PALEVO 1631-0683 Elsevier S1631-0683(16)30122-1 10.1016/j.crpv.2016.10.004 Research article General Palaeontology, Systematics and Evolution Foreword/Avant-propos A morphometric mapping analysis of lower fourth deciduous premolar in hominoids: Implications for phylogenetic relationship between Nakalipithecus and Ouranopithecus Analyse par cartographie morphométrique de la quatrième prémolaire déciduale inférieure chez les hominoïdes : implications pour les relations phylogénétiques entre Nakalipithecus et Ouranopithecus Macchiarelli Roberto Zanolli Clément Morita Wataru a 1 Morimoto Naoki b 1 Kunimatsu Yutaka c Mazurier Arnaud d Zanolli Clément e Nakatsukasa Masato nakatsuk@anthro.zool.kyoto-u.ac.jp b Department of Oral Functional Anatomy, Graduate School of Dental Medicine, Hokkaido University, Hokkaido, Japan Department of Oral Functional Anatomy, Graduate School of Dental Medicine, Hokkaido University Hokkaido Japan Laboratory of Physical Anthropology, Department of Zoology, Graduate School of Science, Kyoto University, Kyoto, Japan Laboratory of Physical Anthropology, Department of Zoology, Graduate School of Science, Kyoto University Kyoto Japan Faculty of Business Administration, Ryukoku University, Kyoto, Japan Faculty of Business Administration, Ryukoku University Kyoto Japan Institut de Chimie des Milieux et Matériaux de Poitiers, UMR 7285, Université de Poitiers, Poitiers, France Institut de Chimie des Milieux et Matériaux de Poitiers, UMR 7285, Université de Poitiers Poitiers France Laboratoire AMIS, UMR 5288 CNRS, Université Toulouse-3, Toulouse, France Laboratoire AMIS, UMR 5288 CNRS, Université Toulouse-3 Toulouse France

Contributed equally to this work.

 16 5-6 S1631-0683(17)X0005-5 Hominin biomechanics, virtual anatomy and inner structural morphology: From head to toe. A tribute to Laurent Puymerail Hominin biomechanics, virtual anatomy and inner structural morphology: From head to toe. A tribute to Laurent Puymerail 655 669 © 2016 Académie des sciences. Published by Elsevier B.V. All rights reserved. 2016 Académie des sciences Full (PDF)

Clarifying morphological variation among African and Eurasian hominoids during the Miocene is of particular importance for inferring the evolutionary history of humans and great apes. Among Miocene hominoids, Nakalipithecus and Ouranopithecus play an important role because of their similar dates on different continents. Here, we quantify the lower fourth deciduous premolar (dp4) inner morphology of extant and extinct hominoids using a method of morphometric mapping and examine the phylogenetic relationships between these two fossil taxa. Our data indicate that early Late Miocene apes represent a primitive state in general, whereas modern great apes and humans represent derived states. While Nakalipithecus and Ouranopithecus show similarity in dp4 morphology to a certain degree, the dp4 of Nakalipithecus retains primitive features and that of Ouranopithecus exhibits derived features. Phenotypic continuity among African ape fossils from Miocene to Plio-Pleistocene would support the African origin of African apes and humans (AAH). The results also suggest that Nakalipithecus could have belonged to a lineage from which the lineage of Ouranopithecus and the common ancestor of AAH subsequently derived.

Afin de mieux comprendre l’histoire évolutive des humains et des grands singes, il est essentiel de caractériser la signature morphologique des hominoïdes africains et eurasiatiques durant le Miocène. Parmi les hominoïdes miocènes, Nakalipithecus et Ouranopithecus ont un rôle crucial en raison de leur existence à des dates similaires sur des continents différents. Nous quantifions ici la morphologie interne de la quatrième prémolaire déciduale inférieure (dp4) des hominoïdes actuels et éteints par des méthodes de cartographie morphométrique et nous examinons les relations phylogénétiques entre ces deux taxons. Nos données suggèrent que les grands singes du Miocène supérieur initial présentent des caractéristiques primitives en général, tandis que les grands singes actuels et les humains montrent des états plus dérivés. Alors que Nakalipithecus et Ouranopithecus partagent un certain degré de similarité morphologique de leur dp4, la dp4 de Nakalipithecus préserve des caractéristiques primitives, tandis que celle d’Ouranopithecus montre des traits plus dérivés. Une continuité phénotypique parmi les fossiles de grands singes africains du Miocène au Plio-Pléistocène semble être compatible avec une origine africaine des grands singes africains et des humains (AAH). Nos résultats suggèrent également que Nakalipithecus pourrait avoir appartenu à la lignée à partir de laquelle celle d’Ouranopithecus et de l’ancêtre commun AAH proviennent.

 Miocene, Hominoid evolution, Micro-CT, 3D morphometrics Miocène, Evolution des hominoïdes, Micro-CT, Morphométrie 3D miscellaneous Handled by Roberto Macchiarelli and Clément Zanolli Introduction

The early Late Miocene is a critical period for the speciation of hominids (great apes and humans). Fossil records indicate that the habitat of the Miocene apes covered Africa and Eurasia, while modern great apes are restricted to Africa and Southeast Asia (Fig. 1). Since Miocene apes were geographically and chronologically widespread, a comparison of African and Eurasian apes while controlling for both time and space is essential to reconstruct the evolutionary history of African great apes and humans (AAH). The fossil record of Miocene apes is increasing but is still very limited, and various questions remain unanswered. A key issue is the taxonomy and phylogeny of the currently known Miocene apes. In this study, we address this issue by focusing on deciduous tooth morphology of two Miocene apes, Nakalipithecus and Ouranopithecus.

