RESEARCH ARTICLE | ANTHROPOLOGY Dental data challenge the ubiquitous presence of Homo in the Cradle of Humankind ment Zanollia,1 , Thomas W. Daviesb,c, Renaud Joannes-Boyaud,e , Ame Cle lie Beaudetf,g,h , Laurent Bruxellesg,i,j , Frikkie de Beerk,l , Jakobus Hoffmank, Jean-Jacques Hublinb , Kudakwashe Jakatam , Lazarus Kgasie,n , Ottmar Kullmero,p , Roberto Macchiarelliq,r, Lei Pans,t,2, Friedemann Schrenko,p , Fre de ric Santosa , Dominic Stratfordg, Mirriam Tawanen, Francis Thackeraym , Song Xings,t, Bernhard Zipfelm , and b,c,u Matthew M. Skinner Edited by Lucas Delezene, University of Arkansas Fayetteville, Fayetteville, AR; received June 17, 2021; accepted April 13, 2022 by Editorial Board Member Richard G. Klein The origins of Homo, as well as the diversity and biogeographic distribution of early Homo species, remain critical outstanding issues in paleoanthropology. Debates about Significance the recognition of early Homo, first appearance dates, and taxonomic diversity within Homo are particularly important for determining the role that southern African taxa Identifying the earliest members may have played in the origins of the genus. The correct identification of Homo remains of the genus Homo is crucial for also has implications for reconstructing phylogenetic relationships between species of understanding when and where Australopithecus and Paranthropus, and the links between early Homo species and selective pressures resulted in its Homo erectus. We use microcomputed tomography and landmark-free deformation- emergence from a Plio- based three-dimensional geometric morphometrics to extract taxonomically informative Pleistocene hominin taxon. Our data from the internal structure of postcanine teeth attributed to Early Pleistocene revision of a large part of the Homo in the southern African hominin-bearing sites of Sterkfontein, Swartkrans, dental fossil record from southern Drimolen, and Kromdraai B. Our results indicate that, from our sample of 23 speci- Africa provides evidence mens, only 4 are unambiguously attributed to Homo, 3 of them coming from Swartk- suggesting a paucity of Homo rans member 1 (SK 27, SK 847, and SKX 21204) and 1 from Sterkfontein (Sts 9). Three other specimens from Sterkfontein (StW 80 and 81, SE 1508, and StW 669) remains and indicates increased approximate the Homo condition in terms of overall enamel–dentine junction shape, levels of dental variation in but retain Australopithecus-like dental traits, and their generic status remains unclear. australopith taxa. Results of the Downloaded from https://www.pnas.org by 158.109.162.13 on February 17, 2023 from IP address 158.109.162.13. The other specimens, including SK 15, present a dominant australopith dental signa- Ba/Ca, Sr/Ca, and elemental ture. In light of these results, previous dietary and ecological interpretations can be mapping of enamel and dentine reevaluated, showing that the geochemical signal of one tooth from Kromdraai (KB also indicate that some of the 5223) and two from Swartkrans (SK 96 and SKX 268) is consistent with that of purported Homo specimens show australopiths. a paleoecological signal similar to that of the australopiths. early Homo j taxonomic assessment j dental structure j geometric morphometrics The South African cave sites of the Cradle of Humankind (Gauteng Province), declared a World Heritage area by the United Nations Educational, Scientific, and Cultural Orga- nization, have yielded hundreds of Pliocene–Pleistocene (Gelasian–Calabrian, https:// stratigraphy.org) hominin remains assigned to the genera Australopithecus, Paranthropus, and Homo. At Sterkfontein, specimens variously assigned to Australopithecus, Paranthro- Author contributions: C.Z. and M.M.S. designed research; pus robustus, and early Homo have been found in western breccias equivalent in age to C.Z. and M.M.S. performed research; C.Z., T.W.D., R.J.-B., A.B., and M.M.S. analyzed data; C.Z., T.W.D., post–member 4 infills (member 5 and the StW 53 infill) (1, 2). Currently available dates R.J.-B., A.B., L.B., F.d.B., J.H., J.-J.H., K.J., L.K., O.K., R.M., for the StW 53 infill and member 5 deposits suggest that the genera associated with these L.P., F. Schrenk, F. Santos, D.S., M.T., F.T., S.X., B.Z., and M.M.S. wrote the paper; L.B. contributed to the revision units existed in close temporal proximity on the southern African landscape during the of stratigraphy and chronology of the sites; F.d.B. Late Pliocene to Early Pleistocene (3), even if a better chronological framework is needed contributed to the 3D scanning and elaboration of the microCT scans; J.H. and K.J. contributed to the microCT to accurately compare specimens from the various sites. Additionally, the identification scanning of the fossil specimens and elaboration of the of specimens belonging to early Homo that are penecontemporaneous with Australopithe- 3D data; J.H., L.K., O.K., R.M., L.P., F. Schrenk., D.S., M.T., F.T., S.X., and B.Z. discussed the implications of the cus and/or Paranthropus at sites such as Kromdraai B, Drimolen, and Swartkrans has led findings; and F. Santos contributed to the statistical analyses. to interpretations of a widespread presence of early Homo throughout the Cradle of Humankind since ∼2.5 Ma (see debates about the geochronology of southern African The authors declare no competing interest. This article is a PNAS Direct Submission. L.D. is a guest hominin sites in refs. 4–7). However, the attribution of a number of these specimens to editor invited by the Editorial Board. Homo has been questioned. In this study, we analyze the dentine shape of postcanine Copyright © 2022 the Author(s). Published by PNAS. tooth crowns, which has demonstrated a strong taxonomic signal at the generic level. We This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 reexamine the taxonomic attribution of the majority of specimens previously considered (CC BY-NC-ND). to represent early Homo, and test the hypothesis of a ubiquitous presence of early Homo 1 To whom correspondence may be addressed. Email: clement.zanolli@gmail.com. in the Early Pleistocene sites of southern Africa. The first species described for Australopithecus, Australopithecus africanus, was erected 2 Deceased May 2020. This article contains supporting information online