New fossils from Kromdraai and Drimolen, South Africa, and their distinctiveness among Paranthropus robustus

doi:10.21203/rs.3.rs-1646149/v1

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New fossils from Kromdraai and Drimolen, South Africa, and their distinctiveness among Paranthropus robustus

https://doi.org/10.21203/rs.3.rs-1646149/v1

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Most fossil hominin species are sampled with spatial, temporal or anatomical biases that render difficult the assessments of their paleodiversity, and may not yield genuine evolutionary signals. We use new fossils from the Kromdraai (Unit P) and Drimolen sites to provide insights into the paleodiversity of Paranthropus robustus and to assess the possible occurrence of “robust” features in Australopithecus africanus. Our focus is first, the shape allometry of the semi-circular canals (SCC), an aspect that has not yet been investigated among southern African australopiths, and second, the morphology of the temporal bone. We find significant differences in P. robustus SCC shape between the sites of Kromdraai, Drimolen and Swartkrans, with some allometric trends. This site-related variation is consistent with other differences in the morphology and overall size of the temporal bone. P. robustus from Kromdraai Unit P is distinctive because of its smaller adult temporal bone and its smaller SCC size combined with its proportionally less developed posterior SCC, independently of age and sex. This SCC pattern is interpreted as either the consequence of allometric scaling of body size, or as a yet unknown ecological/functional diversity in P. robustus. Combined with the occurrence of plesiomorphic features of its temporal bone, the P. robustus palaeodiversity may result from either an adaptive radiation subsequent to the origin of this species, or phyletic evolution.

Bony labyrinth

Temporal bone

Allometry

Paranthropus robustus

Australopithecus africanus

early hominin

human evolution

Assessments of patterns of diversity within early hominin species influence taxonomic and phylogenetic interpretations, and therefore need constant reevaluation when new fossils and analytical methods are available. After the discovery of the holotype of Paranthropus robustus from the site of Kromdraai (Gauteng, South Africa)¹, the taxonomic homogeneity of this species has been discussed² but subsequent fossil discoveries and the use of new methods proposed alternative interpretations. Differences in P. robustus facial morphology were interpreted to reveal phyletic evolution³, whereas variations in cochlear shape provided evidence for directional selection of this species’ hearing organ⁴. While aiming to specify the relationships among the three main P. robustus-bearing sites of Kromdraai, Swartkrans and Drimolen, these studies have also implications for the hypothesis that derived “robust” features may be present in Australopithecus africanus from Makapansgat⁵, Taung⁶ or Sterkfontein^7,8.

Assessments of paleodiversity within P. robustus are limited because its hypodigm is yet biased toward adult cranial remains from the sites of Swartkrans and Drimolen with the notable absence of any such evidence from Kromdraai. Moreover, even though the pervasive effects of size-related shape changes (allometry) on morphological features are widely accepted⁹, they remain unexplored among southern African australopiths. Here, we fill these gaps with new samples from Kromdraai and Drimolen, and by using approaches well suited for allometry¹⁰ and comparisons of shapes^4,11. Following a recent study of cochlear shape in southern African fossil hominins⁴, we first investigate digital endocasts of inner ears (or bony labyrinths, BL) by focusing on the semi-circular canals (SCC) (Fig. 1a). Variation in BL morphology related to allometry is ubiquitous among primates¹² but has not yet been investigated in A. africanus and P. robustus. We therefore newly add allometry to approaches of BL that already proved their utility to reveal species-specific characters among primates^13–15, and genus-level differences between A. africanus and P. robustus⁴. Other features of the temporal bone are also being measured because they have been considered of some taxonomic value to discuss “robust” affinities in A. africanus⁸, and to reveal the more plesiomorphic status of some P. robustus specimens from either Kromdraai¹⁶ or Drimolen¹⁷.

In this study, we address the following four questions. Does the relationship between the SCC shape and size follow allometric trends among southern African early hominins? If so, is this relationship consistent with other morphological differences of the temporal bone? Do these data allow (i) the identification of distinct P. robustus groups consistently with their site and stratigraphic provenience, (ii) the resolution of the taxonomic affinities of other southern African fossil hominins.