Due to their highly mineralized content, dental tissues generally have a greater chance of survival through fossilization than other body parts. Dental morphology has thus routinely been used for functional, phylogenetic, and taxonomic analyses of fossil hominoids (Gómez-Robles et al., 2012, Gómez-Robles et al., 2015, Pilbrow, 2007, Skinner et al., 2009a, Skinner et al., 2008, Skinner et al., 2009b, Suwa et al., 2007 and Suwa et al., 2009). Analyses of tooth morphology are often limited due to dental wear. Recent nondestructive imaging techniques, however, allow us to access the inner structures of teeth. For example, laboratory microcomputed X-ray tomography (μCT) and synchrotron radiation-based imaging enabled extracting the internal structural signature of the teeth of fossil hominins (e.g., Macchiarelli et al., 2006, Macchiarelli et al., 2004, Macchiarelli et al., 2009, Skinner et al., 2015, Smith and Tafforeau, 2008, Smith et al., 2007 and Zanolli et al., 2014).

Teeth in which wear has not reached the dentine provide intact morphological information in terms of the relief of the enamel–dentine junction (EDJ). The EDJ morphology is particularly important for a number of biological reasons. First, it provides insights into development. The EDJ serves as a proxy of the morphogenetic phase of crown formation, where activator–inhibitor signalling and mechanical interactions occur between the inner enamel epithelium and underlying mesenchymal tissues (Jernvall and Jung, 2000, Jernvall and Thesleff, 2012, Kraus and Jordan, 1965 and Morita et al., 2016). Second, the EDJ morphology serves as precursor of the outer enamel surface morphology (Guy et al., 2013, Morita et al., 2014, Skinner et al., 2009a and Skinner et al., 2009b), that is then affected by enamel thickness distribution, reflecting dental functions, such as occlusion and feeding. Third, as various studies have indicated, the EDJ morphology is evolutionarily conserved and useful for estimating phylogenetic relationships among fossil hominoids (Korenhof, 1960, Kraus, 1952, Macchiarelli et al., 2013, Macchiarelli et al., 2006, Olejniczak et al., 2007, Skinner et al., 2010, Skinner et al., 2009a, Skinner et al., 2008, Skinner et al., 2009b, Smith et al., 2000, Smith et al., 1997, Suwa et al., 2007, Zanolli, 2015, Zanolli and Mazurier, 2013, Zanolli et al., 2014, Zanolli et al., 2016 and Zanolli et al., 2015).

While the use of the EDJ confers various advantages, the quantitative evaluation of its morphology is challenging. While the landmark-based geometric morphometrics (Bookstein, 1991) provides a strong means to analyze biological shape variation and has been successfully applied to teeth (e.g., Morita et al., 2014, Skinner et al., 2016, Skinner et al., 2009b, Zanolli et al., 2014 and Zanolli et al., 2012), it is often extremely difficult to identify the homology between different tooth types and between different taxa. The EDJ is feature-rich and exhibits subtle but variably expressed structures such as the accessory cusps, crests, protostylid or Carabelli's trait. Furthermore, even the same tooth within a same individual (intra-individual) or in conspecific individuals can exhibit great variation. To resolve this problem, the authors developed a new method called morphometric mapping (MM) (Morita et al., 2016). This approach is useful for visualizing and quantitatively analysing the EDJ. Methods of tooth MM have great potential to provide new insights in studies of the Miocene apes.

Using MM, we measure the internal 3D tooth structural morphology of Miocene apes and test two hypotheses that have been proposed about the origin of AAH: the African origin hypothesis (H1) (Katoh et al., 2016) and the Eurasian origin hypothesis (H2) (Begun, 2001 and Begun, 2010). The former suggests that stem hominids arose in Africa and African Miocene apes gave rise to the lineage of extant gorillas, chimpanzees, and humans, while species expanded into Eurasia lead to Eurasian Miocene apes and modern orangutans. Thus, this hypothesis postulates phyletic continuity between African Miocene apes (whether known or unknown) and extant great apes and humans. On the other hand, the latter hypothesis suggests that extant large hominids originate from a taxon that migrated from Africa to Eurasia approximately 17 Ma. Based on the rarity (sensu Hull et al., 2015) of fossil apes from the African Late Miocene, H2 postulates that the common ancestor of AAH arose in Eurasia, migrated back to Africa, and gave rise to the lineages of extant African great apes and humans. The discovery of three African fossil apes, Samburupithecus, Chororapithecus, and Nakalipithecus, from the Late Miocene support the African origin hypothesis (Ishida and Pickford, 1997, Kunimatsu et al., 2007 and Suwa et al., 2007; also see Kunimatsu et al., 2016), but no consensus on this issue has yet been reached (Begun, 2005, Begun et al., 2012 and Katoh et al., 2016).

