Reply to ``Comment on ``Ecological niche of

Journal of Human Evolution 117 (2018) 56e60

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Commentary

Reply to “Comment on “Ecological niche of Neanderthals from Spy Cave revealed by nitrogen isotopes of individual amino acids in collagen.” [J. Hum. Evol. 93 (2016) 82e90]” [J. Hum. Evol. 117 (2018) 53e55] e G. Drucker a, d, Naohiko Ohkouchi b, Yuichi I. Naito a, *, Yoshito Chikaraishi b, c, Dorothe e a Bocherens a, d Patrick Semal , Christoph Wißing , Herve €obiologie (Biogeologie), Universita €t Tübingen, Ho €lderlinstraße 12, 72074 Tübingen, Germany Fachbereich Geowissenschaften, Pala Department of Biogeochemistry, Japan Agency for Marine-Earth Science and Technology, 2-15 Natsushima-cho, Yokosuka 237-0061, Japan c Institute of Low Temperature Science, Hokkaido University, Kita-19, Nishi-8, Kita-ku, Sapporo 060-0819, Japan d €t Tübingen, Ho €lderlinstraße 12, 72074 Tübingen, Germany Senckenberg Centre for Human Evolution and Palaeoenvironment (HEP), Universita e Department of Paleontology, Royal Belgian Institute of Natural Sciences, 1000 Brussels, Belgium a

b

a r t i c l e i n f o Article history: Received 18 March 2017 Accepted 20 September 2017 Available online 10 October 2017

We thank O'Connell and Collins for their interest in our research and for their comments on our paper (Naito et al., 2016a). These comments deal essentially with four different aspects of our study, (1) the uncertainty of the estimation of the trophic position (TP), (2) the range of variation of the TP estimates for terrestrial herbivores, (3) the uncertainty in estimating the trophic discrimination factors (TDF) of humans, and (4) some possible discrepancies between the predicted and observed d15N values of some amino acids which are not used in the trophic reconstruction. While we appreciate their comments on the analytical aspects of our isotopic analysis, we believe that most of their arguments do not have a significant impact on our data or our conclusions, as discussed below. 1. Uncertainty in trophic position estimation O'Connell and Collins (2018) pointed out the uncertainty of up to ±0.4 for trophic position (TP) ¼ 3 (primary carnivores), mentioned in Chikaraishi et al. (2011): ±0.32e0.33 units in the case of the Spy DOIs of original article: https://doi.org/10.1016/j.jhevol.2016.01.009, https://doi. org/10.1016/j.jhevol.2017.05.006. * Corresponding author. E-mail address: [email protected] (Y.I. Naito). https://doi.org/10.1016/j.jhevol.2017.09.008 0047-2484/© 2017 Elsevier Ltd. All rights reserved.

Neanderthal specimens based on a newly estimated b value (the difference in d15N values between glutamate and phenylalanine in primary producers: see below) excluding outliers. This uncertainty corresponds to a theoretical value that takes into consideration the propagation of potential errors associated with each parameter, which can, in turn, overestimate for terrestrial animals. Indeed, animals like aphids and ladybugs, considered in Chikaraishi et al. (2011), as well as many herbivorous and carnivorous mammals from archaeological sites, reviewed in Naito et al. (2016b), exhibit reasonable TPs with ranges of 1.8e2.2 for herbivores and 2.8e3.2 for primary carnivores that are consistent with offsets of 0.17 units between measured TP values and expected TP for these animals (Chikaraishi et al., 2011). Extant omnivorous animals, on the other hand, such as wild boars, foxes, and Asiatic black bears and brown bears, show TPs between 2.0 and 3.2, which are expected based on the food habits of these species. In addition, well-controlled laboratory feeding experiments on animal consumers in terrestrial environments, including insects and mammals (Mus musculus) supplied with homogeneous, isotopically characterized diets, demonstrate reasonable TP estimations (Steffan et al., 2013, 2015). However, we acknowledge that the number and types of individuals in feeding experiments such as these are limited, and that those on organisms in marine environments demonstrate a large uncertainty in TP

Y.I. Naito et al. / Journal of Human Evolution 117 (2018) 56e60

estimation, probably due to the uncertainty of trophic discrimination factors (TDF) for several aquatic organisms (McMahon and McCarthy, 2016). Therefore, the core of the discussion here is which types of error to use for TP estimates (theoretically propagated error or accuracy [difference between measured TP and known TP]). Considering that TP estimation has been shown to be accurate for most terrestrial herbivores (TP ¼ 2) and primary carnivores (TP ¼ 3) from a variety of contexts, we do not find the error for TP to be critically large in such contexts. Nevertheless, studies of ancient humans should include comparisons with the animals associated with them to check the validity of TP estimation in this context. This strategy is recommended for and has been done on isotope analyses on bulk collagen as well (e.g., Hedges and Reynard, 2007; Bocherens et al., 2014; Bocherens, 2015). With such precautions and the acknowledgement that the use of amino acid d15N values for paleodietary reconstruction in the late