at based on the Late Pliocene juvenile skull Taung 1 from the eponymous site (8), and the http://www.pnas.org/lookup/suppl/doi:10.1073/pnas. hypodigm was later supplemented by specimens from Sterkfontein, Makapansgat, and 2111212119/-/DCSupplemental. Gladysvale (9). Australopithecus prometheus was first described on the basis of the Published July 5, 2022. PNAS 2022 Vol. 119 No. 28 e2111212119 https://doi.org/10.1073/pnas.2111212119 1 of 11 parietooccipital portion of braincase MLD 1 from Makapansgat africanus (28, 29). For this reason, the validity of this endemic (10). It was also recognized in the Late Pliocene deposits of taxon has not been generally accepted, and the attribution of the Sterkfontein (11). The Early Pleistocene species Australopithecus specimens remains controversial (41). Disagreements partly arose sediba was identified at Malapa and described as showing because previous studies were restricted to the analysis of a few Homo-like features even if the postcanine teeth exhibit an Aus- incomplete mandibles and cranial remains chronologically span- tralopithecus morphology, including birooted lower third premo- ning 700 ka to 500 ka (27). However, the majority of the south- lars and the presence of a marked protostylid on the lower ern African Homo record is represented by dental remains, and molars (12). The type species of Paranthropus, P. robustus, was the analysis of the internal structure of these teeth has the poten- described on the basis of the Early Pleistocene partial skull TM tial to shed light on their taxonomic affiliation, augmenting debate 1517 from Kromdraai (13) and was recognized at several other around the origins and distribution of early Homo in the Cradle sites such as Drimolen, Cooper’s Cave, Gondolin, Sterkfontein, of Humankind. and Swartkrans (9). To reassess the taxonomic attribution of the southern African While less plentiful, remains attributed to early Homo have early Homo dentognathic assemblage, we focus on the morphology been described in four sites of the Cradle of Humankind: of the enamel–dentine junction (EDJ). This interface is preserved Swartkrans, Kromdraai B, Sterkfontein, and Drimolen. The in fully formed tooth crowns between enamel and dentine and first fossil from southern Africa attributed to early Homo was captures the morphology of the membrana praeformativa, a base- the Early Pleistocene mandible SK 15 from Swartkrans member ment membrane serving as the template for the majority of the tax- 2, dated to ∼1.4 Ma (14). It was originally attributed to Telan- onomically relevant aspects of crown morphology (42, 43). Unlike thropus capensis (15) and was later assigned to Homo erectus/ the outer enamel surface, which is often affected by occlusal wear, Homo ergaster (16), but it was recently suggested to belong to the EDJ is generally well preserved and recognized as a reliable tax- Australopithecus (17). Additional specimens, including SK 27, onomic proxy to distinguish between hominid taxa, even at the SK 847, and SKX 21204 from the 2.2 Ma to 1.8 Ma Swartk- subspecies level (44–48). Three-dimensional geometric morpho- rans member 1 (7), and the isolated teeth SKX 257 and 258 metric (3D GM) analyses using landmarks and semilandmarks and SKX 267 through 269 from the chronologically younger show that dentine horn height, crown height, and cervix shape can member 2, are generally recognized as early Homo (18–20). distinguish the postcanine teeth of Australopithecus, P. robustus, and Several specimens from this site, including SK 47, SK 843, SK Late Pleistocene to Holocene Homo (47, 49, 50). The recently 846, and SKX 4446, were previously attributed to Homo (21, developed diffeomorphic surface matching (DSM) method (51), 22), but further analyses suggested that they belong to the P. which models the deformation between shapes, captures the geo- robustus hypodigm (23, 24). metric details of an anatomical structure and is now used as an The associated dentition of KB 5223 from Kromdraai B, advanced analytical tool in morphometrics (51–54). Indeed, DSM Downloaded from https://www.pnas.org by 158.109.162.13 on February 17, 2023 from IP address 158.109.162.13. chronologically assessed to the beginning of the Early Pleisto- analyses can capture the taxonomically relevant aspects of the EDJ cene (25), was attributed to Homo, due, in part, to the small morphology, including both prominent features (such as the den- tooth crown dimensions (26), but was also regarded as mor- tine horns and marginal crests) and more subtle features (like the phologically compatible with P. robustus (27). More than a protostylid and occlusal basin morphology), and might thus dozen specimens from Sterkfontein member 4 (∼2.8 Ma to 2.2 improve on traditional GM analyses that focus only on the shape Ma) and member 5 (2.2 Ma to 2.0 Ma) (4), have been attrib- of the cervix and marginal ridge (53, 55, 56). uted to Homo (SE 255, SE 1508, SE 1579, SE 1937, SE 2396, We use the DSM approach to investigate the EDJ shape of Sts 19, StW 19b, StW 42, StW 53, StW 75, StW 80 and 81, an assemblage of permanent premolars and molars from Dri- StW 84, StW 87, and StW 151) (1, 2, 16, 27), although some molen (DNH 39, DNH 62, DNH 67, and DNH 70), Krom- specimens, including StW 53, were also regarded as representa- draai (KB 5223), Sterkfontein (SE 255, SE 1508, Sts 9, StW tive of A. africanus (28, 29). In addition to the recently 19b, StW 53, StW 80, StW 81, StW 87, StW 151, and StW described calvaria (6), 10 isolated teeth from Drimolen (DNH 669), and Swartkrans (SK 15, SK 18a, SK 27, SK 96, SK 847, 24, DNH 35, DNH 39, DNH 42, DNH 45, DNH 62, DNH SKX 257, SKX 268, and SKX 21204) attributed to, or sug- 67, DNH 70, DNH 71, and DNH 80) were also suggested to gested to represent, Homo (SI Appendix, Supplementary Note 1 represent early Homo (30, 31). and Table S1). We test whether their EDJ morphology more Collectively, this southern African sample of the early Homo closely approximates the condition of Early–Middle Pleistocene hypodigm exhibits a high degree of dentognathic dimensional and Homo from eastern Africa (n = 22/65) and Asia (n = 43/65) or morphological variation, and the attribution to Homo of some the australopith (Australopithecus and Paranthropus from south- specimens remains a matter of