Nakalipithecus was recovered from Nakali, Kenya, and dated to 9.9–9.8 Ma (Kunimatsu et al., 2007). The obtained specimens are a partial mandible with worn teeth and several isolated teeth, suggesting that this species had the size of female gorillas and orangutans. The stable isotope analyses and the associated fossils indicate that the local environment was seasonal sclerophyllous evergreen woodlands (C3-dominated). The Nakali primate fauna contains another large-bodied hominoid and other non-cercopithecoid small catarrhines, as well as cercopithecoids such as colobine monkeys (Y.K., unpublished data). Dentally, Nakalipithecus resembles Ouranopithecus in size and some features but retains less specialized conditions (Kunimatsu et al., 2007).

Ouranopithecus macedoniensis was identified from the early Late Miocene of Greece. It is slightly younger (9.6–8.7 Ma) in chronological age than that at Nakalipithecus (Agustí et al., 2001 and de Bonis and Melentis, 1977). Based on the morphological similarity in the frontal bone (frontal squama and supraorbital tori), premaxilla (clivus and subnasal fossa), palatine, molar morphology, Ouranopithecus is claimed to be similar to the slightly older Eurasian fossil hominoid dryopiths (Begun, 1994, Begun, 2002 and Begun, 2007). It is suggested that Ouranopithecus is close to the ancestry of AAH and australopiths (Andrews et al., 1996, de Bonis et al., 1990, de Bonis and Koufos, 1993, de Bonis and Koufos, 1994, de Bonis and Koufos, 1997, Koufos, 2007 and Koufos and de Bonis, 2004), or that this species underwent convergent evolution as a result of selection for powerful mastication (Begun and Kordos, 1997). However, the phyletic position of this species has not yet been settled (Moyá-Solá and Köhler, 1995). One of the fossil materials that is available for Nakalipithecus and Ouranopithecus is the lower fourth deciduous premolar (dp4). However, relatively little attention has been paid to deciduous dental morphological variation in the context of Miocene hominid evolution.

Generally, it is accepted that the morphology of deciduous dentition is highly conservative (Butler, 1956, Butler, 1971, Dahlberg, 1945, Saunders and Mayhall, 1982, Smith, 1989, Smith and Tillier, 1989 and Suzuki and Sakai, 1973) because it directly reflects genetic variation more strongly (less perturbations caused by environmental noise) than the morphology of permanent teeth due to the earlier and more rapid formation during development (Nanci, 2013). Thus, the dp4 is highly stable in terms of size, morphology and timing of emergence and is considered as the key tooth of the deciduous and permanent molar fields (e.g., Bockmann et al., 2010, Bolk, 1916, Butler, 1956, Butler, 1971 and Saunders and Mayhall, 1982). Recently, the deciduous dentition of fossil hominids has become a focus of some studies (Bailey et al., 2016, Bailey et al., 2014, Bayle et al., 2010, Benazzi et al., 2011b, Benazzi et al., 2011a, Evans et al., 2016, Macchiarelli et al., 2006, Zanolli et al., 2010 and Zanolli et al., 2012).

Applying original techniques to the subtle characters of the dp4 inner structural morphology, we comparatively assessed the structural signal from a multiple morphometric parameters, with the aim of contributing to elucidation of phylogenetic relationships between Nakalipithecus and Ouranopithecus. We also aim to assess the phylogenetic position of these fossil apes from the early Late Miocene compared with extant hominids and extinct Mio-Plio-Pleistocene hominoids. If Nakalipithecus is closer to older species, then H1 is supported. Alternatively, if Ouranopithecus is closer to older species, then H2 is more likely sustained. Specifically, in this study, we analyze the EDJ morphology of dp4 using a novel method, morphometric mapping (MM) (Morimoto et al., 2014, Morimoto et al., 2011, Morimoto et al., 2012, Morita et al., 2016 and Puymerail et al., 2012) to precisely quantify the complex three-dimensional crown morphology.

 Materials and methods

The dental sample comprised 46 mandibular dp4s, including Nakalipithecus (N = 1), Ouranopithecus (N = 1), Miocene African hominoids (N = 8), Plio-Pleistocene australopiths from South Africa (N = 3; including gracile and robust australopiths), and extant great apes and humans (Pan troglodytes: N = 6, Gorilla gorilla: N = 6, Pongo pygmaeus: N = 7, Homo sapiens: N = 14) (Table 1, Fig. 1). Specimens with well-preserved EDJ morphologies were selected for this study (except for the only available A. africanus specimen STS 24 for which the apical extremity of the dentine horns was numerically reconstructed). Both right and left teeth were used to maximize the sample size. For most of the samples, the sex was unknown.

Scans of all specimens were undertaken using μCT scanners at a voxel size between 24 and 72.5 μm. Specimens of Nakalipithecus and other Miocene African fossils were scanned using a peripheral quantitative CT scanner (pQCT: XCT Research SA +) with a tube voltage of 50 kV, a tube current of 50 μA, and a voxel size of 50 μm. Specimen of Ouranopithecus was also scanned at 50 μm (240 kV, 50 μA) with an in-house set-up of the Bundesanstalt für Materialforschung und -Prüfung (BAM) at Berlin (Macchiarelli et al., 2009). Australopiths were scanned using a Nikon XTH 225 ST equipment of the South African Nuclear Energy Corporation (South Africa). Some extant hominids were μCT-scanned using a Viscom X8050-16 system of the University of Poitiers (France) and the rest of the extant great ape specimens were scanned using a ScanXmateA080S μCT scanner with the voxel size 27–63 μm. Micro-computed tomography (μCT) images of left molars were transformed into their mirror images using ImageJ software (National Institutes of Health, Bethesda, MD, USA), and finally, all specimens were regarded as being from the right side. Image segmentation was conducted on each cross section by semi-automatic thresholding methods using Avizo v.6.2 (Visualization Sciences Group Inc., Burlington, MA, USA) and ImageJ software following standard protocols (Coleman and Colbert, 2007, Fajardo et al., 2002 and Spoor et al., 1993).