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Pleistocene is a young field that requires methodological sophistication, we maintain our claim that d15N analysis on individual amino acids can provide important insights into hominin paleoecology, as we have done for Spy Neanderthals (Naito et al., 2016a). 2. Range of TP estimates in terrestrial herbivores O'Connell and Collins' (2018) assertion that TP estimates for the Spy Neanderthals can span between 2.1 and 3.3 is neither robust nor relevant to our study since published b values seem to include less reliable data as well as data that are not realistic for habitats such as those of fossil hominins in the Pleistocene. While b values for terrestrial plants do show variability that has also been recognised by the group in JAMSTEC, most of the data converge on a value around 8‰ (Fig. 1). The cited b values of 3.3‰ and 12.1‰ are based on studies of crops collected from farms with supplements of fertilizer or manure that are nutritionally very different

Figure 1. Histograms showing b values (Dd15NGlx-Phe: d15NGlx e d15NPhe) for terrestrial plants (shown as negative values for convenience). A) All data, BeD) histograms by artificial/ wild that include the category and literature. Data are from the following sources: Chikaraishi et al. (2010a, 2011, 2014); Steffan et al. (2013); Styring et al. (2014); Paolini et al. (2015); Takizawa et al. (2017).

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from conditions for plants in past hominin habitats (Styring et al., 2014; Paolini et al., 2015). Other b values deviating from 8‰ have also been reported for some tree flowers (Takizawa et al., 2017), although it is unlikely that these plant tissues are/were frequently consumed by wild herbivorous mammals. Therefore, those b values strongly deviating from 8‰ should be considered extreme cases, justifying the use of a value of ~8‰ as a representative value for terrestrial plants in human paleodietary studies. The reasonable TP estimates (1.8e2.2) obtained for herbivores from the Pleistocene Belgian cave sites of Spy and Scladina, while using a b value of ~8‰, support this fact (Naito et al., 2016a). In addition, it should be mentioned here that the published data for TDFs, b values, etc. may not be entirely accurate. For example, derivatization techniques for amino acids require great care to produce reliable d15N data (Ohkouchi et al., in press). These techniques, which produce derivatives such as trifluoroacyl-isopropyl ester and pivaloyl-isopropyl ester, have been widely adopted in ecological and geochemical studies, and by JAMSTEC, and they have been highly replicable for original d15N values of amino acids (Metges et al., 1996; Metges and Petzke, 1997; McClelland and Montoya, 2002; Chikaraishi et al., 2007, 2010b; see also Fig. 2). However, they have not all been examined for their reliability. Future work should tackle these technical issues to allow safe comparison of published data.

thus applying a value obtained via animal feeding experiments is a realistic approach that has been applied in many previous dietary isotope studies. According to O'Connell and Collins (2018), our interpretation of the results for the Neanderthal diet based on TPs of 2.7e2.8 for three bone samples is “premature”. We agree that more work is required to clarify and constrain the variability of the relevant parameters, especially for mammals. In addition, it is also necessary to examine whether the consumed fraction of animal (or plant) protein in the diet is linearly and predictably reflected in the d15N values of consumer body tissues as well as their TPs (Webb et al., 2016). Nevertheless, similar TPs (2.7e2.8) have been observed for Holocene Jomon populations, in the Japanese archipelago, who are well-known for their use of terrestrial plants, such as nuts (Imamura, 1996; Habu, 2004; Crawford, 2011; Naito et al., 2013a, 2016b), and for modern humans with vegetarian diets (Ohkouchi et al., 2014). Despite the uncertainty and preliminary aspect in our approach, this evidence, in addition to direct evidence of plant remains trapped in tooth calculus of Spy Neanderthals, demonstrates a dietary habit that could include plants on a regular basis (Henry et al., 2011). Our attempts to produce quantitative results are still preliminary and should be confirmed by further studies. 4. Discrepancy in d15N values of proline and hydroxyproline in collagen

3. Uncertainty in estimating the TDF of humans We recognise that there is uncertainty associated with our estimate of “up to ~20%” protein contribution from plants for Spy Neanderthals (Naito et al., 2016a) and we therefore appreciate O'Connell and Collins' (2018) critique of this estimate. Such uncertainty may be derived from trueness and variability in the b value of terrestrial plants, as discussed above, and those of TDFs for humans. Directly estimating TDFs for humans is a challenge, and

Observed discrepancy in d15N values of proline and hydroxyproline in collagen analysed in JAMSTEC is neither essential nor significant for our study on Spy Neanderthals. First, these two amino acids were not used for TP estimation. Secondly, analytical conditions in JASMTEC for glutamate and phenylalanine used for the TP estimation, as well as other amino acids, have been quite robust in terms of baseline resolution on GC chromatogram and reproducibility of original d15N values for derived amino acids

