Bringing up Baby: Developmental Simulation of the Adult Cranial Morphology of Rungwecebus Kipunji

Home , Arunachal macaque, Gelada, Kipunji, Mandrill, Yellow baboon

THE ANATOMICAL RECORD 293:388–401 (2010)

Bringing Up Baby: Developmental Simulation of the Adult Cranial Morphology of Rungwecebus kipunji

MICHELLE SINGLETON,1* KIERAN P. MCNULTY,2 STEPHEN R. FROST,3 2 3 JOHN SODERBERG, AND EMILY H. GUTHRIE 1Department of Anatomy, Midwestern University, Downers Grove, Illinois 2Department of Anthropology, Evolutionary Anthropology Laboratory, University of Minnesota, Minneapolis, Minnesota 3Department of Anthropology, University of Oregon, Eugene, Oregon

ABSTRACT Rungwecebus kipunji is a recently discovered, critically endangered primate endemic to southern Tanzania. Although phenetically similar to mangabeys, molecular analyses suggest it is more closely related to Papio or possibly descended from an ancient population of baboon-mangabey hybrids. At present, only a single kipunji specimen, an M1-stage juvenile male, is available for study; thus, the cranial morphology of the adult kipunji is unknown. In this study, we used developmental simulation to estimate the adult kipunji’s 3D cranial morphology. We examined variation in cercopithecine developmental vectors, applied selected vectors to the juvenile cranium, and compared the resulting simulated adults to actual adult male papionins. Differences between papionin developmental vectors were small and statistically insignificant. This uniformity suggests conservation of an ancestral papionin developmental program. Simulated kipunji adults were likewise extremely similar. As a group, the simulated adults were morphometrically distinct from other papionins, corroborating the kipunji’s generic status. Simulated adults were phenetically most similar to Lophocebus aterrimus but were distinguished from all adult papionins by the same unique traits that characterize the kipunji juvenile: a tall neurocranium, broad face, short nasal bones, concave anteorbital profile, and dorsally rotated palate. This concordance between juvenile and estimated-adult morphologies confirms that papionin cranial shape is largely established before M1 eruption. The estimated kipunji adult’s neurocranium strongly resembles that of Papio, providing the first cranial evidence supporting their phylogenetic relationship. If the kipunji does indeed have a hybrid origin, then its phenetic affinity to L. aterrimus favors Lophocebus as the proto-kipunji’s paternal lineage. Anat Rec, 293:388–401, 2010. VC 2009 Wiley-Liss, Inc.

Key words: mangabey; Lophocebus; Papio; hybrid origin; permutation test

Additional Supporting Information may be found in the online *Correspondence to: Michelle Singleton, Department of Anat- version of this article. omy, Midwestern University, 555 31st Street, Downers Grove, IL 60515. Fax: 630-515-7199. E-mail: [email protected] Institutional sponsor: NYCEP, Midwestern University, The University of Minnesota, The University of Oregon, The Field Received 28 May 2009; Accepted 2 October 2009 Museum of Natural History, The University of Illinois– DOI 10.1002/ar.21076 Urbana-Champaign; Grant sponsor: NSF; Grant number: Published online 4 December 2009 in Wiley InterScience (www. IIS-0513660. interscience.wiley.com).