A three-dimensional surface model was generated from segmented images. First, the outline of the cusp tip and intercuspal ridges of each tooth was manually digitized on the surface model using MeshLab 1.3.3 software (http://meshlab.sourceforge.net/), and the least-squares plane of the occlusal table was computed. Each deciduous premolar was then positioned with the least-squares plane parallel to the xy-plane of the Cartesian coordinate system and centred on the centroid of the occlusal table. Then, the xy-plane shifts to a cervical plane that was calculated from the digitized cervical line.

In this study, the following three morphometric parameters were used: the mean curvature of the EDJ surface (c) was calculated analytically for each vertex of the 3D model, whereas the height from the cervical plane (h) and the radius from the centroid of the cervical line (r) were calculated directly from the 3D coordinates of the surface mesh. These three parameters represent the following morphological features: c, surface relief; h, height and relative positions of cusps; r, relative diameter in a horizontal direction.

For each specimen, these three variables (c, h, and r) were sampled from each cross-sectional outline and around the entire EDJ surface. The EDJ surface was digitally sectioned equiangularly (L = 300) by a plane orthogonal to the xy-plane and through the centroid. In each cross section, the outline that runs from the point located just above the centroid of the coordinate system to the point at the level of the xy-plane (equal to cervix) was parameterized by elliptic Fourier analysis (EFA) equidistantly (K = 300). EFA was used to reduce noise and define parametric outline functions (Kuhl and Giardina, 1982). They were mapped onto a polar coordinate system (d, θ), where d denotes the normalized position along each cross-sectional outline (d = 0→1: centroid→cervix) and θ denotes the anatomical direction [θ = 0°→360°: buccal (0°)→mesial (90°)→lingual (180°)→distal (270°)→buccal (360°)]. The EDJ could be visualized using 2D morphometric maps M (d, θ), and the distributions c (d, θ), h (d, θ), and r (d, θ) could be represented as K × L matrices, where K and L denote the numbers of elements along d and θ, respectively (K = L = 300).

The effects of scaling were corrected by normalization of the variables c, h, and r using cervical size (i.e., the square root of the summed squared distances of each value of r at the cervix [d = 300]). This is analogous to the ordinary geometric morphometric method (Bookstein, 1991). Each row of the K × L matrix for each specimen was weighted by the length of each cross-sectional outline and the value of r for each element.

For the comparative analysis of the morphometric maps M i of all specimens (i = 1, 2, …, N), differences between specimens in orientation around the centroid (θ) had to be minimized. First, all specimens were pre-aligned manually to orient them in an anatomical direction similar to that described above. Second, optimal fitting was achieved by iteratively minimizing inter-specimen distance in Fourier space through rotation around θ (z-axis). Furthermore, 2D-Fourier transforms F(M i ) of all M i were calculated (M has natural periodicity in θ), resulting in K × L sets of Fourier coefficients that represented a specimen's shape of the EDJ surface as a point in the multidimensional Fourier space. Major patterns of shape variation among fossil and extant specimens were inspected using principal component analysis (PCA) on the low-frequency domain of Fourier coefficients (i.e., low-pass filtering in Fourier space [see Morita et al., 2016, for details]) of the mean configurations of extant genera (i.e., the eigen analysis was carried out on the group means) employing the covariance matrix. To take into account the intraspecific variation of the extant samples, PC scores for all of the original individuals were computed a posteriori using vector products. This method is also called between-group PCA (bgPCA) (Almécija et al., 2013, Gunz et al., 2012 and Mitteroecker and Bookstein, 2011). To facilitate visual inspection and morphological interpretation of the results of bgPCA, morphometric maps were reconstructed by transforming an arbitrary point in bgPC space into its corresponding sets of Fourier coefficients and then applying an inverse transformation. Morphometric maps were visualized using a false-colour mapping scheme. All calculations were performed in MATLAB 8.1 (MathWorks, Natick, MA, USA) (see Morita et al., 2016, for details). Phenotypic distances among specimens were calculated as Euclidean distances in morphospace. The neighbour-net diagram was computed with SplitsTree4 (Huson and Bryant, 2006). The phylogeny of extant anthropoid taxa was based on the consensus tree downloaded from the 10 kTree website (ver. 3; http://10ktrees.fas.harvard.edu/) (Arnold et al., 2010) and modified with Mesquite v. 3.05 (Maddison and Maddison, 2015) to condense the tips at the generic level. We reconstructed the evolutionary history of dp4 in extant and extinct hominoids by projecting the phylogenetic tree into morphospaces (= bgPC shape space) using the R package “phytools” (Revell, 2012).

 Results

Fig. 2 shows a visual comparison of the 3D representation of dp4 EDJ morphology and its corresponding MM for Nakalipithecus and Ouranopithecus specimens. The EDJ surface and MM show marked features that are associated with the characteristics of the enamel surface. Hence, we used anatomical terms for the enamel surface to indicate EDJ features.