Figure 2. An example of d15N measurements of individual amino acids in JAMSTEC. A) A regression line between known (‰, vs. AIR) and measured values (‰, vs. reference N2 gas) for 10 amino acid standards measured together with samples from Spy and Scladina caves (precision ¼ 0.3e0.6‰ [1s] when these samples were measured. Error bars are not shown for each amino acid standard because they hide behind symbols. The proline standard was not measured in the study (though the same quality of measurements of this amino acid has been achieved in JASMTEC). Note that our measurement quality is comparable to that reported in Styring et al. (2012) (slope [y ¼ 0.93x þ 0.25], r2 ¼ 0.996, precision ¼ better than 0.8‰ [1s]). B) Gas chromatogram (m/z 28 ion current) displaying the separation of collagen amino acids from a Spy Neanderthal specimen (SPY-94a), measured on a HP-Ultra 2 capillary column (50 m 0.32 mm internal diameter; 0.52 mm film thickness). Note that alanine and glycine (retention time: 1530e1720 s in the figure) do not appear on the chromatogram because they are measured separately from other amino acids because of the large peak size of glycine in collagen.


based on appropriate temperature settings for oxidation/reduction, carrier gas flow rate, etc. (see Fig. 2: e.g., Chikaraishi et al., 2010b). Further, it has been shown that these conditions are at least as good as those used in Bristol (Styring et al., 2012). Therefore, measurement problems are very unlikely to be involved in the discrepancy. The reason for the isotopic difference between proline and hydroxyproline is still unclear, but it might, for example, relate to site taphonomy for archaeological samples or animal phylogeny (Szpak, 2011). The collagen samples reported by Styring et al. (2010) that O'Connell and Collins (2018) likely use in their figure 1 are from Holocene samples from South Africa and are therefore younger than those from the sites studied in Naito et al. (2010a, 2010b, 2013a, 2013b, 2016a, 2016b, 2016c) and Itahashi et al. (2014), except for sites associated with the Okhotsk culture in Naito et al. (2010a). This is especially true for the Belgian site Spy, which dates to approximately 40,000 years ago (Semal et al., 2009). O'Connell and Collins' (2018) claim that no significant isotopic difference can be assumed between these two amino acids as hydroxyproline in collagen is formed from proline via the posttranslational modification, but how the metabolisms of hydroxyproline and proline in collagen affects their nitrogen isotopic composition is not well understood: e.g., the influence of catabolic processes on bone collagen (Murphy and Nagase, 2008). Because comparisons with modern terrestrial animals show that TP estimations based on d15N values of glutamate and phenylalanine (not taking into account d15N values of proline or hydroxyproline) correctly evaluate the trophic position of most of the fossil terrestrial animal remains, we are confident that our approach provides robust results and conclusions. More generally, relying on predictable patterns between trophic categories based on modern equivalents to evaluate the preservation of these proxies for unknown specimens is a common strategy used in various other approaches; using isotopic proxies from fossil material, for instance for carbon and oxygen isotopic values in the carbonate fraction of bioapatite (e.g., Lee-Thorp et al., 1989; Bocherens et al., 1996, 2011; Lee-Thorp and Sponheimer, 2003). Therefore, we believe that the approach using glutamate and phenylalanine is appropriate for current studies on fossil hominins as long as appropriate quantitative errors are recognized. However, approaches using multiple amino acid d15N values including proline for TP estimation should be used with caution, due to the open questions about the d15N values of other amino acids and the fact that its robustness has not yet been demonstrated. Further, we assert that the proline-hydroxyproline discrepancy does not undermine the validity of our Neanderthal paleoecological and paleodietary reconstruction, even if some future work on this new isotopic method may refine these reconstructions. Acknowledgements We are thankful to Gillian Wong for her thorough English language editing. References Bocherens, H., 2015. Isotopic tracking of large carnivore palaeoecology in the mammoth steppe. Quaternary Science Reviews 117, 42e71. Bocherens, H., Koch, P.L., Mariotti, A., Geraads, D., Jaeger, J.-J., 1996. Isotopic biogeochemistry (13C, 18O) of mammal enamel from African Pleistocene hominid sites: implications for the preservation of paleoclimatic isotopic signals. PALAIOS 11, 306e318. Bocherens, H., Sandrock, O., Kullmer, O., Schrenk, F., 2011. Hominin palaeoecology in Late Pliocene Malawi: insights from isotopes (13C, 18O) in mammal teeth. South African Journal of Science 107, 95e100. Bocherens, H., Drucker, D.G., Madelaine, S., 2014. Evidence for a 15N positive excursion in terrestrial foodwebs at the middle to upper Palaeolithic transition in south-western France: implication for early modern human palaeodiet and palaeoenvironment. Journal of Human Evolution 69, 31e43.

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