We investigate a sample of southern African early hominins (n = 25), including two undescribed fossils recovered from Drimolen in the late 1990s (DNH 34 and DH 60) and four P. robustus specimens from Kromdraai Unit P ^4,18,19 (Fig. 1a, Methods, Text S1, Figs S8-13, Table S1). The sample from Kromdraai Unit P includes the first nearly complete temporal bone of a P. robustus child known (KW 10840), and craniodental remains of the first fully adult specimen from Kromdraai (KW 9900) (Text S1, Table S2). We expand the knowledge of paleodiversity within P. robustus by contributing additional evidence of its outer and inner temporal bone morphology (Text S1). We also use this new evidence to assess statistically the presence of “robust” features in A. africanus, including its holotype from Taung²⁰, fossils from Jacovec Cavern (StW 578), Members 2 (StW 573) and 4 at Sterkfontein, and Makapansgat (MLD 31, a specimen recovered from the dumps). The only undisputed early Homo specimen represented in our sample (SK 847) was recovered from Swartkrans Member 1 Hanging Remnant. We also discuss whether our results help to resolve the taxonomic assignation of two specimens from Sterkfontein here considered as “indeterminate”: StW 53 from Member 5A, and StW 151 with less certain stratigraphic provenience (Methods, Text S1). Finally, we add four samples (n = 10, for each one) representing modern humans (Homo sapiens), bonobos (Pan paniscus), common chimpanzees (P. troglodytes) and gorillas (Gorilla gorilla) (Table S1), in order to discuss the potential effects of sexual dimorphism and allometry on the patterns of variation observed among early hominins.

The semicircular canals

We first use the SCC, two distinct Principal Component Analyses (PCA) (Fig. 1b,d) and one multidimensional scaling (MDS) (Fig. 1e) obtained from computational anatomy¹¹ (Methods) to assess the taxonomic affinities of the yet undescribed DNH 34 and DNH 60 specimens from Drimolen²¹ (Text S1). As detained in previous studies of dental surfaces¹¹ and cochlear shapes⁴, deformation-based methods from computational anatomy use a non-linear geometric framework to define size-independent differences between SCCs illustrated by MDS. The DNH 34 and DNH 60 specimens are first considered as “indeterminate” and projected onto the two PC1/PC2 (Fig. 1b,d) and the MDS1/MDS2 (Fig. 1e) representations to identify their closest neighbours. The first PCA (Fig. 1b) is applied after a Generalized Procrustes Analysis (GPA) of 5 landmarks and 7 semilandmarks per semi-circular canal (SCC) (Fig. 1a) (Methods). The second PCA (Fig. 1d) is obtained from 12 indices and 10 angles of the BL (Methods, Text S1, Table S3). All the three analyses (the two PCAs and MDS) unambiguously associate both DNH 34 and DNH 60 with P. robustus rather than with early Homo (SK 847). P. robustus and SK 847 are most differentiated (at p < 0.01) along the largest component variance (PC1) in one PCA (Fig. 1d), or the second largest one (PC2) in the other PCA (Fig. 1b). The MDS (Fig. 1e) (with a stress value of 0.0068 which indicates a very good representation quality) is consistent with the PCA results and shows a very good fit of the specimens in their groups (Methods). As shown by the comparison between the minimum and maximum values along PC2 in the shape space (Fig. 1c), DNH 34 and DNH 60 share significant features with P. robustus, when compared to early Homo (SK 847) (Fig. 1b). In early Homo, the posterior SCC (PSC) is posteriorly expanded (Fig. 1c). The horizontal SCC (HSC) is reduced in diameter and its posterior part is superiorly displaced (Fig. 1c). Finally, the lateral extremity of the anterior SCC (ASC, near the center of the ampulla, landmark 5) is inferiorly displaced (Fig. 1c). Among the 12 indices and 10 angles of the BL, the posterior semi-circular canal index 1 (PSCI1 that divides the arc length of the PSC situated below and above the HSC) (Methods) is the measurement that most differentiates P. robustus from SK 847 (i.e., with the most significant correlation with PC1, r = 0.88, p < 0.001).