contention (e.g., refs. 1, 2, 6, 19, ern Africa) pattern (for the list of the comparative material, see 20, 22, 24, 27, and 32–40). Indeed, this assemblage has been SI Appendix, Table S2). The reference Homo sample includes referred to as Homo sp., Homo habilis, and H. erectus/H. ergaster, specimens attributed to H. erectus/H. ergaster or early Homo but many of the specimens differ in dental morphology from the and that are widely accepted to unambiguously belong to our eastern African remains allocated to the latter two species (27). genus during the early period of its evolution. This casts doubts on the nature and identity of the southern Afri- can Early Pleistocene material regarded as Homo. In addition, a Results new species Homo gautengensis (40) was erected to accommodate most of the Early Pleistocene southern African Homo-like speci- As illustrated in the assemblage from Swartkrans (Fig. 1), for all mens. StW 53 was defined as the holotype, along with a number tooth positions, there are aspects of EDJ morphology of the of paratypes (SE 255, SE 1508, StW 19b/33, StW 75-79, StW southern African purported Homo specimens that are more typ- 80, StW 84, StW 151, SK 15, SK 27, SK 45, SK 847, SKX 257/ ical of Australopithecus and Paranthropus (SI Appendix, Figs. 258, SKX 267/268, SKX 339, SKX 610, SKW 3114, and DNH S1–S8). To quantitatively assess EDJ shape variation and reas- 70). However, the type specimen of H. gautengensis, StW 53, has sess taxonomic attribution of the purported Homo teeth, we been generally regarded as representing H. habilis (27), and has conducted DSM GM analyses of the EDJ using specimens of also been shown to be morphologically indistinguishable from A. Australopithecus, Paranthropus, and Homo whose taxonomic 2 of 11 https://doi.org/10.1073/pnas.2111212119 pnas.org Downloaded from https://www.pnas.org by 158.109.162.13 on February 17, 2023 from IP address 158.109.162.13. Fig. 1. The EDJ of the postcanine teeth of the purported Homo specimens from Swartkrans compared with those of Early Pleistocene Homo (KNM-ER 1590 M1 and M2, Sangiran 4 M3, and KNM-ER 992 lower postcanine teeth), Australopithecus (Taung M1 and M1, StW 183 M2, StW 128 M3, StW 498 P3, StW 104 P4, StW 133 M2, and StW 312 M3), and Paranthropus (TM 1517 upper molars and P3, P4, M1, and M3, and SK 1 M2). Specimens belonging to the same individual are encased by a plain line, and specimens that likely belong to the same individual are enclosed by a dotted line. identity at genus level is well established and undisputed as ref- more regular cervical outline in the molars. Australopithecus is erence samples. In all between-group principal component distinguished from Paranthropus by a more developed lingual analyses (bgPCA) based on the deformation fields computed by than buccal aspect in upper molars, a less distally positioned DSM, the three hominin genera are well discriminated, despite protoconid in the P3, a shorter P4 EDJ crown, and a more a slight overlap between Australopithecus and Paranthropus expanded buccal shelf in the lower molars. for the M3 and P4 (Figs. 2 and 3). The cross-validated bgPCA The purported Homo specimens were projected into the (cv-bgPCA) confirm the general distinction of the three groups. bgPCA and CVA, and the typicality probabilities were com- In this case, while the australopiths overlap for the premolars puted (Table 1). With a few exceptions, the results of the and third molars, Homo is largely well distinguished (SI bgPCA and CVA are consistent. Specimens from Drimolen Appendix, Figs. S9 and S10). Additionally, we conducted (DNH 39, DNH 62, DNH 67, and DNH 70) and Kromdraai canonical variate analyses (CVA) on subsets of the principal B (KB 5223) are statistically attributed to Paranthropus, sharing components, and results are consistent with those of the with the teeth of this genus a more mesiodistally compressed bgPCA and cv-bgPCA (SI Appendix, Figs. S11 and S12). As lingual aspect of the EDJ than the buccal one in the upper shown by the statistical analyses, all cv-bgPCA and CVA of PC molars (Fig. 2) and a developed lower molar protostylid (Fig. scores exhibit high degrees of classification accuracy for the 3). The specimen SK 15 also shows strong affinities with Para- comparative specimens (SI Appendix, Tables S3 and S4), and nthropus (of the three lower molars analyzed, the M2 and left group differences are not purely allometric, as they remain large M3 fall within Paranthropus, while the right M3 is intermediate and significant after controlling for allometry (SI Appendix, between Paranthropus and Australopithecus). The P3/4 SK 18a, Table S5). With respect to the australopiths, Homo generally that was suggested to belong to the SK 15 mandible, also dis- shows a taller EDJ crown with a smaller EDJ central basin in plays an intermediate signal, and is closer to Australopithecus if the premolars, and a proportionally larger basin with respect to it is a P4 (Figs. 1 and 3). The specimens SE 255, StW 19b, the crown base (except in the third molars) and a rounder or StW 87, and StW 151 from Sterkfontein and SKX 257 and PNAS 2022 Vol. 119 No. 28 e2111212119 https://doi.org/10.1073/pnas.2111212119 3 of 11 Fig. 2. Bivariate plot of the bgPCA scores based on the DSM deformation fields for the M1 (A), M2 (B), and M3 (C). The totality of the variance refers to between-group variation. Symbols highlighted in bold represent the holotype specimens of Australopithecus (Taung) and Paranthropus (TM 1517). Filled trian- gles indicate African Homo, while open triangles represent Asian Homo. SKX 268 from Swartkrans are distinctly classified within was erected, Leakey et al. (59) proposed several morphological Australopithecus (even if the M2 of StW 151 is closer to the dental features distinguishing Homo from Australopithecus, Paranthropus morphology). The M3s of StW 53 exhibit a including a bicuspid P3, smaller postcanine teeth (in particular mosaic of Paranthropus and Australopithecus morphology, while the M3, generally smaller than the M2 or M1), anterior teeth the M3 shape is compatible with the Australopithecus variation. proportionally less reduced than the postcanine elements, and less Conversely, the specimens SK 27 and SKX 21204 from buccolingually expanded premolars and molars (essentially