In a Nakalipithecus specimen (Fig. 2A), MM of the surface curvature (c-M) captured well-defined anatomical features: five cusps (protoconid, metaconid, hypoconid, entoconid, and hypoconulid), ridges that are located between the cusps and delimit the occlusal table, and the mesial transverse crest. The Nakalipithecus dp4 has a weakly developed buccal cingulum, located on the mesiobuccal face of the protoconid and small depressions at the bases of the buccal grooves and at the distobuccal side between the hypoconid and the hypoconulid. MM of height (h-M) from the cervix captured the relative location and distribution of the cusps, showing a high and relatively large metaconid, a relatively small mesial fovea and a broad talonid basin. MM of the radius (r-M) from the centroid of the cervical line gave a comprehensive view of the horizontal dimensions of the EDJ, showing elongation in mesiobuccal, distobuccal, and distolingual directions.

In an Ouranopithecus specimen (Fig. 2B), c-M demonstrated five cusps and well-developed ridges between cusps. The mesial fovea is relatively large with a secondary ridge running in the buccolingual direction. A small cingulum and relatively large depression are present on the buccal valley between the protoconid and the hypoconid. The h-M showed low cusps relatively to Nakalipithecus, especially to the hypoconulid, and a broad talonid. The trigonid and talonid are located distantly. The r-M also captured a larger dimension in a distal direction.

MM-based shape variation of the entire sample was explored using bgPCA in which all three bgPC axes were computed (see Supplementary Figs. S1–S6 to see the contribution of each morphometric parameter). In this shape space, extant great apes are well divided from each other (Fig. 3 and Fig. 4; see also Supplementary Fig. S7 for a 3D plot). The bgPC1 (41% of variance) largely separates Pongo from other hominids. A positive value along bgPC1 is associated with the following features: sharp metaconid and hypoconid and concave mesial fovea (c-M); high metaconid and hypoconid (h-M); and larger dimension in a mesial direction (r-M). A negative value along bgPC1 is associated with the following features: sharp buccal and lingual inter-cusp ridges and interrupted occlusal outline at mesial and distal ridges (c-M); high protoconid and lingual two cusps (h-M); and mesiobuccal–distolingual elongation (r-M). The bgPC2 (35% of variance; Fig. 3) captured differences between Homo and other hominids from higher and more pointed five cusped dp4s to teeth with less developed hypoconulid. A positive value along bgPC2 is associated with the following features: clear relief, that is, pointed cusps and incised grooves (c-M); five well-defined cusps, especially on talonid with higher hypoconid and entoconid (h-M); and larger in hypoconid and entoconid directions (r-M). On the other hand, the negative value along bgPC2 is associated with the following features: pointed cusps (except for the hypoconulid) and a reduced talonid basin (c-M); four cusps with a notably high metaconid and diminished hypoconulid (h-M); and larger in a distal direction and constricted at the middle of buccal and lingual side (r-M).

Further, bgPC3 (Fig. 4) separates Pan from the other taxa. A positive value along bgPC3 (24% of variance) is associated with the following features: sharp in both cusp tips and inter-cusp ridges (c-M); higher cusps (h-M); and larger in a mesiobuccal direction (r-M). Conversely, a negative value along bgPC3 is associated with the following features: lower dentine horns and marginal ridges tips (c-M); significantly lower cusps (h-M); and larger in a distolingual direction (r-M).

Considering the shape space defined by bgPC1 and bgPC2 (Fig. 3), Nakalipithecus and Ouranopithecus do not overlap with the range of extant great apes. On the other hand, they differ from each other along bgPC2. They occupy close positions in bgPC1 and bgPC3 space (Fig. 4) and are located close to the distribution of Pan. Miocene African fossil hominoids as well as Plio-Pleistocene australopiths occupy a general mean position among extant hominids (see also Supplementary Figs. S8 and S9: PC plots with only fossil specimens).

Between-taxon distances were evaluated by calculating the Euclidean distances in morphospace (bgPC1-bgPC3), and distance-based similarity patterns were visualized as a neighbour-net diagram (Fig. 5). Most of the conspecific specimens of extant great apes are connected to each other and form taxon-specific clusters. The clusters of extant great apes are located distant from each other. On the other hand, Early and Middle Miocene African hominids (MAH: Proconsul, Ugandapithecus, Afropithecus, and Nacholapithecus) are located close to the centre of this diagram, except for two Ugandapithecus specimens. The Nakalipithecus specimen is not located so distant from the centre of this diagram, while the Ouranopithecus specimen is situated more peripherally, in the Pan cluster.