We then consider the A. africanus (n=11) and P. robustus (including DNH 34 and DNH 60) (n=11) specimens on the basis of their site and stratigraphic provenience (Methods), early Homo (SK 847), StW 53 and StW 151, and one sample of modern H. sapiens (n=10). Turning to statistical assessments of SCC variation (Fig. 2), we compute Procrustes and Mahalanobis distances after GPA²², and we illustrate them by a between-group PCA (bgPCA) (Fig. 2a) and a Canonical Variate Analysis (CVA)²³ (as a type of discriminant analysis, Fig. S1), respectively (Methods). We also compute MDS from computational anatomy (Fig. 2b, stress value of 0.069) (Methods). StW 53 and StW 151 are projected onto each of the three biplots (Figs 2a,b, Fig. S1). Because spurious patterns of differences between groups may arise when a bgPCA²⁴ or a CVA²² is applied on small sample sizes relative to the number of variables, we assess this problem by varying the number of semilandmarks used to represent each SCC (1, 7, 18 or 48) (Methods, Fig. S2). Because the results do not change when using more semilandmarks (Fig. S2), we consider that 7 semilandmarks appropriately sample each SCC (Fig. 2a). The first two bgPCs (Fig. 2a) and the MDS (Fig. 2b) separate H. sapiens and early Homo on the one hand, and P. robustus specimens from Kromdraai Unit P on the other. All the other southern African australopiths occupy an intermediate position, with some overlap between A. africanus and P. robustus from Drimolen/Swartkrans (Fig. 2a) or from Kromdraai Unit P/Drimolen (Fig. 2b). Importantly, a Monte-Carlo test (10000 permutations) (Methods) reveals that the differences reported in Figure 2a reach statistical significance (p < 0.001) and that 46.3% of the total inertia comes from these differences (Fig. S3). Moreover, the fossil hominins illustrated in Figure 2b fit very well in their respective groups, consistently with their site and stratigraphic provenience, as indicated by the goodness of fit (0.069). The CVA also illustrates a separation between the three P. robustus samples (Fig. S1). Permutation tests (Methods) indicate that 100% of P. robustus specimens from Kromdraai Unit P and Swartkrans are correctly classified in their respective groups (Table S4). Two out of three P. robustus specimens from Drimolen are correctly classified (one is clustered with Australopithecus). The CVA results (fig. S1) are therefore consistent with those of the bgPCA (Fig. 2a) and MDS (Fig. 2b).

In the bgPCA (Fig. 2a), the MDS (Fig. 2b) and the CVA (Fig. S1), StW 53 and StW 151 fall closer to A. africanus than to early Homo (Fig. 2). When compared to early Homo (Fig. 3a,b,c), P. robustus from Drimolen (Fig. 3b) and Swartkrans (Fig. 3c), the SCC mean shape sampled from Kromdraai Unit P is distinctive mainly because of its proportionally less developed PSC. Consistently with the results presented in Fig. 1c, the mean configuration from Kromdraai Unit P is typical of P. robustus when compared to SK 847.

When fossil hominins are projected onto the bgPC1/bgPC2 morphospace computed by using the four modern species only, most australopiths fall within the SCC variation of common chimpanzees (Fig. S4), but some appear closer to gorillas than to the latter. The MDS does not discriminate better these groups (Fig. S5).

We next separate the SCC shape differences caused by allometry (CAC) from those that are not (RSC1)¹⁰ (Fig. 2c). The SCC shape in P. robustus from Kromdraai Unit P is distinctive either because of allometric scaling (along CAC) when compared to Drimolen, or independently of size (along RSC1) when compared to Swartkrans and KB 6067 (Fig. 2c). In the fossil sample, there is a significant linear correlation between CAC and centroid size (CS) (Fig. 2d), a relationship that even improves when the specimens are grouped according to their species and site/stratigraphic provenience to create an additional covariate (Wilcoxon tests, p < 0.05). The CAC versus RSC1, and CS versus CAC relationships observed among fossil hominins are confirmed when the four modern species are added to the model (Fig. S6). The lower CAC values observed in P. robustus from Kromdraai Unit P correspond to its smaller CS (mean and individual values) (Figs. 2d and 4a). However, none of the differences in CS between the fossil samples are significant (Fig. 4a, Table S5). On the contrary, most of the differences in CS between modern species are significant, with no sexual dimorphism (Fig. 4a, Table S5). Therefore, allometry is important in differentiating SCC shape in P. robustus specimens from different site/stratigraphic provenience, but size differences are caused neither by sexual dimorphism, nor by ontogeny. For instance, within A. africanus, its holotype (Taung child) shows higher CAC and CS values than StW 504 (Fig. 2d), a presumably large male²⁵. In P. robustus from Kromdraai Unit P, the KW 9900 adult also shows lower CAC and CS values than the KW 9700 child (Fig. 2d).