VC 2009 WILEY-LISS, INC. DEVELOPMENTAL SIMULATION OF Rungwecebus 389 INTRODUCTION The kipunji, Rungwecebus kipunji (Cercopithecinae: Papionini), is a critically endangered primate endemic to the Udzungwa Mountains and Southern Highlands of Tanzania (Rovero et al., 2009). Following its discovery in 2003, the kipunji was referred to the mangabey genus Lophocebus (Jones et al., 2005; Ehardt and Butynski, 2006), but subsequent molecular phylogenetic analyses found it to be the sister taxon to the baboon genus Papio (Davenport et al., 2006; Olson et al., 2008). To accommo- date this new papionin, the monotypic genus Rungwece- bus was created (Davenport et al., 2008). Most recently, further mitochondrial studies (Burrell et al., 2009; Zinner et al., 2009) have found evidence that the kipunji originated through hybridization between female yellow baboons (Papio hamadryas cynocephalus) and as yet unidentified, mangabey-like sires. The genetic material used in these studies derives Fig. 1. Papionin phylogenetic relationships. Following Harris (2000), from a single, juvenile male voucher specimen. This the Lophocebus-Papio-Theropithecus (LPT) clade is shown as an specimen’s immaturity prevents the evaluation of many unresolved trichotomy. Nuclear sequences suggest a sister relation- characters considered diagnostic for African papionins ship (arrow) between Rungwecebus and Papio (Davenport et al., 2006; (Fleagle and McGraw, 2002; Davenport et al., 2006; Olson et al., 2008); however, kipunji mitochondrial sequences cluster McGraw and Fleagle, 2006; Gilbert, 2007), hindering within Papio and closest to southeastern populations of Papio hamadryas cynocephalus (Burrell et al., 2009; Zinner et al., 2009). conventional assessments of its morphological affinities. Singleton’s (2009) comparative analysis of subadult papionin cranial shape found the kipunji juvenile to be mangabeys (Cercocebus, Lophocebus), mandrills and morphometrically distinct from other papionins at a drills (Mandrillus), baboons (Papio), and geladas (Thero- level consistent with generic status and to have stronger pithecus) (Szalay and Delson, 1979; Strasser and Delson, phenetic affinities to macaques and Cercocebus than to 1987). Molecular phylogenetic analyses have consistently its closest relatives, Lophocebus and Papio. shown the mangabeys to be diphyletic; Cercocebus and Singleton’s findings raise at least as many questions Mandrillus constitute a sister clade to the Lophocebus- as they answer, foremost among them the nature of Papio-Theropithecus (LPT) clade, whose internal phylo- adult kipunji morphology. Although several studies have genetic relationships remain unresolved (Barnicot and shown that taxonomic differences in primate cranial Hewett-Emmett, 1972; Cronin and Sarich, 1976; Hewett- shape are established early in development (O’Higgins Emmett et al., 1976; Disotell, 1994; Van Der Kuyl et al., and Collard, 2002; McNulty et al., 2006; Singleton, 1995; Harris and Disotell, 1998; Page et al., 1999; Har- 2009), it has also been demonstrated that convergence of ris, 2000). ontogenetic trajectories can diminish or eliminate juve- Though it no longer references a monophyletic group, nile shape differences between papionin species (Leigh, the term ‘‘mangabey’’ has persisted as convenient short- 2007). If shape differences cannot be assumed to be sta- hand for ecomorphologically similar, medium-sized Afri- ble throughout ontogeny, alternate methods must be can papionins characterized by moderate facial used to infer the adult morphology of isolated juvenile prognathism, well-developed suborbital fossae, hard- specimens. Morphometric simulation can be a powerful object diets, bunodont molars, and varying degrees of tool for evaluating taxonomic affinities of unknown arboreality (Kingdon, 1997). The mangabey phenon con- specimens (Richtsmeier and Walker, 1993; McNulty trasts with the large-bodied, long-faced, predominantly et al., 2006; Baab and McNulty, 2008). We adopted this terrestrial ‘‘baboons,’’ Mandrillus, Papio, and Theropi- approach here and estimated adult kipunji cranial mor- thecus. The extensive homoplasy implied by the phyloge- phology by applying a range of cercopithecine develop- netic distribution of these ecomorphs has made African mental vectors to the original kipunji specimen. papionins a touchstone for the study of primate morpho- Simulated adults were compared with adult males of logical variation and evolution (Lockwood and Fleagle, other papionin species and to each other to evaluate the 1999; Collard and O’Higgins, 2001; Singleton, 2002; effect of developmental model choice, characterize adult Leigh, 2007). kipunji cranial morphology, and explore the kipunji From its initial description in 2005, the kipunji was adult’s phenetic affinities. identified as a mangabey. Jones et al. (2005), citing the kipunji’s noncontrasting eyelids and arboreal habitus, BACKGROUND referred it to Lophocebus as a new species, L. kipunji, Papionin Phylogeny and Rungwecebus but noted differences from Central African Lophocebus species in pelage, tail carriage, and vocalization. This Systematics matter was revisited when the first physical voucher The Old World monkey tribe Papionini (Primates: Cer- specimen, an M1-stage juvenile male