Fig. 6 (bgPC1 vs. bgPC2) and Fig. 7 (bgPC1 vs. bgPC3) show a phylo-morphospace projection of the phylogeny of extant great apes onto bgPCs of dp4 in extant and fossil species, reconstructing hypothetical ancestral morphologies as internal nodes. MAH are largely located close to the basal node of the great apes (=stem hominid). Plio-Pleistocene australopith specimens are also located close to the basal node. The inferred Gorilla-Pan-Homo last common ancestor (GLCA) and Homo-Pan last common ancestor (CLCA) are located close to each other. The Nakalipithecus specimen is located closer to both GLCA and CLCA than Ouranopithecus. Fig. 8 shows the average MM of dp4 EDJ of four extant great ape species, MAH, and the inferred states of stem hominids, GLCA and CLCA. The average shape of Homo is characterized by greater surface relief with high talonid cusps, particularly the entoconid, and a large radius in the lingual direction. The average shape of Pan is characterized by concave mesial fovea, high and sharp trigonid cusps, and a large diameter in the mesiobuccal direction. The average shape of Gorilla is characterized by high and pointed lingual cusps and a large diameter in the distolingual direction with a buccolingual constriction at the middle. The average shape of Pongo is characterized by a broad and concave talonid basin, high and mesially located metaconid, and a large diameter in the mesial direction. The average shape of MAH is characterized by five pointed cusps, a narrow and concave mesial fovea, and a large dimension in the mesiobuccal direction associated to a small dimension in the lingual direction. MM of the inferred stem hominid reconstructed from morphospace resembles MAH in exhibiting five well-developed cusps and a concave mesial fovea. The inferred morphologies of GLCA and CLCA are similar to each other, but GLCA exhibits a larger diameter in the distal direction while CLCA has a higher protoconid and entoconid than GLCA.

 Discussion

The data of MM-based PCA showed that the dp4 morphologies of the extant great apes were well divided from each other (Fig. 3 and Fig. 4). Most specimens of Miocene African hominoids were located around the grand mean of extant hominids, while extant taxa were located more peripherally in the morphospace. The similarity pattern of living and fossil individuals was also visualized as a neighbour-net diagram (Fig. 5). The neighbour-net diagram showed that Miocene hominoids were located around the central node of the diagram, whereas specimens of extant taxa were located peripherally.

Given that extant great ape taxa exhibit a great degree of diversification and that chronologically distant Plio-Pleistocene hominins and Miocene hominoids exhibit close dp4 morphology, it is not likely that modern great apes retain primitive morphology of the dp4. Alternatively, the analysis of dp4 morphology suggests that modern great apes and humans represent a derived state and that Plio-Pleistocene hominins represent a primitive state retained from Miocene African hominids. This is consistent with the evolutionary scenario proposed in various recent studies of fossil hominins (Almécija et al., 2015, Almécija et al., 2013, Lovejoy et al., 2009, Moyá-Solá et al., 2009 and Nakatsukasa and Kunimatsu, 2009).

Our results indicate that extant hominids evolved taxon-specific features of dental morphology from the ancestral state in Miocene African hominoids, probably reflecting taxon-specific adaptations, for example, a preference for ripe fruit in Pan (Janmaat et al., 2016 and Yamagiwa and Basabose, 2006) and folivory with seasonal frugivory in Gorilla (Remis, 1997). These differences in dietary habitat would also be related to enamel thickness and its distribution (Kono, 2004 and Vogel et al., 2008). In the morphospace analyzed in this study, extant great apes and humans exhibit diversified morphologies (Fig. 3 and Fig. 4). It is likely that morphological diversification is associated with such specialization in the direction from centroid to taxon-specific clusters in the morphospace (Fig. 3 and Fig. 4). While such an adaptive scenario sounds reasonable, caution is warranted. Since form-function relationships of dp4 morphology are yet to be investigated in detail, it remains elusive to what extent the taxon-specific dp4 morphologies reflect phylogenetic effects that are not directly related to functional adaptations.

In the context of interpreting the mandibular tooth morphology of Ouranopithecus, Koufos and de Bonis (2004) used the degree of “molarization” in dp4 as a criterion to determine primitive versus derived states. They suggested that a greater degree of molarization of dp4 (i.e., more molar-like dp4) indicates a derived feature. Further, they concluded that the dp4 of Ouranopithecus represents a derived state since it has a more molarized dp4 than chimpanzees and gorillas (see also Macchiarelli et al., 2009). Our MM-based approach shows a new perspective on evolutionary history. Fig. 8 shows that MAH in general have five cusps, which are similarly developed and low. The ancestral morphologies reconstructed as internal nodes also retain this hypothetical generalized five-cusp state. On the other hand, extant great apes and humans also show five cusps but each species exhibits specific cusps that are more developed than the others (Homo, talonid; Pan, trigonid; Gorilla, lingual; Pongo, metaconid). This indicates that each of the extant species evolved taxon-specific features independently from the generalized dp4 shape represented in MAH.

The MM-based PCA also gave insights into the evolution of dp4 morphology in hominins. Plio-Pleistocene Australopithecus and Paranthropus are not included in the range of extant hominids but occupied similar locations to the Miocene African hominoids in the morphospace (Fig. 3 and Fig. 4). This indicates that the deciduous teeth of Plio-Pleistocene hominins retain low-specialized morphology and are different from those of modern Homo, which show derived features. The robust australopiths (Paranthropus) have been traditionally considered to be specialized herbivores, consuming harder and relatively brittle food (Grine and Kay, 1988). Recent isotopic and dental microwear analyses, however, revealed their broad range of food resources in adulthood (Balter et al., 2012, Scott et al., 2005, Sponheimer et al., 2013 and Sponheimer and Lee-Thorp, 1999).

Our data show that gracile and robust australopith individuals exhibit overall similar dp4 morphologies (Fig. 3 and Fig. 4). This contrasts with considerable differences of masticatory structures in gracile and robust australopiths. The similar dp4 EDJ morphology of Australopithecus and Paranthropus may reflect the evolutionary degree of conservation of deciduous teeth. It may also reflect genetic cohesiveness among southern African australopiths.

