Incorporation of animal-based foods into early hominin diets has been hypothesized as a major catalyst of important evolutionary events, including brain expansion. However, direct evidence of the onset and evolution of animal resource consumption in hominins remains elusive. The nitrogen-15-to-nitrogen-14 ratio of collagen provides trophic information about individuals in modern and geologically recent (<200,000 year) ecosystems, but diagenetic loss of this organic matter precludes studies of greater age. In contrast, nitrogen within tooth enamel is preserved for millions of years. Here, we report enamel-bound organic nitrogen and carbonate carbon isotope measurements of Sterkfontein Member 4 mammalian fauna, including seven Australopithecus specimens. Our results suggest a variable but plant-based diet (largely C₃) for these hominins. Therefore, we argue that Australopithecus at Sterkfontein did not engage in regular mammalian meat consumption.

All data are available in the main text or the supplementary materials. Data was analyzed using the Stable Isotopes Bayesian Ellipses (SIBER) package (version 2.1.) and the Turner et al. (2010) statistical code. The R code is included in the Supplementary Materials. All specimens are housed at the Evolutionary Study Institute (ESI) at the University of Witwatersrand (Johannesburg, South Africa) under the curatorship of Dr. Zipfel. All specimens are accessible to researchers following a fossil access application process, which is also managed by Dr. Zipfel.

Tooth enamel from 43 specimens from Pliocene Sterkfontein Member 4 mammalian fauna was analyzed for stable carbon (δ¹³C_enamel) and nitrogen (δ¹⁵N_enamel) isotopes (Table S1). Analyzed taxa include Australopithecus sp. (n = 7), two non-hominin primates Papionini (n = 8) and Cercopithecoides (n = 1), bovids (n = 12; including browsing (n = 11) and grazing (n = 1) taxa), felids (n = 4), canids (n = 5) and hyenids (n = 6). For information on location (square and depth within the Sterkfontein deposits), excavation details, and authentication see Table S1, for discussion of ages see SM.

Tooth enamel was drilled on site at the ESI using a handheld Dremel with a 0.7 or 0.9 mm diamond ball head (<3,000 RPM). Sampling was conducted carefully to minimize damage to diagnostic morphological features. Australopithecus sp. molars from the 1966 to 1996 excavations were sampled and specimens which were incomplete or already damaged (e.g., cut in half for previous studies) were preferentially chosen.

Bulk enamel samples (5 to 15 mg) were taken longitudinally, between the cervix and cusp of the tooth, avoiding the underlying dentin. The surficial enamel was discarded to avoid contamination with any adherent sediment and the remaining enamel was homogenized. In general, bulk enamel samples integrate approximately three months to one year of dietary signal, depending on taxon, tooth type and size, and enamel thickness. Images of the seven Australopithecus specimens after sampling are shown in Fig. S7. The last forming tooth was targeted for analysis whenever possible, usually representing adult, post-weaning diet, see section “Tooth Formation” in the SM.

δ¹⁵N_enamel of mineral-bound nitrogen in tooth enamel was analyzed with the “oxidation-denitrification method”, following the protocol published in Leichliter et al. (2021) and Moretti et al. (2023). An aliquot of the same sample powder was analyzed for stable carbon isotope analyses with the “cold trap method” after Vonhof et al. (2020). All measurements were performed at the Max Planck Institute for Chemistry (MPIC) in Mainz, Germany. For details see SM.

Isotope ratios are reported on the international scales of Vienna Pee Dee Belemnite (VPDB; δ¹³C) or AIR (δ¹⁵N), where δ¹³C or δ¹⁵N (in ‰) = (R_sample/R_standard-1) x 1,000, and R_sample and R_standard are ratios of heavy to light isotopes of the sample and the standard, respectively.

References: 

J. N. Leichliter et al., Nitrogen isotopes in tooth enamel record diet and trophic level enrichment: results from a controlled feeding experiment. Chemical Geology 563, 120047 (2021).

T. Lüdecke et al., Carbon, nitrogen, and oxygen stable isotopes in modern tooth enamel: A case study from Gorongosa National Park, central Mozambique. Frontiers in Ecology and Evolution 10,  (2022).

S. Moretti et al., Analytical improvements and assessment of long-term performance of the oxidation–denitrifier method. Rapid Communications in Mass Spectrometry 38, e9650 (2024).

T. F. Turner, M. L. Collyer, T. J. Krabbenhoft, A general hypothesis-testing framework for stable isotope ratios in ecological studies. Ecology 91, 2227-2233 (2010).

H. Vonhof et al., High‐precision stable isotope analysis of <5 μg CaCO₃ samples by continuous‐flow mass spectrometry. Rapid Communications in Mass Spectrometry 34, e8878 (2020).