The oval window

We give particular attention to repeatable measurements of the oval window area (OWA) (Methods) in order to prevent spurious results, as emphasized previously²⁶. In P. robustus, the specimens from Kromdraai/Drimolen show a significantly lower OWA than in Swartkrans (p < 0.05, Wilcoxon). An example of the difference in OWA between specimens from Kromdraai/Drimolen (DNH 22) and Swarkrans (SK 879, with its 59% larger OWA) is illustrated in Figure S7. The OWA mean value from Kromdraai/Drimolen (n = 8) falls 3.5 standard deviations below the smaller OWA value from Swartkrans (Fig. 4b,d,e). Ontogeny does not influence differences in OWA among australopiths, larger values being often measured in juveniles than in adults (e.g., KW 10840 and StW 98), as already noticed in modern humans and African apes²⁷. There are significant differences in OWA between, on the one hand larger-bodied modern humans and G. gorilla (with no significant difference between them), and P. troglodytes or P. paniscus on the other (Fig. 4b), with no sexual dimorphism within any of these four modern species (Table S5). Among australopiths, there is no correlation between OWA and CS (p = 0.62). The CS versus OWA biplot shows a separation between the three P. robustus samples, the Kromdraai Unit P specimens falling in the lower left quadrant with lower CS and OWA values (Fig. 4d).

The temporal bone

The right temporal bone of KW 9900 represents the first fully adult P. robustus cranial remain from Kromdraai (Text S1, Figs S9-S12). It was found associated with its malleus and a heavily worn maxillary dentition (Fig. S12, Table S2). The overall size of the KW 9900 temporal bone is notably smaller than in TM 1517 (Fig. S10) and any of the P. robustus specimens from Swartkrans, as indicated by the length of its tympanic bone (22.1 mm), the area its external auditory meatus (88.4 mm²), and the degree of projection of its postglenoid process relative to the zygomatic root (12.5 mm) (Text S1). The external auditory meatus and the inferior projection of the postglenoid process of KW 9900 and DNH 7, a presumably P. robustus female from Drimolen, show relatively similar dimensions. The length of the tympanic bone is smaller in KW 9900 than in DNH 7. The morphology of the KW 9900 temporal bone also departs from the pattern yet described for P. robustus in two important features (Text S1). First, in lateral view, the most inferior point on the preserved remnant of the mastoid process (or mastoid tip) projects anteroposteriorly below the external auditory meatus (Fig. S11). This reflects an anteriorly positioned mastoid tip in relation to the asterion-porion distance. Our minimum estimate of this distance yields a “mastoid tip position index” not higher than 30%, which is lower than that of the two other measurable P. robustus crania, and more similar to the condition in A. africanus from Makapansgat (MLD 37/38) and A. afarensis (A.L. 444-2)¹⁷ (Text S1). Second, unlike in the P. robustus holotype (TM 1517) and in specimens from Swartkrans, the relatively thin inferolateral edge of the KW 9900 tympanic bone (2.7 mm) is slightly recessed from the lateral margin of the mastoid process and the suprameatal crest, with no peculiar lateral extension at this level (Fig. S11). Moreover, when seen in inferior view (Fig. S11, KW 9900 does not evince a widened and flared lateral portion of its tympanic bone (or “trumpet” shape), a feature that has been considered as typical of P. robustus²⁸.

We find a site-related diversity in P. robustus SCC shape that is partly attributable to allometric scaling (Fig. 2c). Some SCC shape differences between P. robustus from Drimolen and Kromdraai Unit P (Fig. 2c) are caused by the allometric effect of a smaller CS in the latter sample (Fig. 4a). The SCC shape uniqueness of the Kromdraai Unit P P. robustus (Fig. 3) is consistent with the well-known allometric trend in which larger-bodied primates possess a PSC proportionally more developed¹². However, the smaller CS in P. robustus from Kromdraai Unit P is not necessarily associated with changes in body size for several reasons. First, SCC shape is also influenced by locomotor agility in primates in general²⁹. Second, since SCC size was found to be significantly larger in G. gorilla males than in females¹⁵, the allometric differences between the Kromdraai Unit P and Drimolen P. robustus samples might reflect sexual dimorphism. Turning to CS variation among the four extant species investigated in our study, we nevertheless find no sexual dimorphism in Pan, modern humans, and even G. gorilla. Despite this evidence, one may argue that P. robustus specimens with small and large CS might represent females from Kromdraai Unit P and males from Drimolen, respectively. There is however no definite evidence on differences in sampling biases (e.g., predation-biased) between Kromdraai Unit P and Drimolen. It therefore seems reasonable to consider a random sampling of sex ratio in both sites, P. robustus females being also represented at Drimolen¹⁷. Moreover, in the likely under-representation of large males in the P. robustus hypodigm, the full extent of size variation in this sexually dimorphic species³⁰ is probably unknown³¹. Because the distinctive SCC shape measured in P. robustus from Kromdraai Unit P is also size-independent (i.e., not entirely caused by allometry, as indicated by MDS - Fig. 2b - and the CAC versus RSC1 biplot, Fig. 2c), the pattern of variation in this species may have another underlying cause than sexual dimorphism in body size, as suggested in recent studies^3,4,16.