The Nakalipithecus specimen resembles Early and Middle Miocene African hominoids in the bgPC1 and bgPC2 axes (Fig. 3) and is located close to the central node of the neighbour-net diagram (Fig. 5). This indicates that Nakalipithecus retains dp4 ancestral morphology that is shared with earlier African fossil hominoids. These results suggest the African origin of Nakalipithecus. On the other hand, the Ouranopithecus specimen is far from the African Mio–Pliocene hominoid group, including Nakalipithecus and australopiths in Fig. 3, and is located more distant from the stem of the diagram (Fig. 5). This indicates that dp4 of Ouranopithecus is more derived than that of Nakalipithecus, though morphological similarities between Nakalipithecus and Ouranopithecus have found in size and some dentognathic features, such as mandible, permanent upper canine, and lower premolars (Kunimatsu et al., 2007). In fact, Nakalipithecus and Ouranopithecus specimens resemble each other along bgPC1 and bgPC3 (Fig. 4) and are located relatively close to each other in the neighbour-net diagram (Fig. 5). Assuming that this similarity and character state between them reflect phylogeny, Nakalipithecus could be a member of taxa from which the lineage to which Ouranopithecus belongs is derived. This “ancestral-descendant” relationship between Nakalipithecus and Ouranopithecus is consistent with the currently known chronology of these taxa (Nakalipithecus: 9.9–9.8 Ma, Ouranopithecus: 9.6–8.7 Ma). The alternative interpretation is convergence, i.e., the structural signal captured in this study indicates independent dietary adaptations in these taxa rather than phyletic history. If this were the case, then Ouranopithecus might be a relatively derived taxon among the Miocene Eurasian ape lineage. To draw more definitive conclusions on ape evolution during the Miocene, further analyses should be performed that encompass further data, especially on Eurasian hominids, such as dryopiths and Sivapithecus. In any case, morphological similarity among African fossils from Miocene hominoids to Plio-Pleistocene australopiths suggests that African apes maintain the phenotypic identity without genetic contribution from Eurasian lineages, which is consistent with the African origin hypothesis (H1) of AAH. Considering the ancestral state of morphology, locality, and chronology, Nakalipithecus may not be a direct ancestor itself but could be one of the stem species lineages for membership in the African great ape and human clade.

We analyzed the dp4 morphology of fossil and living hominids using novel geometric morphometric methods. Phenotypic continuity in African fossils from Miocene to Plio-Pleistocene indicated in this study is consistent with the African origin of AAH. Dental character states and moderate morphological similarity among Nakalipithecus and Ouranopithecus would reflect their “ancestral-descendant” relationship, but further comparative studies are needed to clarify phylogenetic relationship of Miocene apes.

 Acknowledgements

This paper is a tribute to Laurent Puymerail, who has prematurely passed away, leaving a great contribution to the three-dimensional morphometric mapping in paleoanthropology. The authors thank G. Suwa, R. Kono, T. Domon and S. Takahashi for thoughtful discussion and comments. We are also grateful to Y. Kikuchi for collecting datasets. We thank L. de Bonis and R. Macchiarelli for having provided the μCT record of Ouranopithecus macedoniensis discussed in this paper. We also thank S. Potze for the access to the Australopithecus and Paranthropus specimens stored at the Ditsong National Museum of Natural History of Pretoria, South Africa, within the context of an ongoing research project led by J. Braga. We appreciate the Government of Kenya and the National Museums of Kenya for permitting us to examine fossil materials. We appreciate the assistance from the JSPS Nairobi Research Station during our research in Kenya. This work was in part supported by JSPS KAKENHI Grant Nos. 18255006, 22255006, 24000015, 25257408, and 16H02757 to M.N., and 15H05609 to N.M.

 Supplementary data

The following are the supplementary data to this article:

Variation along between-group principal components (bgPCs) 1 and 2 using only c-map.

Variation along between-group principal components (bgPCs) 1 and 3 using only c-map.

Variation along between-group principal components (bgPCs) 1 and 2 using only h-map.

Variation along between-group principal components (bgPCs) 1 and 3 using only h-map.

Variation along between-group principal components (bgPCs) 1 and 2 using only r-map.

Variation along between-group principal components (bgPCs) 1 and 3 using only r-map.

Three-dimensional variation along between-group principal components (bgPCs) 1, 2, and 3 using all three (c, h, and r) maps.

Variation along principal components (PCs) 1 and 2 using only fossil specimens.

Variation along principal components (PCs) 3 and 4 using only fossil specimens.

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Chrono-spatial distribution of hominids and large-bodied hominoids used in this study.

Distribution chrono-spatiale des hominidés et hominoïdes étudiés dans le présent travail.

Three-dimensional model of the enamel–dentine junction (stereo picture) and corresponding morphometric maps [surface curvature (c), height from the cervical line (h), and radius from the centroid of the occlusal table (r), from left to right] of Nakalipithecus (mirrored from original specimen) (A) and Ouranopithecus (B). Scale bar: 5 mm. prd: Protoconid, med: metaconid, mtc: mesial transversal crest, hyd: hypoconid, end: entoconid, hld: hypoconulid, mf: mesial fovea, tab: talonid basin, b: buccal, m: mesial, l: lingual, d: distal.