due to Swartkrans, as well as Sts 9 from Sterkfontein, fall well within the reduction or absence of the protostylid in Homo). However, Homo, showing a taller EDJ crown than in the australopiths these features are also variably found in australopith specimens, (Figs. 2 and 3). The M3 of SK 847 has a mesiodistally com- sometimes in combination (for example, the Australopithecus pressed shape that is similar to that of Homo, but also a trapezoi- specimen Sts 52 has a smaller M3 relative to the M2 and a bicus- dal outline reminiscent of the Australopithecus condition. The pid P3), whereas some specimens attributed to Homo (e.g., the classification of the specimens StW 80 and 81 from Sterkfontein H. habilis specimen OH 16) show a larger M3 than M2 and a Downloaded from https://www.pnas.org by 158.109.162.13 on February 17, 2023 from IP address 158.109.162.13. varies depending on tooth position. The P3 and M3 EDJ are mesiodistally elongated P3 (36, 59). Wood and Collard (57) sug- comparable to Homo but outside the variation range displayed gested that fossil specimens/species should be included in Homo by the comparative sample used in this study, while the P4 only if their teeth are more similar in morphology and relative exhibits an australopith morphology, and the M2 is intermediate proportions to the modern human condition than they are to the between Homo and Australopithecus. The specimens SK 96 from australopiths. However, modern human tooth morphology differs Swartkrans, as well as SE 1508 and StW 669 from Sterkfontein, from most of the earlier species of Homo: The P3 and P4 have a are less definitive, as their EDJ shape is intermediate between all small to absent metaconid and have a high crown, the M2 and three genera (Figs. 2 and 3 and SI Appendix, Figs. S11 and S12). M3 variably display only three cusps (lacking a hypocone), and We also scored nonmetric morphological features of the EDJ the M2 and M3 often have only four cusps (SI Appendix, in the purported early Homo and comparative samples, showing Supplementary Note 2). This is largely due to the trend for reduc- that there is an overlap in the presence/absence and degree of tion of the size and prognathism of the face, the jaw, and tooth expression of most nonmetric dental features recorded at the EDJ dimensions that occurred through the Late Pleistocene (60). of the three hominin genera. None of these traits in isolation can Conversely, Early to Middle Pleistocene Homo species have be considered diagnostic of Homo (SI Appendix, Supplementary more-complex lower premolars (with a large metaconid and an Note 2 and Table S6). extended talonid), four-cusped upper molars, and five-cusped Finally, we investigated whether tooth size (assessed here using lower molars, making them more suitable for comparison with the EDJ surface area as a proxy) is a diagnostic feature to distin- the Late Pliocene to Early Pleistocene record and the identifica- guish early Homo from the australopiths. With the exceptions of tion of early Homo. SK 15, StW 19b, StW 81, and StW 53 that have relatively large In the bgPCA, cv-bgPCA, and CVA of PC scores conducted EDJ areas (in the upper range of Australopithecus and Paranthro- here, fossil Homo, Australopithecus, and Paranthropus are generally pus), the size of most of the purported early Homo specimens is in well discriminated, further demonstrating the relevance of the the lower range of the australopiths and within the range of Early EDJ for taxonomic diagnosis (44, 45, 48, 53). The Early to Mid- to Middle Pleistocene Homo (SI Appendix, Fig. S13). In addition, dle Pleistocene reference sample includes both African and Asian even if Early to Middle Pleistocene Homo tends to have smaller specimens that group closely and are mixed together in the plots tooth dimensions than the australopiths, there is overlap with Aus- despite their wide chronogeographic distribution. Due to the tralopithecus for most tooth positions. A summary of the results inherent nature of the fossil record, hominin taxa (including early for the metric, nonmetric, and size analyses is presented in SI Homo species) are represented by a limited number of specimens, Appendix, Tables S6–S8 along with an overall assessment of the which complicates assessment of intrataxonomic and intertaxo- taxonomic affiliation of each of the purported Homo specimens. nomic variation, and, ultimately, taxonomic attribution (27, 57). However, in the absence of molecular data for early hominins, Discussion studying morphology remains the only way to diagnose extinct taxa. Among the 23 southern African specimens previously sug- The definition of the genus Homo on paleontological grounds gested to belong to Homo examined in this study (Table 2), only remains a conundrum (57, 58). When the species H. habilis three are unequivocally attributed to Homo (SK 27, SKX 21204, 4 of 11 https://doi.org/10.1073/pnas.2111212119 pnas.org Downloaded from https://www.pnas.org by 158.109.162.13 on February 17, 2023 from IP address 158.109.162.13. Fig. 3. Bivariate plot of the between-group principal component analysis (bgPCA) scores based on the DSM deformation fields for the P3 (A), P4 (B), M1 (C), M2 (D), and M3 (E). The totality of the variance refers to between-group variation. Symbols highlighted in bold represent the holotype specimens of Australo- pithecus (Taung) and Paranthropus (TM 1517). Filled triangles indicate African Homo, while open triangles represent Asian Homo. and Sts 9). SK 847 morphology is closer to Homo, even if the showing that it shares synapomorphies of the Homo clade but overall EDJ configuration retains Australopithecus features (Fig. 2 probably represents a more primitive species than H. erectus s.l and SI Appendix, Fig. S3) and can also be confidently attributed (18, 39). The specimens StW 80/81, representing a single indi- to the former genus. This is compatible with the analysis of the vidual, preserve crushed and fragmentary portions of the mandib- morphology of the face and partial cranium of this specimen ular bone with little morphological information (1). The EDJ of PNAS 2022 Vol. 119 No. 28 e2111212119 https://doi.org/10.1073/pnas.2111212119 5 of 11 the postcanine teeth of StW 80/81 displays affinities with Homo, Table 1. Typicality probabilities