with permanent copithecinae) encompasses two geographically segre- incisors erupting (see Fig. 2), provided genetic material gated subtribes (see Fig. 1). The predominantly Asian for phylogenetic analysis (Davenport et al., 2006; Olson macaque radiation (Macacina) is the sister taxon to the et al., 2008). Mitochondrial sequences (12s rRNA, COI, predominantly African Papionina, which encompasses and COII) consistently linked the kipunji with Papio to 390 SINGLETON ET AL. mata turning smoothly toward the skull posteriorly, and a mandibular corpus that deepens anteriorly. However, the specimen’s age prevents evaluation of key morphological traits such as the relative size of the permanent P4 and the configurations of the temporal and nuchal lines (McGraw and Fleagle, 2006; Gilbert, 2007). Like- wise, the specimen’s postcrania are too immature for reliable assessment of characters that distinguish the African clades (Fleagle and McGraw, 1999, 2002; Daven- port et al., 2006). In contrast with many qualitative traits, which often cannot be evaluated until late in ontogeny, morphometric comparisons are possible at any developmental stage. Singleton’s (2009) geometric morphometric analysis of Fig. 2. Cranium of the first physical voucher specimen of Rungwe- the kipunji juvenile’s cranial shape used a developmen- cebus kipunji, FMNH 187122, an M1-stage juvenile male. Figure repro- tally equivalent comparative sample to evaluate its mor- duced and adapted from Singleton (2009) with permission of the phometric distinctiveness and phenetic relationships. Journal of Human Evolution and Elsevier. She found the kipunji to be phenetically distant from Lophocebus and Papio and closer to Cercocebus and Macaca, two outgroups of the LPT clade. Overall, phe- the exclusion of Lophocebus. Results based on nuclear netic distances between the kipunji and other papionins sequences were mixed, with two sequences (X-chromo- were comparable with those between recognized genera. some intergenic region, LPA) strongly supporting the Although the kipunji exhibits some traits (e.g., anterior Papio-kipunji sister relationship, one (1, 3-GT) providing malar position, a sinuous zygomatic arch) associated weak support, and two (TSPY, CD4) failing to resolve with Lophocebus, its unrestricted suborbital fossa, para- the LPT clade. These results, as well as the apparent sagittally oriented zygomatic arch, and long auditory absence of shared features between the kipunji and tube are not typical of this genus (Groves, 1978). Fur- Papio, led Davenport et al. (2006) to erect a new genus, thermore, kipunji features such as a relatively tall neu- Rungwecebus. rocranium; broad face and cranial base; short nasal More recently, two independent studies (Burrell et al., bones and concave anteorbital profile; and dorsally 2009; Zinner et al., 2009) have shown that kipunji mito- rotated palate were unique among papionin juveniles chondrial sequences (COI, COII) nest within the Papio (Singleton, 2009). clade (see Fig. 1). Specifically, its haplotypes group with Singleton’s (2009) analysis provides independent cor- southern populations of yellow baboons (Papio hama- roboration for the generic status of Rungwecebus, but it dryas cynocephalus) from the geographic vicinity (325 begs the question of adult kipunji morphology. Her find- km) of Mt. Rungwe. In these studies, divergence esti- ing that shape differences among papionin species mates for the kipunji and P. h . cynocephalus mitochon- described by Groves (1978), among others, are already drial lineages range from 0.65 Ma (95% CI: 1.34–0.25 present by M1 eruption is consistent with prior cranio- Ma; Burrell et al., 2009) to 0.35 Ma (95% CI: 0.67–0.09 metric studies (O’Higgins and Collard, 2002; McNulty Ma; Zinner et al., 2009). Both research groups interpret et al., 2006), but one cannot simply assume the kipunji these findings as evidence of ancient hybridization juvenile’s distinctive cranial features would persist between yellow baboon females and, based on the kipun- unchanged into adulthood. Leigh (2007), for example, ji’s phenotype, males of an unidentified mangabey spe- found that differences in juvenile neurocranial propor- cies. Burrell et al. (2009) favor Lophocebus as the source tions between Papio and Mandrillus diminish through of the paternal population but, on the basis of reported ontogeny due to convergence of their growth trajectories. phenetic similarities (Singleton, 2009), do not rule out This finding is particularly relevant given the kipunji’s Cercocebus. Zinner et al. (2009) postulate a Papio-Rung- apparent phylogenetic link to Papio. Pending availability wecebus divergence (i.e., a true sister-group relationship) of an adult specimen for morphometric analysis, alterna- followed by introgressive hybridization between an iso- tive means must be used to assess the kipunji’s adult lated southeastern yellow baboon population and proto- cranial form. kipunji males. In both scenarios, the modern kipunji is hypothesized to have descended from a population of Developmental Simulation reproductively viable, intergeneric hybrids. Phylogenetic relationships within the LPT clade are The dilemma posed by the kipunji juvenile is analo- unresolved due primarily to the extremely short (esti- gous to that encountered by paleontologists, who must mated < 400,000 years) divergence intervals (interno- infer the adult morphology of juvenile fossils to deter- des) between its constituent taxa (Disotell, 1994; Harris mine their taxonomic affinities (e.g., Tobias, 1978; Alem- and Disotell, 1998; Harris, 2000, 2002). This uncertainty, seged et al., 2006; McNulty et al., 2006). In such cases, coupled with the limitations of the available kipunji developmental simulation of adult cranial shape has sequences, makes resolution of the kipunji’s nuclear- proved useful for exploring ontogenetic differences DNA relationships problematic; thus, morphological cor- among species and for testing taxonomic hypotheses roboration of the molecular results is extremely desira- (Richtsmeier et al., 1993; Richtsmeier and Walker, 1993; ble. Davenport et al. (2006) described the kipunji Ackermann and Krovitz, 2002; McNulty et al., 2006). juvenile cranium as possessing classic Lophocebus fea- McNulty et al.’s (2006) simulation of the Taung child’s tures such as relatively narrow zygomatic breadth, zygo- adult facial morphology using extant great ape DEVELOPMENTAL SIMULATION OF Rungwecebus 391 TABLE 1. Study sample by dental stage Abbr. dp4 M1 M2 Adult # Total Allenopithecus nigroviridis Ann 0 3 5 8 16 Chlorocebus aethiops Cax 0 7 4 8 19 Macaca mulatta Mmx 4 4 5 5 18 Cercocebus agilis Cga 0 6 7 4 17 Cercocebus atys Cta 1 5 6 10 22 Cercocebus torquatus Ctt 1 5 1 5 12 Mandrillus leucophaeus Mln 0 0 0 4 4 Mandrillus sphinx Msp 0 2 5 7 14 Theropithecus gelada Tgn 0 0 4 7 11 Lophocebus albigena Laj 4 4 4 12 24 Lophocebus aterrimus Laa 4 5 7 10 26 Papio hamadryas kindae Phk 0 6 11 4 21 Rungwecebus kipunji Rkn 0 1 0 0 1 Dental stage defined by full eruption of the nominal tooth. developmental models is the most recent exemplar of frequently unknown, use of mixed-sex samples was nec- this approach. They found that even when developmen- essary to obtain reasonable juvenile sample sizes. This is tal trajectories are significantly different, adults created justified by prior findings that male and female ontoge- using extant juveniles of one species and the develop- netic trajectories do not typically diverge until late in mental trajectory of a different species consistently clas- ontogeny (O’Higgins and Jones, 1998; O’Higgins and sified to the original juvenile’s species (McNulty et al., Collard, 2002; Leigh, 2006). As the only known kipunji 2006). These findings demonstrate the robustness of de- specimen is a male, this study focused on male develop- velopmental simulation as a method of examining spe- mental patterns and adult females were not sampled. cies-level morphological differences. In this study, we used a similar developmental simulation to infer the adult cranial morphology of Rungwece- Data Collection and Processing bus. To select appropriate models, we first examined Three-dimensional craniometric landmarks and cra- differences among male cercopithecine developmental nial contours were collected and processed following a patterns and then simulated adult kipunji cranial shape previously published protocol (Singleton, 2009). Contours using a range of these models. The resulting simulated were reduced to equal numbers of equidistant semi-land- kipunji adults were compared with adult male papionins marks using Resample (Raaum, 2006) and treated as and with each other to: (1) explore variation in papionin standard landmarks during superimposition. The final developmental patterns; (2) assess the effect of develop- landmark configurations, comprising 112 landmarks and mental model choice; (3) characterize adult kipunji cra- 41 semi-landmarks (see Singleton, 2009), were aligned nial morphology; and, (4) evaluate the adult kipunji’s using generalized Procrustes analysis (GPA) in Mor- phenetic affinities. pheus (Slice, 1998). This procedure removes the effects of translation, rotation, and scale, thus bringing all specimens into a common shape space (Gower, 1975; MATERIALS AND METHODS Rohlf and Slice, 1990; Dryden and Mardia, 1998). Fol- Materials lowing superimposition, missing landmarks were esti- The study sample comprised 205 cercopithecine crania mated either by reflection of antimeres or by representing all papionin genera and two cercopithecin substitution of stage-specific species-mean coordinates outgroups (see Table 1). The majority were wild-shot (see Slice, 1996; Singleton, 2002). Reflection and estima- specimens of known provenience, but zoo specimens tion were executed using SAS 9.1 IML modules (SAS judged to present normal (i.e., nonpathological) morphol- Institute, Cary, NC). Species adult means were com- ogy were included for taxa (e.g., Mandrillus, Theropithe- puted for males only; subadult dental-stage means were cus) otherwise poorly represented in museum collections. computed using pooled-sex samples, which provide rea- Criteria for specimen selection included cranial symme- sonable estimates for subadults of both sexes (Singleton, try, absence of dental anomalies, and normal bone tex- 2009). Complete configurations were then subjected to a ture and density. Specimens were drawn from the second GPA alignment. As Procrustes-aligned coordi- collections of the Royal Museum for Central Africa (Ter- nates have nearly perfect correspondence with their Eu- vuren), the Royal Belgian Institute of Natural Sciences clidean tangent-space projections (Marcus et al., 2000; (Brussels), the Tulane University Museum of Natural McNulty et al., 2006; Singleton, 2009), aligned coordi- History (New Orleans), the American Museum of Natu- nates were used in all analyses. ral History (New York), and the Field Museum of Natu- ral History (Chicago). Calculation and Comparison of Developmental Specimens were assigned to developmental categories Vectors on the basis of dental eruption. Dental stages were defined by eruption of the nominal tooth to full occlu- The developmental trajectory for each papionin species sion. Thus, in an M1-stage individual, M1s are fully was approximated by linear regression of Procrustes- erupted and wearing but M2s are not yet in occlusion. aligned coordinates, that is, cranial shape, on a four- Because subadult specimens are scarce and their sexes state categorical (dummy) variable representing dental 392 SINGLETON ET AL. stage (Zelditch et al., 2004). The resulting developmental Visualization of Simulated Adults vector consists of regression coefficients and summarizes To aid in the visualization of the simulations, an ana- shape change correlated with cranial development from tomically accurate surface model of the juvenile kipunji the dp4 stage (1) through dental maturity (4). Although cranium, FMNH 187122, was created using a NextEn- pre-M1 developmental patterns have no direct bearing gine desktop 3D laser scanner. Multiple scan views were on the kipunji developmental simulation, species vector VR aligned and merged in Rapidform XOR (Rapidform, comparisons were made using the full (dp4–adult) devel- Inc.) to produce a single, complete model. Estimates of opmental sequence, wherever possible, to obtain the adult kipunji cranial shape were visualized by applying most robust possible representations of papionin devel- the morphing function of Landmark 3.0.0.6 (Wiley et al., opmental patterns. Use of dental stage, rather than size, 2005) to this model. For visualization purposes, symmet- as a surrogate for ontogeny facilitates comparison of rical landmark configurations were created for the juve- individuals at comparable ontogenetic stages irrespective nile and simulated kipunji adults (SKAs). Each of absolute cranial size. In the present study, the use of landmark configuration was mirror imaged by exchang- dental stages is not only desirable but also necessary to ing the coordinates of its right and left landmarks after simulate development of the kipunji, whose adult cranial first adding missing-data values as placeholders for the size is presently unknown. nonexistent left semi-landmarks. Mirror images were Differences in developmental trajectories between cer- superimposed in Morpheus and averaged to yield a sin- copithecine species were measured as the angle between gle, symmetrical configuration with semi-landmarks on their developmental vectors, which equals the arccosine both right and left sides. The surface model and sym- of the vector correlation (dot product) (Zelditch et al., metrical landmark configurations were then loaded into 2004). Small angles indicate little or no divergence Landmark, and the juvenile kipunji landmarks were between species developmental vectors. Permutation transferred to the model surface. Three-dimensional rep- tests were used to determine the statistical significance resentations of the adult kipunji cranium were created of these differences. Following McNulty et al. (2006), by morphing the juvenile model to each adult landmark pairwise tests were computed using randomized groups configuration, using corresponding landmarks to direct of equal size and with equal numbers of specimens in the transformation and thin-plate splines to interpolate each dental stage but including individuals from both the shape transformations between landmarks (Book- species. The angular difference between the permuted stein, 1991; Wiley et al., 2005). Comparisons were made groups’ developmental vectors was calculated for 10,000 among SKAs to examine the influence of different devel- permuted samples. For a single comparison, the opmental vectors on the estimated adult shape. observed angle would be significant if it exceeded 95% of Shape differences between SKAs and actual adults of the permuted values (Good, 2000; Zelditch et al., 2004). other species were also explored graphically by superim- However, to compensate for the present study’s multiple position of symmetric cranial landmark configurations in comparisons, a Bonferroni-adjusted significance level of Morpheus (Slice, 1998). Although less detailed than the P < 0.001 was required for statistical significance (Sokal surface visualizations, these wireframe representations and Rohlf, 1981). Finally, cluster analysis (Ward, 1963) facilitate the identification of regional shape differences of the matrix of pairwise angles was used to explore between superimposed configurations. Comparisons of the taxonomic patterning of developmental-vector the average adult kipunji shape to mean adult-male differences. shapes of other papionins were used to assess the kipunji’s phenetic resemblance to other species.