Three distinct periods are represented by the P. robustus specimens yet discovered within the revised stratigraphy of the Kromdraai site³². Unit P represents the oldest of the three periods, and an unknown interval of time separates it from the stratigraphically younger, immediately overlying Unit Q-R (or “Member 3”) where KB 6067 was discovered¹⁶. Paleomagnetic data suggest that both Unit P and Q-R are older than the Olduvai SubChron (1.95 Ma) and that the P. robustus holotype from Kromdraai (TM 1517) may postdate this period¹⁶. KB 6067 was confirmed to be P. robustus on the basis of its “robust”-like, and size-independent cochlear shape⁴. Its SCC shape is distinctive when compared to its conspecifics from Kromdraai Unit P, Drimolen and Swartkrans (Fig. 2). When compared to P. robustus from Swartkrans, the smaller OWA in KB 6067 was interpreted as a possible primitive condition for this species, with some similarities to some A. africanus specimens¹⁶. In this regard, the new P. robustus samples from Kromdraai Unit P and Drimolen underline the importance of OWA in the analysis of this species’ paleodiversity. Comparisons of OWA values, consistently with our results on SCC CS and shape, indicate a site-related diversity in P. robustus. The smaller SCC CS in the Kromdraai Unit P specimens is associated with a significantly smaller OWA in the Kromdraai/Drimolen sample, when compared to Swartkrans (Fig. 4, Table S7). As in the case of SCC CS, differences in OWA are not necessarily associated with changes in body size because allometry does not explain the larger OWA in larger-bodied catarrhine species²⁷. The smaller OWA in Kromdraai/Drimolen P. robustus and in A. africanus (Fig. 4) can be interpreted as more plesiomorphic, and is consistent with the overall small size of the heavily worn maxillary dentition and the temporal bone of KW 9900 by P. robustus standards. KW 9900 defines the low end of variation in P. robustus in two MD diameters of its teeth (P4 and M2) (Table S2). Moreover, three dimensions (length of the tympanic, area of the external auditory meatus and degree of projection of the postglenoid process) of the KW 9900 temporal bone are smaller than in any of the P. robustus specimens from Swartkrans. Two out of three features also show lower dimensions in KW 9900, when compared to DNH 7, a presumably P. robustus female from Drimolen¹⁷. When compared to the subadult specimen and holotype of P. robustus from Kromdraai, the stratigraphically older KW 9900 specimen also appears particularly small in its dental and temporal bone dimensions (Text S1). The differences between the larger TM 1517 specimen and its conspecifics from Swartkrans (e.g., SK 46 and SK 83), on the one hand, and the smaller DNH 7 complete skull from Drimolen, on the other hand, have been interpreted as consistent with sexual dimorphism within P. robustus³⁰. If these differences in size are interpreted exclusively in terms of sexual dimorphism, this would imply that KW 9900 simply represents a small female of P. robustus.

As for the morphology of the temporal bone (i.e., beyond size), the KW 9900 P. robustus small specimen from Kromdraai Unit P evinces features that are unlike those of any small or large specimen from Swartkrans, and Drimolen. For instance, the KW 9900 anteriorly positioned mastoid tip is unlike the two other adult P. robustus fossils from Swartkrans (SKW 18/SK 52) and Drimolen (DNH 7) in which this trait can be assessed (Text S1), and more similar to the plesiomorphic condition observed in A. afarensis and A. africanus¹⁷. Moreover, the absence of a lateral extension, widening and flaring of the lateral portion of the KW 9900 tympanic bone (Fig. S10) is shared with the DNH 7 skull. This feature is also interpreted as a symplesiomorphy in comparison with the fossils from Swartkrans¹⁷, consistently with the low OWA in KW 9900 (Fig. 4). These observations will need to be supplemented by new approaches applied to the most complete specimens of P. robustus, including its holotype from Kromdraai. For the moment, in the absence of high resolution (i.e., with pixel size lower than 50 microns) and sufficiently contrasted micro-CT data, several important inner features of TM 1517 (including its OWA and cochlear shape) cannot be measured accurately.

