age of 13540 ± 40 years before present (BP) (UCIAMS-127363) [18]. Bottom-right: gelatin obtained by heating KOH-extracted collagen in pH 2 HCl (weak acidity) at 90 °C for 1 hour giving an age of 11,495 ± 30 years BP (UCIAMS-127364) [18]. Purified gelatin gave ages of 11685 ± 50 years BP by ultrafiltration after a solvent rinse (OxA-37337), 11440 ± 30 years BP by XAD purification (UCIAMS-127351) and 11445 ± 55 years BP by hydroxyproline isolation (OxA-X-2736-10) [19]. Significance: conservation glue (Butvar) makes the geological age of KOH-extracted collagen ~ 2 millennia older than truth until its gelatin has been extracted, and humic contamination likely makes the ultrafiltered gelatin ~ 2 centuries older than dating all collagen amino acids (XAD) or only hydroxyproline.


In my paper, I complemented the questionnaire survey with a literature review of bone-contamination issues for 14C dating. I propose the following three areas of future development:

  1. Universal standards: A certification agency seems much needed to oversee quality control across laboratories. The overarching principles of such an agency could be to enforce procedural consistency across 14C laboratories, to promote a network of research sites specialising on specific aspects of dating (e.g., types of samples like bone, charcoal, shell, etc.), and to supervise and speed up the transfer of chemistry developments from research to application at 14C laboratories. 
  2. Affordable accuracy: Molecular-level (primary and secondary amino acids) dating is expensive (costly reagents, equipment, highly skilled personnel) and not widely available. We need to make current protocols cheaper, and/or to develop alternative, affordable chemical protocols to isolate and date amino acids. Genomics illustrate that innovation cheapens technology and cheaper technology triggers innovation, so sequencing a genome has plummeted from US$100M to US$1K in 20 years. 
  3. Collective effort: Researchers must be more conceptually involved in how 14C data are generated, and journal Editors should not accept research manuscripts using 14C data without clear and replicable data-quality filters (including sample pretreatment). Provoking paper titles like “big data little help in megafauna mysteries” with regard to Quaternary extinctions [20] convey the message that data quality is as important as the most sophisticated modelling to make global inferences about past biological events.

During peer review

Before my successful submission to Royal Society Open Science (RSOS), my manuscript was rejected at the Editorial level three times for the following reasons: (i) “your manuscript falls outside the scope of this journal” (Scientific Reports, decision date = 20/07/2020), (ii) “not convinced that the findings presented have the potential significance for publication in eLife” (28/07/2020), and (iii) “not feel that your study represents the strength of advance that we require for publication in PLoS Biology” (29/05/2020). Of course, I hold no grudge against those decisions — rejections are normal outcomes in a scientist’s work. But the above rationale of rejection contradicts the facts that (i) these journals publish research built on 14C dates and (ii14C dating is propelling advances in multiple scientific disciplines [21]. After all: what can be more important for scientific research than finessing data quality?

One of the two RSOS reviewers pondered that “I worry that it [my article] will scare potential users of radiocarbon labs into never attempting to radiocarbon date bone” (RSOS has made reviewers’ comments and my responses to them available here). I conclude this blog with my answer to that statement:

The overarching goal of my manuscript is to promote improvements rather than discrediting the field of 14C dating. In that direction, I have now emphasised the critical (current/future) role of 14C dating in scientific research in the concluding section of my article: 

14C dating has meritoriously established itself as one of the most powerful tools for dating cultural and palaeontological deposits from the late Quaternary [22, 23]. The method is conceptually simple and well understood. Along with its prominence in the Quaternary sciences, its importance in modern research has been, and will be even more, heightened by the growing application of palaeoarchives and fossil materials to understand ongoing global ecosystem shifts and anthropogenic impacts on biodiversity and the environment [5, 6].”


Acknowledgements

My paper was accepted for publication last December 2020, only days before moving from Australia to Spain. I thank my dear colleagues at the Australian Centre for Ancient DNA (ACAD) for the inspiring exchanges we have shared in the last three years. I also appreciate the supreme feedback provided by Matthew Collins, Dizzy Gillespie, and Tom Stafford during the conception and writing of the paper.

I thank the 132 authors who participated in the questionnaire survey, and Alex Bayliss, Corey Bradshaw, Tom Higham, Greg McDonald, Kieren Mitchell, Paula Reimer, Mike Richards, John Southon and Chris Turney for their generous input at different stages of conception and completion of my study. Photographs kindly provided by Esteban Álvarez-Fernández (Cervus), Gavin Prideaux (Procoptodon), Tom Stafford (Equus), Natalia Villavicencio (Hippidion), Christine Oberlin (Robert Longin) and Mike Richards (Erle Nelson). My salary was funded by Australian Research Council Discovery Project DP170104665, and publication fees were paid by the School of Biological Sciences (University of Adelaide, Australia).

Salvador Herrando-Pérez

References

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[2] Villavicencio, Werdelin 2018 The Casa del Diablo cave (Puno, Peru) and the late Pleistocene demise of megafauna in the Andean Altiplano. Quaternary Science Reviews 195: 21-31 doi:10.1016/j.quascirev.2018.07.013

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[7] Seddon et al. 2014 Looking forward through the past: identification of 50 priority research questions in palaeoecology. Journal of Ecology 102: 256-267 doi:10.1111/1365-2745.12195

[8] Walker 2005 Quaternary dating methods. Chichester, West Sussex, England: Wiley.

[9] Collins et al2002 The survival of organic matter in bone: a review. Archaeometry 44: 383-394 doi:10.1111/1475-4754.t01-1-00071

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[12] Brown et al1988 Improved collagen extraction by modified Longin method. Radiocarbon 30: 171-177 doi:10.1017/S0033822200044118

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[14] Gillespie et al1984 Radiocarbon dating of bone by accelerator mass spectrometry. Journal of Archaeological Science 11: 165-170 doi:10.1016/0305-4403(84)90051-7

[15] Stafford et al1987 Study of bone radiocarbon dating accuracy at the University of Arizona NSF Accelerator Facility for radioisotope analysis. Radiocarbon 29: 24-44 doi:10.1017/S0033822200043538

[16] Ho et al1969 Radiocarbon dating of petroleum-impregnated bone from tar pits at Rancho La Brea, California. Science 164: 1051-1052 doi:10.1126/science.164.3883.1051

[17] Rodríguez-Rey et al2016 A comprehensive database of quality-rated fossil ages for Sahul’s Quaternary vertebrates. Scientific Data 3: 160053 doi:10.1038/sdata.2016.53

[18] Waters et al2015 Late Pleistocene horse and camel hunting at the southern margin of the ice-free corridor: Reassessing the age of Wally’s Beach, Canada. Proceedings of the National Academy of Sciences 112: 4263-4267 doi:10.1073/pnas.1420650112

[19] Devièse et al2018 Increasing accuracy for the radiocarbon dating of sites occupied by the first Americans. Quaternary Science Reviews 198: 171-180 doi:10.1016/j.quascirev.2018.08.023

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[23] Taylor, Bar-Yosef 2016 Radiocarbon dating. An archaeological perspective. New York, USA: Routledge.


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