of the investigated as well as some features reminiscent of the australopith condition specimens computed for the bgPCA and CVA of shape (mesiodistally elongated P4 and M3, and developed protostylid analyses on the M2). This combination of Australopithecus-like and Homo- like traits detected in some specimens investigated here can be HOM AUS PAR interpreted in different ways: 1) These individuals could represent bgPCA CVA bgPCA CVA bgPCA CVA some of the earliest members of the genus Homo, retaining some 1 primitive features of the dentition; 2) they could belong to an M Australopithecus group evolving directly or convergently toward DNH 39 0.19 0.25 the Homo condition; or 3) they could be an australopith-Homo DNH 62 0.34 0.08 hybrid of the kind hypothesized by Thackeray (61). With respect DNH 70 0.37 0.11 to the first and second scenarios, StW 80/81 comes from member SE 255 0.82 0.18 5 west of Sterkfontein, likely dating to 1.7 Ma to 1.4 Ma (1). If SK 27 L 0.16 0.42 this individual represents an early member of our genus with a SK 27 R 0.18 0.55 morphology reminiscent of Australopithecus, it could imply that a SKX 268 0.24 0.07 stem group of early Homo remained genetically isolated for some StW 151 L 0.39 0.68 StW 151 R 0.28 0.58 time from H. erectus/H. ergaster. If it represents Australopithecus, StW 669 0.19 0.01 it is more recent than A. sediba, and future studies of the EDJ of M2 the postcanine dentition could resolve whether StW 80/81 mor- SE 1508 0.06 0.39 phology is compatible with A. sediba. The third hypothesis can- SK 27 0.80 0.75 not be discarded either, and, while the influence of interbreeding StW 151 0.29 0.52 on hominin tooth morphology is poorly understood, molecular M3 analyses such as paleoproteomics should be soon able to test this SK 847 0.22 0.02 and investigate phylogenetic relationships of Pliocene and Early StW 19 0.50 0.30 0.24 0.09 Pleistocene hominins (62). StW 53 L 0.46 0.26 The case of the specimens SE 1508 and StW 669, from StW 53 R 0.50 0.76 Sterkfontein, is more ambiguous, as they have a central position P3 in the bgPCA and/or CVA plots (Fig. 2 and SI Appendix, Fig. SK 18a 0.21 0.16 S11), indicating the need for more caution in the interpretation SK 96 0.03 0.07 of their group affiliation. The EDJ of the M2 SE 1508 is simi- SKX 21204 0.36 0.89 Downloaded from https://www.pnas.org by 158.109.162.13 on February 17, 2023 from IP address 158.109.162.13. lar to that of SK 27, albeit with a lower and broader crown StW 80 0.05 0.74 base with respect to the occlusal basin. In the DSM analyses, P4 these differences drive the specimen away from the Homo SK 18a 0.26 0.32 morphology represented in our sample and make it closer to SKX 21204 0.31 0.32 Paranthropus. However, together with the marked similarities StW 80 0.25 0.58 with SK 27, the position of the dentine horns and relatively StW 87 0.11 0.06 StW 151 L 0.79 0.31 simple crown morphology (i.e., absence of accessory traits) sug- StW 151 R 0.38 0.73 gest that SE 1508 could represent early Homo. The M1 StW M1 669 shows external crown morphological features similar to DNH 67 0.99 0.99 those of H. habilis (63), and preliminary analyses of the internal KB 5223 0.14 0.12 tooth structure also show affinities with Homo (64). However, SKX 257 0.52 0.63 our comparative analyses reveal that the EDJ retains some Sts 9 0.84 0.47 Australopithecus-like features, such as a markedly waisted occlusal StW 151 L 0.71 0.77 outline, a buccal shelf, and a mesial lateral aspect that is strongly StW 151 R 0.60 0.76 oriented inwardly (as in StW 283, for example). While an attri- M2 bution to early Homo cannot be rejected, the specimen may rep- SK 15 0.81 0.53 resent Australopithecus with derived features resembling the StW 80 0.19 0.62 Homo condition. M3 All the other purported early Homo specimens analyzed SK 15 L 0.61 0.10 here show a dominant australopith signature of the EDJ mor- SK 15 R 0.31 0.42 phology, and statistical analyses suggest that their inclusion StW 53h 0.37 0.01 in the Homo hypodigm is poorly supported based on EDJ For each specimen, typicality probabilities are shown only for the group with which that morphology. As many of these are isolated teeth, additional specimen was affiliated. Values in bold are below the 0.05 threshold and are regarded analyses will be necessary to more clearly establish their taxo- as outliers for the groups included here. AUS, Australopithecus; HOM, Early and Middle Pleistocene Homo; PAR, Paranthropus. nomic status (e.g., proteomic and ancient DNA evidence, and/or isotopic data [see below]). This also implies that the species H. gautengensis, created to accommodate the large S12), as also indicated by the typicality probabilities (Table 1). morphodimensional variation of the southern African “early The bgPCA probabilities are below the statistical threshold of Homo” material (40), is invalid, as it includes specimens belong- classification within any of the comparative groups (P < 0.05), ing to Homo (e.g., SKX 21204 and SK 27), Australopithecus suggesting that SK 96 could either be an outlier for any of the (e.g., SE 255 and StW 53), and Paranthropus (e.g., KB 5223 comparative genera or represent another genus that is not repre- and DNH 70). sented in the reference samples. It was suggested by Davies et al. SK 96 has a relatively central position in the bgPCA (Fig. 3), (50) that SK 96 EDJ shares similarities with Paranthropus and while the CVA place it close to Paranthropus (SI Appendix, Fig. also approximates that of Homo naledi. The taxonomic 6 of 11 https://doi.org/10.1073/pnas.2111212119 pnas.org Table 2. Summary of taxonomic evidence for specimens previously attributed to Homo Bone Revised Specimen Tooth EDJ area Nonmetric traits cv-bgPCA cv-CVA morphology assessment DNH 39 RM1 UND UND PAR PAR n/a PAR DNH 62 LM1 UND UND PAR PAR n/a PAR DNH 67 RM1 AUPITH UND PAR PAR n/a PAR DNH 70 LM1 UND UND PAR PAR n/a PAR KB 5223 LM1 AUPITH AUPITH PAR PAR n/a PAR SE 255 RM1 UND UND AUS AUS n/a AUS SE 1508 RM2 HOM UND INT PAR n/a PAR? StW 19b RM3 AUPITH UND AUS AUPITH n/a AUS StW 53 LM3 AUPITH UND AUPITH PAR HOM? AUPITH StW 53 RM3 AUPITH UND AUPITH PAR HOM? AUPITH StW 53 LM3 AUPITH UND AUS AUS HOM? AUS StW 80 RP3 UND UND HOM HOM UND HOM StW 80 RP4 HOM? UND AUS? AUS UND AUS/HOM StW 80 RM2 UND UND AUS? AUS UND AUS? StW 