In the present study, the SCCs of StW 53 and StW 151 show closer similarities with A. africanus than with SK 847, the only undisputed early Homo specimen in our sample. StW 53 has been attributed to early Homo³³, but previous analyses revealed unique features of its bony labyrinth¹³, its Australopithecus-like cochlear shape⁴ and late fusion between its maxillary and incisive bones³⁴. When other cranial features have been considered sufficiently diagnostic to retain StW 53 within early Homo³³, they have been visually assessed and classified into vague categories (e.g., ‘narrow’ versus ‘moderate’, or ‘short’ versus ‘short/moderate’). We therefore argue that more fully automate morphological analyses of StW 53 cranial remains are needed to test statistically the taxonomic status of this specimen. From the results of the present study, we conservatively assume that it does not represent early Homo because of its too plesiomorphic BM.

The cochlea of early Homo resembles that of A. africanus and, in both of these two taxa, it is intermediate between P. robustus on the one hand, and modern humans, chimpanzees and gorillas on the other⁴. Importantly, SK 847 displays a mosaic of features unlike australopiths that combines modern human-like SCCs^13,16 and an Australopithecus-like cochlear shape⁴. The cochlear shape yields a different evolutionary signal than the SCC among southern African early hominins. The uniquely derived and invariant cochlea of P. robustus reveals a strong selection for its morphology early in the evolutionary history of this species⁴. This evidence may be seen as incongruent with the SCC pattern of diversity in P. robustus and also reveals a modular (or mosaic) evolution of the bony labyrinth in southern African early hominins.

More reliable radiometric dates and consistent biostratigraphic comparisons between Kromdraai, Drimolen and Swartkrans will allow further interpretations of the P. robustus paleodiversity described in this study. At this stage, we consider that the morphological differences in both size and shape between the samples from Kromdraai, Drimolen and Swartkrans can equally illustrate either an adaptive radiation subsequent to the origin of P. robustus, or phyletic evolution within its lineage (i.e., a transformation from an ancestral state to a derived one).

Details on the provenience, the sex and age of the specimens in our samples, and the micro-CT systems used to scan them, are provided in Table S1. Among the 25 southern African early hominins, one juvenile (DNH 34) and one adult (DNH 60) specimen from Drimolen were previously unpublished, and one adult (KW 9900) and three juvenile (KW 9600, KW 9700, KW 10840) P. robustus specimens from Kromdraai Unit P were described only for their cochlear shape⁴. The DNH 34 juvenile status is indicated by the degree of the opening of its subarcuate fossa (5.6%) which is equivalent to that measured in the KW 9600 and KW 10840 specimens⁴. Here, we describe these six new specimens from Drimolen and Komdraai Unit P by focusing on their taxonomically diagnostic features. Among the other fossil hominins in our sample, all have already been published. However, the BM of some of these published specimens is newly reconstructed and investigated in the present study. This is the case for one P. robustus (SK 83) and two A. africanus (Taung child, MLD 31) specimens. As demonstrated both previously⁴ and in the present study, there are no differences in BM size and shape between adult and juvenile specimens among hominid or hominin species. We therefore combine juvenile and adult specimens in our samples.

The majority of the sample is represented by the right BMs. In cases of damage of the right side, the left BM is reconstructed after mirroring, with no loss of size and shape information during this process. Mesh surfaces of each BM were obtained in Avizo Standard 8.1.1 (www.thermofisher.com) from micro-CT data resliced in a plane that best fitted the horizontal SCC. The measurement protocol of the SCC began with the computation of the centerlines of the SCCs from the surface models with the ‘Skeletonization’ pack and the ‘Autoskeleton” module. A set of five landmarks was digitized on each centerline skeleton (Fig. 1a) and the placement error was estimated (see below). Landmarks 1 and 2 (Ld1 and Ld2, respectively) are located on the horizontal SCC (HSC) and correspond to the intersections between the plane that best fit the anterior SCC (ASC) and, respectively, the lateral and medial ends of the HSC’s centerline. Landmark 3 (Ld3) is located on the center of the ampulla of the posterior SCC (PSC). Landmark 4 (Ld4) is the bifurcation point of the common crus and landmark 5 (Ld5) is the center of the ampulla of the ASC. We also placed one landmark at the apex of the cochlear curve (Landmark 6, Ld6).

We then assess the size and shape of each BM either by a geometric morphometric method (GMM) (see below), by a deformation-based method from computational anatomy (see below), or by using 35 variables that include one area, six arc lengths, six linear distances (line segments), 12 indices and 10 angles detailed in Table S3. Among these 35 variables, five have been defined in previous studies (Table S2). All these 35 variables but one (OWA) are measured directly on each centerline skeleton by using the best-fitting planes of the HSC (HSCP) and ASC (ASCP), and the surface mesh. All these measurements are provided in Supplementary Material (Data S1.xls).