Modèle en trois dimensions de la jonction émail–dentine (image stéréo) et cartes morphométriques correspondantes [la courbure de surface (c), la hauteur de la ligne cervicale (h), et le rayon depuis le centroïde de la surface occlusale (r), sont indiqués de gauche à droite] de Nakalipithecus (symétrique du spécimen original) (A) et Ouranopithecus (B). Barre d’échelle : 5 mm. Prd : Protoconide, med : métaconide, mtc : crête transversale mésiale, hyd : hypoconide, end : entoconide, hld : hypoconulide, mf : fovéa mésiale, tab : bassin du talonide, b : buccal, m : mésial, l : lingual, d : distal.

Variation along between-group principal components (bgPCs) 1 and 2. Morphometric maps (c, h, and r, from top to bottom and left to right) visualizing ±1 s.d. along each bgPC axis. b: Buccal, m: mesial, l: lingual, d: distal.

Variation le long des composantes principales (bgPCs) 1 et 2 de l’analyse intergroupes. Les cartes morphométriques (c, h, et r, de haut en bas et de gauche à droite) représentent ± 1 écart-type le long de chaque axe bgPC. b : Buccal, m : mésial, l : lingual, d : distal.

Variation along between-group principal components (bgPCs) 1 and 3. Morphometric maps (c, h, and r, from top to bottom and left to right) visualizing ±1 s.d. along each bgPC axis. b: buccal, m: mesial, l: lingual, d: distal.

Variation le long des composantes principales (bgPCs) 1 et 3 de l’analyse intergroupes. Les cartes morphométriques (c, h, et r, de haut en bas et de gauche à droite) représentent ± 1 écart-type le long de chaque axe bgPC. b : Buccal, m : mésial, l : lingual, d : distal.

Neighbour-net diagram of all specimens based on Euclidean distances in morphospace.

Diagramme Neighbour-net de tous les échantillons, en fonction de leur distance euclidienne dans le morpho-espace.

Phylo-morphospace of hominid dp4s. The phylogeny of extant great apes is projected into a plot defined by between-group principal components (bgPCs) 1 and 2 of the covariance matrix among extant species means. Internal node morphologies [stem hominid, extant African ape and human ancestor (GLCA), and the chimpanzee/human last common ancestor (CLCA)] were reconstructed using squared-change parsimony.

Espace morpho-phylogénétique des dp4s hominidés. La phylogénie des grands singes actuels est projetée dans une parcelle définie par les composantes principales (bgPCs) 1 et 2 de l’analyse intergroupes basée sur la matrice de covariance entre les moyennes d’espèces existantes. La morphologie des nœuds internes [représentant l’hominidé souche, ancêtre africain des grands singes existants et des humains (GLCA) et le dernier ancêtre commun chimpanzé/humain (CLCA)] a été reconstruite à l’aide de la méthode squared-change parsimony.

Phylo-morphospace of hominoid dp4s. The phylogeny of extant great apes is projected into a plot of between-group principal components (bgPCs) 1 and 3. Internal node morphologies [stem hominid, extant African ape and human ancestor (GLCA), and the chimpanzee/human last common ancestor (CLCA)] were reconstructed using squared-change parsimony.

Espace morpho-phylogénétique des dp4s d’hominidés. La phylogénie des grands singes actuels est projetée dans une parcelle définie par les composantes principales (bgPCs) 1 et 3 de l’analyse intergroupes basée sur la matrice de covariance entre les moyennes des espèces existantes. La morphologie des nœuds internes [représentant l’hominidé souche, ancêtre africain des grands singes existants et des humains (GLCA) et le dernier ancêtre commun chimpanzé/humain (CLCA)] a été reconstruite à l’aide de la méthode squared-change parsimony.

Average morphometric maps (c, h, and r from left to right) of Homo, Pan, Gorilla, Pongo, and Early and Middle Miocene African Hominoids (MAH). The stem hominid, extant African ape and human ancestor (GLCA), and the chimpanzee/human last common ancestor (CLCA) were reconstructed from the inferred ancestral state using bgPC scores. Arrows indicate marked cusps (Homo: talonid, Pan: trigonid, Gorilla: lingual, Pongo: metaconid).

Cartes morphométriques moyennes (c, h, et r de gauche à droite) de Homo, Pan, Gorilla, Pongo et d’hominoïdes africains du Miocène inférieur et moyen (MAH). L’hominidé souche, ancêtre africain des grands singes existants et des humains (GLCA) et le dernier ancêtre commun chimpanzé/humain (CLCA) ont été reconstruits à partir de l’état ancestral inféré en utilisant les résultats des bgPC. Les flèches indiquent les cuspides les plus marquées (Homo : talonide, Pan : trigonide, Gorilla : cuspides linguales, Pongo : métaconide).

Deciduous lower second molars used in this analysis.

Liste des secondes molaires lactéales inférieures étudiées dans ce travail.

Table 1 Taxon Specimens Nakalipithecus KNM-NA 46435 Ouranopithecus RPl 83 Proconsul KNM-RU 1865 Ugandapithecus KNM-ME 10 KNM-LG 1460 KNM-SO 451 KNM-RU 1767 Afropithecus KNM-MO 26 Nacholapithecus KNM-BG 15331 KNM-BG 15334 Australopithecus Sts 24 Paranthropus SK 61 SK 63 Homo (N = 14) Pan (N = 6) Gorilla (N = 6) Pongo (N = 7)