81 RM3 AUPITH UND INT HOM UND HOM StW 87 RP4 HOM? UND AUS? AUS n/a AUS StW 151 LM1 UND UND AUS AUS UND AUS StW 151 RM1 UND UND AUS AUS UND AUS StW 151 RM2 HOM UND PAR PAR UND AUPITH StW 151 LP4 HOM UND AUS AUS UND AUS StW 151 RP4 HOM UND AUS? AUS UND AUS StW 151 LM1 UND AUPITH? AUS AUS UND AUS StW 151 RM1 UND AUPITH? AUS AUS? UND AUS StW 669 RM1 UND UND AUS? AUS n/a AUS? Sts 9 RM1 AUPITH UND HOM HOM n/a HOM SK 15 RM2 AUPITH UND PAR PAR HOM? PAR SK 15 LM3 AUPITH UND PAR PAR HOM? PAR Downloaded from https://www.pnas.org by 158.109.162.13 on February 17, 2023 from IP address 158.109.162.13. SK 15 RM3 AUPITH UND AUS? AUS? HOM? AUPITH SK 18a LP3 or LP4 HOM? UND AUS AUPITH HOM? AUPITH SK 27 LM1 UND UND HOM HOM HOM HOM SK 27 RM1 UND UND HOM? HOM HOM HOM SK 27 RM2 HOM UND HOM HOM HOM HOM SK 96 LP3 UND UND AUPITH PAR? n/a AUPITH SK 847 LM3 AUPITH UND HOM AUS? HOM HOM SKX 257 RM1 UND UND AUS AUS n/a AUPITH SKX 268 RM1 UND AUPITH AUS AUS n/a AUPITH SKX 21204 RP3 HOM UND HOM HOM HOM HOM SKX 21204 RP4 HOM UND HOM HOM HOM HOM Bold and nonbold rows separate the different individuals represented here. AUS, Australopithecus; AUPITH, australopith; HOM, Early and Middle Pleistocene Homo; INT, intermediate; PAR, Paranthropus; UND, undiagnostic. attribution of this specimen thus remains problematic. If SK 96 Paranthropus condition (67). If SK 15 belongs to P. robustus, then belongs to Paranthropus, it differs from the morphology of it either suggests high levels of variation in mandible size/shape P. robustus, and it raises questions regarding possible affinities with implications for sexual dimorphism within the genus or a with H. naledi. There is a chronological gap of more than 1 My higher degree of taxonomic diversity than currently recognized in between the Swartkrans member 2 specimen and the Rising the genus. The currently available evidence from dental structural Star Cave hominins, and phylogenetic relationships of H. naledi organization suggests that SK 15 possibly belongs to Paranthropus, with other hominin taxa are still uncertain (50, 65), and could but its taxonomic status should be investigated further. be a focus of future studies. Our results also indicate that SK Based on morphology and dimensions of the craniodental 15, holotype of the nomen oblitum T. capensis (15), as well as remains, the specimens StW 53 and StW 151 were both sug- SK 18a that is supposedly associated with this mandible, actually gested to represent early Homo or Australopithecus individuals represent an australopith taxon and not Homo. Although gener- more derived toward Homo than the rest of the australopith ally regarded as a representative of H. erectus s.l (9), the specimen hypodigm (2, 28, 29, 68). StW 53 preserves partial aspects of is extremely robust, with proportions of the mandibular corpus the face and cranium (69). The most recent reconstruction that markedly differ from those of H. erectus/H. ergaster and shows a small endocranial volume and an intermediate mor- other Pleistocene Homo species (66). The EDJ of SK 15 molars phology between Australopithecus and H. habilis (37), even if it shows strong affinities with Paranthropus but differs in size and has been criticized for the anatomically unrealistic shape, indi- in relevant morphological aspects from the species P. robustus cating that the reconstruction of the cranium would be closer (e.g., absence of protostylid and accessory features) (Fig. 1). to Australopithecus (29). The juvenile specimen StW 151 only Interestingly, SK 15 molar root morphology has been linked with preserves small bone fragments of the jaw and cranium that are H. naledi and is, to some extent, also comparable with the not very informative for taxonomy (68). Our results show PNAS 2022 Vol. 119 No. 28 e2111212119 https://doi.org/10.1073/pnas.2111212119 7 of 11 Fig. 4. Chronostratigraphic distribution of the investigated purported Homo remains. In light of the results of the present study, only four specimens are likely to represent Homo (green ticks), four are possibly Homo (light green question marks), and the others more likely belong to Australopithecus or Paranthropus (red crosses). that EDJ morphology of both specimens is on the margins of teeth than Australopithecus, on average (but only partially, as Australopithecus variation or even approximates the Paranthropus indicated by the small determination coefficient and SI condition. Interestingly, the analysis of bony labyrinth shape Appendix, Fig. S13 and Table S7). In fact, some of the investi- shows that the two specimens represent the two extremes of the gated specimens show small dimensions, close to those of Australopithecus variation, where StW 53 appears as an outlier of Homo, but the EDJ shape unambiguously displays an australo- the taxon (70). pith morphology (e.g., DNH 70 and StW 151), and vice versa All the Drimolen specimens investigated here that were sug- (SK 27 and SK 847 have large molars but a Homo-like EDJ gested to represent early Homo (DNH 39, DNH 62, DNH 67, shape). This result thus indicates that tooth size alone should and DNH 70) show EDJ morphology that is more similar to not be used for taxonomic diagnosis in this lineage. P. robustus. Interestingly, the M1 KB 5223, whose crown shows Following our revision, among the 23 specimens previously small dimensions comparable to those of early Homo (26) and labeled as Homo, only between 4 and 7 specimens very likely Downloaded from https://www.pnas.org by 158.109.162.13 on February 17, 2023 from IP address 158.109.162.13. smaller than in P. robustus, likely belongs to the latter taxon. belong to this taxon. It is possible that some of the southern Indeed, the EDJ of this specimen displays a marked protostylid African purported Homo specimens that, in our analysis, exhibit that is similar to that of P. robustus. It is noteworthy that the an australopith-like signal actually do belong to Homo. This analysis of enamel microstructure of KB 5223 revealed an over- would mean that, dentally speaking, the typical tooth morphol- all pattern compatible with Paranthropus (71). The above speci- ogy of specimens attributed as H. erectus/H. egaster around 2 Ma mens from Drimolen are relatively small and are dated to did not emerge with the genus or was not ubiquitous among all around 2 