We measure the external cochlear length (ECL) and the oval window area (OWA) by following the measurement protocol described in references 16, 26 and 27. We also measure the transverse labyrinthine index (TLI), the inclination of the ampular line and the cochlear basal turn relative to the orientation of the horizontal (or lateral) semi-circular canal (APA < LSCm and COs < LSCm, respectively), as defined in reference 13. Three curves are digitized on the HSC (between Ld1-Ld2), the PSC (between Ld3-Ld4) and the ASC (between Ld4-Ld5) in order to measure the three corresponding arc lengths (HSCL, PSCL and ASCL, respectively). On the PSC, we measure the arc lengths below and above the plane that best fit the HSC (PSCL.B and PSCL.A, respectively). We also measure six line segments (linear distances) between the following landmarks: (i) Ld3-Ld6 (HELPAM), (ii) Ld4-Ld6 (HELCRS), (iii) Ld5-Ld6 (HELAAM), (iv) Ld3-Ld5 (or “ampular line”) (PAMAAM), (v) Ld4-Ld3 (joining two extremities of the PSC) (CRSPAM), (vi) Ld4-Ld5 (CRSAAM).

We compute the following 11 indices: (i) the ratio dividing the arc length of the PSC (arc length between Ld3-Ld4) situated below and above the HSC (or Posterior Semi-circular Canal index 1, PSCI1), (ii) the ratio dividing the line segment (linear distance) between Ld3 and the plane best-fitting HSC (HSCP) by the Ld3-Ld4 line segment (PSCI2), (iii) the ECL/HSCL, ECL/PSCL, ECL/ASCL, HSCL/PSCL, HSCL/ASCL, PSCL/ASCL indices, (iv) the HEL1 ratio between HELPAM (Ld3-Ld6) and HELCRS (Ld4-Ld6), (v) the HEL2 ratio between HELPAM (Ld3-Ld6) and HELAAM (Ld5-Ld6), (vi) the CRS2 ratio between CRSPAM (Ld3-Ld4) and PAMAAM (Ld3-Ld5). Finally, we measure the following eight angles: (i) between the Ld2-Ld4 and CRSPAM (Ld4-Ld3) line segments (CRS1), (ii) between the PAMAAM and CRSPAM line segments (CRS3), (iii) between the Ld1-Ld2 and Ld2-Ld4 line segments (inclination of the common crus) (CRS4), (iv) between the Ld3-Ld2 and Ld2-Ld4 line segments (inclination of the common crus) (CRS5), (v) between the Ld2-Ld1 and Ld1-Ld5 line segments (inclination of the anterior ampulla) (AAM1), (vi) ) between the Ld4-Ld1 and Ld1-Ld5 line segments (inclination of the anterior ampulla) (AAM2), (vii) between the Ld1-Ld2 and Ld1-Ld6 line segments (inclination of the cochlea) (CO1), (viii) between the Ld1-Ld5 and the Ld1-Ld6 line segments (inclination of the cochlea) (CO2).

In order to assess measurement errors, each variable was measured twice on all the fossil specimens with more than a one-day interval between each trial. For each BM, the repeats cluster closely together. The distance between the repeats of the same BM are always significantly closer to one another than they are to any other specimen. We also observe a high reproducibility between the repeats (Wilcoxon signed-rand tests, p< 0.01), including for OWA that requires the most careful consideration (Fig. S4).

For GMM, we used the R packages Morpho 4.0.5 (see https://cran.r-project. org/web/packages/Morpho/index.html) and Geomorph version 4.0.0 (see https:// cran.r-project.org/package=geomorph). We considered four varying sets of semilandmarks (1, 7, 18 or 48) to represent each SCC. Therefore, we conducted four different analyses by using a total of either 8, 26, 59 or 149 (semi)-landmarks (Fig. S1).

We first performed a Generalized Procrustes analysis (GPA) (with scaling). Following superimposition, we summarized variation in shape space by using a Principal Components Analysis (PCA) and a between-group PCA (bgPCA). We use a permutation test (or Monte-Carlo test, or randomization test) in the R ade4 package (see https://cran.r-project.org/web/packages/ade4/index.html) in order to assess the statistical significance of the bgPCA. The statistical significance is evaluated with the ‘randtest.between’ function by simulating 999 permutations.

