Heavy Reliance on Plants for Romanian Cave Bears Evidenced by Amino Acid Nitrogen Isotope Analysis Yuichi I

Heavy Reliance on Plants for Romanian Cave Bears Evidenced by Amino Acid Nitrogen Isotope Analysis Yuichi I

www.nature.com/scientificreports OPEN Heavy reliance on plants for Romanian cave bears evidenced by amino acid nitrogen isotope analysis Yuichi I. Naito 1,2*, Ioana N. Meleg3*, Marius Robu 3, Marius Vlaicu3, Dorothée G. Drucker 4, Christoph Wißing1, Michael Hofreiter5, Axel Barlow 5,6 & Hervé Bocherens 1,4 Heavy reliance on plants is rare in Carnivora and mostly limited to relatively small species in subtropical settings. The feeding behaviors of extinct cave bears living during Pleistocene cold periods at middle latitudes have been intensely studied using various approaches including isotopic analyses of fossil collagen. In contrast to cave bears from all other regions in Europe, some individuals from Romania show exceptionally high δ15N values that might be indicative of meat consumption. Herbivory on plants with high δ15N values cannot be ruled out based on this method, however. Here we apply an approach using the δ15N values of individual amino acids from collagen that ofsets the baseline δ15N variation among environments. The analysis yielded strong signals of reliance on plants for Romanian cave bears based on the δ15N values of glutamate and phenylalanine. These results could suggest that the high variability in bulk collagen δ15N values observed among cave bears in Romania refects niche partitioning but in a general trophic context of herbivory. Bears represent the largest terrestrial members within the Carnivora alive today and the vast majority of them have carnivorous or omnivorous feeding habits. Until around 25,000 years ago, the coldest period in the Pleistocene, additional, now extinct bear species were living1–4, among which the so-called cave bears, a very large type of bear that formed the sister lineage of extant brown bears and polar bears (e.g., ref. 5). Te paradox of the cave bear is that their diet has been said to be herbivorous despite their large body sizes while extant herbivorous Carnivora species are smaller6,7. Afer their divergence from the brown bear lineage 1.2–1.6 million years ago, cave bear populations showed substantial morphological and genetic variability and multiple forms have been recognized8, although their taxonomic status and the relationships among them continue to be debated9. Te possible causes of the extinction of these bears are also intensively debated, involving climate change, human impacts, and (lack of) fexibility in feeding behavior10–15. Understanding cave bear feeding behavior is therefore important as it might give insights into the extinction of this species, and also it could be relevant for the conser- vation of extant, herbivorous carnivoran species that are under threat of extinction (e.g., binturong, red panda, giant panda16,17). More recent studies have shown mixed results based on diferent lines of evidence including anatomical prop- erties like craniodental morphologies, tooth wear analyses, mortality patterns (e.g., sex ratio), etc. wherein the conclusions were highly context dependent and difered by sample-sets18–23. Tis is also the case for stable carbon and nitrogen isotope analyses (δ13C and δ15N) on collagen extracted from bone/teeth24–31. Relatively low δ15N values of most of these bears so far indicate their highly-plant-dependent feeding habits with possible exceptions for some groups in today’s Romania that exhibited relatively high δ15N values30,32,33. Te δ15N value of bulk bone/ 1Department of Geosciences, Biogeology, University of Tübingen, Hölderlinstraße 12, 72074, Tübingen, Germany. 2Nagoya University Museum, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan. 3“Emil Racoviță” Institute of Speleology, Romanian Academy, Calea 13 Septembrie, nr. 13, 050711, Sector 5, Bucharest, Romania. 4Senckenberg Centre for Human Evolution and Palaeoenvironment (S-HEP), University of Tübingen, Hölderlinstraße 12, 72074, Tübingen, Germany. 5Institute for Biochemistry and Biology, Faculty for Mathematics and Natural Sciences, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476, Potsdam, OT Golm, Germany. 