Ma (6), and KB 5223 from Kromdraai comes from early Homo groups. However, the diversity of Australopithecus-like early Early Pleistocene sediments (25), again suggesting that a and Paranthropus-like signals that we have found in the southern smaller morph of P. robustus than that from Swartkrans coex- African purported Homo hypodigm suggests that it is unlikely isted in southern Africa for some time. However, the chronol- that all of these specimens belong to Homo. We propose that, in ogy of the deposits from these sites is debated, and the small addition to the four that display a Homo signal, a number of Paranthropus dental specimens from Drimolen and Kromdraai other individuals that show australopith affinities (e.g., SE 1508, that were previously identified as Homo could represent the StW 80 and 81, and StW 669) should be investigated further early condition of the P. robustus morphocline (72, 73). and compared with the hominin specimens from Olduvai Gorge, Most of the Early Pleistocene southern African teeth that our Tanzania, currently under study for internal tooth structure (74). analyses identify as misattributed to Homo are smaller than The presence of another hominin taxon not included in the refer- those typical of Australopithecus and Paranthropus, while those ence sample and represented by one or more of these intermedi- compatible with Homo in shape are relatively large (SI ate specimens cannot be discarded either. This revision of the Appendix, Fig. S13). It is possible that, besides the smaller purported early Homo dental material reduces its frequency and dimensions, the external crown shape initially used to classify representation in the Early Pleistocene southern African fossil these teeth is also less diagnostic. In this respect, allometric record (Fig. 4), and this has implications for the interpretation of changes could be considered as a potential factor. In our PCA, the fossil hominin paleodiversity, paleoecology, and dietary bgPCA, and CVA, allometric changes could be considered as a behavior at macroregional scale (75). potential factor. However, even after controlling for allometry, As an example, if a specimen is regarded as Homo whereas it group differences remain large (R2 ranging from 9 to 29%) and belongs to Australopithecus or Paranthropus, the interpretations of significant (SI Appendix, Table S5). This suggests that differ- the biological signal that can be extracted from the mineralized ences between EDJ morphology of the specimens mostly repre- tissues are erroneous. This leads to the false impression that sent shape variation and are only modestly influenced by size. Homo was more variable in growth, development, morphology, This study also demonstrates that, at many postcanine tooth behavior, and ecology than it really was, and prevents under- positions, crown dimensions overlap in Australopithecus, Homo, standing of how it biologically differed from other hominin gen- and Paranthropus (SI Appendix, Fig. S13). Using the natural era. To illustrate this, we analyzed here the geochemical profile logarithm of the EDJ as a proxy for crown size, Early to Middle of three teeth previously regarded as Homo: SK 96, whose taxo- Pleistocene Homo generally has significantly smaller postcanine nomic identity remains uncertain, and KB 5223 and SKX 268, teeth than Paranthropus (except for the M1) and slightly smaller reattributed, in this study, to Paranthropus and Australopithecus, 8 of 11 https://doi.org/10.1073/pnas.2111212119 pnas.org layering architecture of tooth formation, mimicking the profiles obtained by a previous study on Australopithecus (77). SKX 268 preserves part of the nursing sequence, which is comparable with the breastfeeding timing previously proposed for Australopithecus (77). Lactation behavior—with the infant relying more on solid food during periods of abundance, allowing the mother to replen- ish her energetic and calcium reserves to support lactation during periods of food scarcity—was proposed to represent a versatile adaptive trait to survive challenging ecological niches (77). Taxo- nomically misattributing SKX 268 would thus skew our under- standing of the intrataxic/intertaxic variability of this feature. Regardless of the robustness of the biogeochemical studies arising from the southern African hominin fossil record, accu- rate interpretations are contingent on correct taxonomic classifi- cation of hominin dental specimens. Our results highlight the need to continually interrogate taxonomic attributions of Early Pleistocene hominins in light of new evidence, such as shifts in first appearance dates [i.e., necessitated by the classification of the Ledi-Geraru mandible as Homo (78)], the unresolved taxo- nomic status of numerous specimens from Omo, Ethiopia (79), and Koobi Fora, Kenya (36), and the recent discovery of rather primitive Homo dentitions from Rising Star Cave, South Africa (65), Liang Bua, Indonesia (80), and Callao Cave, Philippines (81). This will facilitate paleobiological reconstructions and our understanding of the timing, location, and nature of the selec- tive pressures that resulted in a shift between australopith-like and Homo-like grades during hominin evolution. Materials and Methods Downloaded from https://www.pnas.org by 158.109.162.13 on February 17, 2023 from IP address 158.109.162.13. Thirty-seven southern African postcanine teeth previously attributed to early Homo, as well as one isolated lower molar, Sts 9, generally attributed to Australopithecus but recognized here as Homo (SI Appendix, Table S1), were compared with Australopithecus (n = 123), Paranthropus (n = 97), and African and Asian Early to mid-Middle Pleistocene Homo (n = 66; SI Appendix, Supplementary Note 1 and Table S2). We combined nonmetric trait and DSM GM approaches to investi- Fig. 5. Ba/Ca (A) and Sr/CA (B) ratios and elemental mapping of SK 96, KB gate the EDJ shape of the purported early Homo sample, and we conducted 5223, and SKX 268 compared with the distributions of Australopithecus and geochemical analyses on three of the investigated specimens (for more detailed Paranthropus. Asterisks denote that data for taxa distribution were calcu- information, see SI Appendix, Supplementary Material and Methods). lated using a combination of new data