To complete our analysis, we additionally analyze our sample using a deformation-based method from computational anatomy. Instead of relying on sparse corresponding features such as GMM, this method estimates and analyzes dense deformations (diffeomorphisms) between non-homologuous curve and shapes. For pairwise alignment of the SCCs, we employ deformetrica 4.0 (www.deformetrica.org)³⁵. The alignment is driven by the metric of currents, which enables a comparison of non-homologous curves. The difference between the SCCs of two specimens is then modelled as the amount of diffeomorphic (smooth and invertible) deformation needed to align them. A more detailed description of the method and application to the shape analysis of fossil hominins can be found in reference 11. Each diffeomorphism is modelled as a vector field which describes the displacements of a regular grid of control points, deforming the underlying space. The displacements of each control point represent the estimated amount of deformation between two curves. For our analysis, we compute a symmetric distance matrix from the control point displacements by calculating the each pairwise distance as the average L2-norm of the displacement vectors. We performed a non-metric Multidimensional Scaling (MDS)³⁶ with an embedding dimension of two, in order to embed the high-dimensional data in a low-dimensional embedding space using the scikit-learn library (version 0.24.2) in Python 3.8.10.

Since we investigate multiple species across a wide range of sizes, the first modes of variation on the PCA and bgPCA, likely represent a combination of size-correlated shape differences and shape differences among taxa unrelated to size^10,22. We therefore investigate the relationships between SCC shape and size changes (allometry). The common allometric component (CAC) represents the pooled within-group direction of covariation (after removing inter-group variation) between the shape variables on the log centroid size^10,22. The RSC scores describe the residuals of the pooled regression analysis, i.e., the non-allometric component. The first principal component of this analysis is referred to as residual shape component 1 (RSC1)¹⁰. We explore variation in the common allometric component (CAC) and residual shape components (RSCs)¹⁰. Finally, we employ the first few PC axes in the calculation of Canonical variate analysis (CVA) that maximizes the between-group variance relative to the within-group variance in order to differentiate a priori defined groups. In all the statistical analyses, the StW 53 and StW 151 specimens are considered as indeterminate and projected onto the (bg)PC1/(bg)PC2 representations to identify their closest neighbours. This is also the case for the DNH 34 and DNH 60 specimens when we first investigate their taxonomic (i.e., before their attribution to P. robustus) (Fig. 1). We differentiate nine a priori defined groups of fossils (Table S1) in the statistical analyses. We define four P. robustus groups: (i) one from Kromdraai Unit Unit P (KW 9600, KW 9700, KW 9900, KW 10840), (ii) one specimen from Kromdraai Unit Q-R (KB 6067), (iii) three from Drimolen (DNH 22, DNH 34, DNH 60) and (iv) three from Swartkrans (SK 83, SK 879, SKW 18/SK52). We also define four A. africanus groups: (i) the holotype from Taung, (ii) one specimen from Makapansgat (MLD 31, (ii) two samples from Sterkfontein in order to distinguish the specimens attributed to this species on a consensual basis (Sts 5, Sts 19, StW 98, StW 329) from those (StW 252/255/259, StW 498, StW 504/505, StW 573 and StW 578) that have been referred by one worker⁷ to a second species with purported robust australopith affinities.

Ethical approval

All the steps of the present study were performed in accordance with relevant guidelines and regulations. No data used in this study involved experimentation, risk or constraint added by the research. Only museum specimens were included in this study and we obtained permissions to access them.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on request. Some datasets and codes supporting the current study have not yet been deposited in a public repository because they are part of further investigation.

Acknowledgments

We thank the South African Heritage Resources Agency (SAHRA), the Evolutionary Studies Institute, University of the Witwatersrand, for curating fossils from Kromdraai and the Ditsong National Museum of Natural History for giving us access to the fossils under their care. We also thank Sifelani Jirah for access to specimens and Frikkie de Beer, Jakobus Hofman for scanning.

This work was supported by the French Ministry of Foreign Affairs (Commission des fouilles), the Centre National de la Recherche Scientifique (CNRS), the Institut des Déserts et des Steppes (Paris), the “Patrick Mathieu Recherche et Conseil” company, the Institut Picot de Lapeyrouse (Toulouse) and the “AESOP plus” programme of Erasmus Mundus (European Union).

Author contributions statement

J.B., G.C and V.Z. contributed to the data collection and analysis. J.B. and V.Z. conceived, designed, and performed the experiments. J.B. and V.Z. wrote the paper. All the authors analyzed the results and reviewed the manuscript.

Competing interests

The authors state here that there are no financial and non-financial competing interests.

Availability of Data and Materials

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No competing interests reported.

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New fossils from Kromdraai and Drimolen, South Africa, and their distinctiveness among Paranthropus robustus

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