6School of Science and Technology, Nottingham Trent University, Clifton Lane, Nottingham, NG11 8NS, UK. *email: ynaito@ num.nagoya-u.ac.jp; [email protected] SCIENTIFIC REPORTS | (2020) 10:6612 | https://doi.org/10.1038/s41598-020-62990-0 1 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 1. Map showing locations of the cave sites in Romania mentioned in the text. teeth collagen is known to increase from prey to its consumer in a food web, thus being an important indicator to evaluate the trophic position of organisms34,35. Other factors also infuence δ15N values of collagen, such as varia- bility in the consumed plants and environmental factors including climate36–38, leading to possible spatiotemporal shifs of the isotopic baseline due to heterogeneities among local environments39–42. Hence, some herbivores may exhibit similar δ15N values as predatory species in the same context30,43–45. To solve this uncertainty, a new isotopic approach has been developed. Indeed, it has been demonstrated that δ15N values of individual amino acids (AA) can be classifed into several categories, “trophic” and “source” AAs that exhibit more 15N enrichment and less/little 15N enrichment, respectively, in each trophic step of food webs46–53. It is worth mentioning that the comparison of the “source” AAs like phenylalanine (Phe) with the “trophic” AAs like glutamate (Glx) enables the evaluation of animal trophic position regardless of baseline δ15N fuctuations in ecosystems54. We aim at testing the two hypotheses that high collagen δ15N values observed for cave bears in the Romanian region were attributed to (i) omnivorous/carnivorous feeding behavior associated with a trophic level efect as a corollary or (ii) consumption of plants with high bulk δ15N values. To this end, δ15N values of individual AAs in collagen were measured for adult cave bears from several sites in this region (Fig. 1). One cave lion and one horse have been analyzed together with the adult cave bear collagen to represent end-members for trophic positions for a terrestrial carnivore and a terrestrial herbivore in the Late Pleistocene. Results Judging from the C content (26.0–46.1%), N content (9.1–16.2%) and C/N atomic ratio (3.3–3.5), all the cave bear as well as the cave lion and horse bone collagen are well preserved (Table 1)55,56. Large variations in δ13C and δ15N values of bulk collagen for the cave bears were observed: −19.9 to −22.3‰ for δ13C and 5.2 to 9.8‰ for δ15N values, respectively (Fig. 2). Te bears sampled from each cave corresponded to diferent time horizons. Out of six cave bear samples, two have been molecular dated (USR10 from Măgura cave and USR67 from Răsufătoarei cave), and the other four have been radiocarbon dated (Table 1). Based on calibrated 14C age and the mean of the posterior samples for molecular dated samples, ages of the samples from Măgura fell within the interval 28–31 ka BP, while the oldest bears were from Cioclovina Uscată with 14C ages of c. 43 and 48 cal ka BP. Te samples from Răsufătoarei cover a wider time-span (20–49 ka BP), due to the wide confdence interval depicted based on molecular tip dating, given that USR67 is the only representative within its clade. In each cave and related timespan, both high (above 8‰) and lower δ15N values of bulk collagen have been recorded (see more details in Table 1). Strong correlations between the δ15N values of several AAs and those of bulk collagen were observed for the 2 investigated cave bears, among which phenylalanine showed the strongest one (R = 0.926, P < 0.01). Other AA 2 15 such as hydroxyproline (R = 0.704, P < 0.05) also showed strong correlations with bulk collagen δ N values. Te 15 15 scatterplot presenting δ NPhe vs. δ NGlx values shows that some cave bear individuals from the Romanian caves exhibited isotope values similar to those analyzed in previous studies for cave bears from Late Pleistocene Belgian sites, though three individuals with bulk collagen δ15N values above +8‰ analyzed in our study exhibited higher SCIENTIFIC REPORTS | (2020) 10:6612 | https://doi.org/10.1038/s41598-020-62990-0 2 www.nature.com/scientificreports/ www.nature.com/scientificreports Uncalibrated 14C Calibrated dates (95.4%) (cal Common name/ date/Molecular tip BC)/95% credibility interval Lab. C/N Lab. ID Species name Site date* (yrs BP) for molecular estimates* number d13C (‰) d15N (‰) %C %N atom USR10 Cave bear/− Măgura 30,866* 28,885-32,518* — −22.0 6.3 26.0 9.1 3.3 USR11 Cave bear/− Măgura 24,615 ± 101 28,392-28,891 ETH-87394 −22.1 8.1 36.8 12.3 3.5 USR22 Cave bear/− Cioclovina Uscată 40,567 ± 594 43,125-45,230 ETH-87395 −22.0 5.2 33.9 11.8 3.3 USR25 Cave bear/− Cioclovina Uscată 43,119 ± 807 44,990-48,401 ETH-87396 −22.3 8.9 36.7 12.9 3.3 USR65 Cave bear/− Răsufătoarei 31,005 ± 183 34,541-35,333 ETH-95736 −21.8 9.8 38.3 13.5 3.3 USR67 Cave bear/− Răsufătoarei 38,313* 19,932-49,692* — −19.9 5.4 46.1 16.2 3.3 Reference fauna Horse /Equus LOM 16 Lommersum −20.9 4.2 38.8 13.7 3.3 ferus Cave lion Leo 01 /Panthera North Sea 37110 ± 1070 −19.5 8.3 36.8 13.1 3.3 spelaea Table 1. Collagen samples for cave bears from the three Romanian caves and reference fauna analyzed in this study. a b 20 10 Mammoth(Belgium) ) 15 ) Mammoth(Crimea) (‰ ‰ ( Cave lion (Thisstudy) 8 x Gl 10 N Horse(This study) Collagen 15 N δ Goyetcave 15 δ 6 5 Scladina cave This study 4 0 -23-22 -21-20 -19 0510 15 20 13 15 δ CCollagen (‰) δ NPhe (‰) cd 10 10 ) ) ‰ ( (‰ n 8 2 8 2 ge R =0.642 agen R =0.926 a l l l l o Co C N N 5 1 15 δ 6 δ 6 4 4 810121416 810121416 15 15 δ NPhe (‰) δ NGlx (‰) Figure 2.

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