and a previously published datasets (80, 81), with the average values of KB 5223 (triangles) and SKX 268 Data Availability. All study data are included in the article and/or SI Appendix. (crosses) extracted from Balter et al. (76). SK 96 profile analysis was real- The microCT scans of fossil specimens are available by research application to ized on the enamel for this study, as well as the geochemical maps of KB 5223 and SKX 268. All error bars are 2 sigma. the relevant curatorial institution. ACKNOWLEDGMENTS. For their contribution with scanning, granting access respectively (Fig. 5). We also conducted elemental mapping of to, and/or sharing data of some of the fossil hominin specimens included in Sr/Ca and Ba/Ca ratios on the latter two specimens. Our geo- this study, we acknowledge J. W. Adams, C. Argot, F. Bernardini, J. Braga, chemical results confirm those of a previous analysis of KB 5223 L. Fiorenza, D. Grimaud-Herve, J. Kibii, H. Lelievre, L. Mancini, A. Mazurier, and SKX 268 that aimed to infer early Homo dietary preferences E. Mbua, S. Potze, F. Semah, C. Tuniz, and H. Widianto, as well as the online (76). Both SK 96 and KB 5223 have low Sr/Ca ratios, close to platform European Synchrotron Radiation Facility heritage database for paleon- the mean value of TM 1517, holotype of P. robustus, with a range tology, evolutionary biology, and archaeology (paleo.esrf.eu). For scientific discussion and comments, we thank R. J. Clarke, F. Detroit, J. Dumoncel, F. E. of variation compatible with the values of penecontemporaneous Grine, S. Schlager, A. Urciuoli, and B. A. Wood. For technical support and access browser taxa (76). The elemental maps of KB 5223 are highly to tomographic scans and associated digital data, we thank the following institu- affected by diagenesis, showing large areas of uniform density and tions: University of the Witwatersrand (Johannesburg), Ditsong National few structured areas indicative of a biogenic signal (i.e., of biologi- Museum of Natural History (Pretoria), Max Planck Institute for Evolutionary cal origin, as opposed to diagenetic that is related to taphonomy Anthropology (Leipzig), Centre de Microtomographie Universite de Poitiers, and fossilization processes). In the M1 SKX 268, the Ba/Ca and South African Nuclear Energy Corporation SOC Ltd. (Pelindaba), and International Sr/Ca ratios are higher than in the other two specimens, more Centre for Theoretical Physics (Trieste). We thank the South African Heritage closely approximating the condition of the Australopithecus. The Resources Agency for authorizing the study of South African fossil material. overall distribution of Ba/Ca in the enamel indicates a dominance We acknowledge support from the National Center for Scientific Research of biogenic signal (some of the highest concentrations follow the (CNRS)/IN2P3 Computing Center (Lyon, France) for providing computing and striation pattern of the Retzius lines), while the Sr/Ca ratio is data processing resources needed for this work. We thank the editor-in-chief, the more influenced by diagenetic processes (with low and high values editor, A. Cardini, R. J. Clarke, and two anonymous reviewers for their construc- distributed homogeneously). Conversely, for both variables, the tive comments that helped to improve previous versions of this article. This dentine shows pristine, biogenic banding typical of the growth research was funded by Erasmus+ Programme of the European Union Bakeng PNAS 2022 Vol. 119 No. 28 e2111212119 https://doi.org/10.1073/pnas.2111212119 9 of 11 se Afrika, French CNRS, AESOP+ program; National Research Foundation (South of Cambridge, Cambridge CB2 1TN, United Kingdom; gSchool of Geography, Archaeology and Environmental Studies, University of the Witwatersrand, Johannesburg 2000, South Africa); and Max Planck Society. This research benefited from the scientific frame-  de Paleontologia Miquel Crusafont, Universitat Auto Africa; hInstitut Catala  noma de work of the University of Bordeaux’s IdEx “Investments for the Future” program/ Barcelona, 08193 Bellaterra, Spain; iUMR 5608 CNRS, TRACES, Maison de la Recherche, Universite Toulouse Jean Jaure s, 31058 Toulouse Cedex 9, France; INRAP, French Institute j GPR “Human Past.” The participation of M.M.S. was supported by funding from for Preventive Archaeological Researches, 30900 Nîmes, France; kSouth African Nuclear the European Research Council under the European Union’s Horizon 2020 Energy Corporation SOC Ltd., Pretoria 0001, South Africa; lDepartment of Anthropology & Development Studies, University of Johannesburg, Auckland Park 2006, South Africa; research and innovation program (Grant Agreement 819960). m Evolutionary Studies Institute, University of the Witwatersrand, Johannesburg 2000, South Africa; nDitsong National Museum of Natural History, Pretoria 0001, South Africa; o Department of Paleoanthropology, Senckenberg Research Institute and Natural History Museum Frankfurt, Frankfurt 60325, Germany; pDepartment of Paleobiology and Environment, Institute of Ecology, Evolution, and Diversity, Goethe University Frankfurt, Author affiliations: aUniv. Bordeaux, CNRS, MCC, PACEA, UMR 5199, F-33600 Pessac, 60323 Frankfurt, Germany; qUMR 7194 CNRS, Muse  um National d’Histoire Naturelle, France; bDepartment of Human Evolution, Max Planck Institute for Evolutionary 75005 Paris, France; rDe partement Ge  osciences, Universite  de Poitiers, 86000 Poitiers, Anthropology, 04103 Leipzig, Germany; cSchool of Anthropology and Conservation, France; sKey Laboratory of Vertebrate Evolution and Human Origins, Institute of University of Kent, Canterbury CT2 7NZ, United Kingdom; dGeoarchaeology Vertebrate Paleontology and Paleoanthropology, Chinese Academy of Sciences, 100044 and Archaeometry Research Group, Southern Cross GeoScience, Southern Cross Beijing, China; tCenter for Excellence in Life and Paleoenvironment, Chinese Academy of University, Lismore, NSW 2480, Australia; ePalaeo-Research Institute, University of Sciences, 100044 Beijing, China; and uCentre for the Exploration of the Deep Human Johannesburg, Auckland Park 2006, South Africa; fDepartment of Archaeology, University Journey, University of the Witwatersrand, Johannesburg 2000, South Africa 1. 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