Developmental Simulation Morphometric Affinities of Simulated Adults Cercopithecine developmental trajectories were applied to the juvenile kipunji cranial landmark configu- A principal components analysis (PCA) of actual ration to create a series of estimated kipunji adults. Ju- papionin adults and SKAs was used to locate simulated venile samples of Mandrillus leucophaeus and adults relative to other species and to each other in a Theropithecus gelada were either unavailable or too reduced-dimension cranial morphospace. Quantitative sparse to provide reliable simulations; however, adult comparisons of cranial shape differences between SKAs males of these species were included in subsequent com- and adult males of other species were based on the Pro- parative analyses. To model the kipunji’s transformation crustes distance, which is equivalent to the Euclidean from M1-stage juvenile to adult male, developmental distance in shape space between two optimally superim- vectors of the remaining species were recalculated posed specimens (Bookstein, 1991). Procrustes distances excluding dp4 juveniles. Just as bivariate regression were calculated between each actual adult specimen and coefficients express change in a dependent variable per its species mean, and descriptive statistics were calcu- unit change of the independent variable, the multivari- lated from these to determine the level of shape varia- ate developmental vector summarizes cranial shape tion expected within species. Procrustes distances transformation at each landmark coordinate between between SKAs and the average simulated adult were successive dental stages. To represent shape change compared with these species distributions to assess the from M1 eruption (stage 2) to adulthood (stage 4), the consistency of the adult estimates and the effects of dif- developmental coefficient vector is multiplied by a factor ferent developmental models on the estimated adult of two, the number of stage changes. Addition of this morphology. augmented vector to the landmark coordinates of the The phenetic affinities of individual SKAs were tested kipunji juvenile cranium represents its simulated adult using three different Procrustes distance-based criteria. landmark configuration (McNulty et al., 2006). First, we determined affinity according to the nearest DEVELOPMENTAL SIMULATION OF Rungwecebus 393 TABLE 2. Angles between species developmental trajectories Ann Cax Cga Ctt Cta Laa Laj Mmx Msp Phk Tgn A. nigroviridis – 0.6651 0.5975 0.2056 0.2056 0.4691 0.1349 0.4939 0.1609 0.4708 0.2379 Ch. aethiops 18.96 – 0.6202 0.2604 0.2782 0.4101 0.3561 0.5700 0.1701 0.4221 0.2064 C. agilis 22.70 22.39 – 0.0902 0.8869 0.8223 0.3221 0.6432 0.0629 0.2246 0.0764 C. torquatus 26.12 26.30 21.80 – 0.0334 0.3050 0.0657 0.1732 0.0914 0.2249 0.2479 C. atys 22.29 21.18 14.45 20.16 – 0.4569 0.0588 0.3462 0.1080 0.3048 0.1316 L. aterrimus 20.96 20.84 18.10 20.91 14.71 – 0.0439 0.9652 0.1457 0.6354 0.1823 L. albigena 27.73 23.60 23.73 28.96 21.73 22.13 – 0.3077 0.2869 0.4740 0.3436 M. mulatta 21.24 21.79 20.15 21.45 18.07 16.05 22.39 – 0.0774 0.6858 0.1118 M. sphinx 43.55 43.03 43.61 37.21 41.30 41.75 39.39 40.98 – 0.0656 0.4510 P. h. kindae 29.78 29.28 26.57 27.20 23.50 24.35 27.32 23.55 38.96 – 0.1249 T. gelada 46.56 44.96 47.08 43.93 45.27 44.71 43.75 42.18 41.41 41.02 – Below diagonal: angular differences (in degrees) between species developmental trajectories. Above diagonal: P values for interspecific angular differences (10,000 permutations); no angles were significant at the Bonferroni-adjusted level of P ¼ 0.001. adult specimen to each SKA. Second, we identified the Similarity among SKAs is confirmed by comparisons species whose mean adult-male shape was closest to of Procrustes distances between individual SKAs and each SKA. Finally, to allow for potential differences the mean simulation (Table 3), which reflect a level of among species’ vectors in the magnitude of developmen- shape variation similar to that found within papionin tal shape change, we employed a ‘‘non-discrete’’ simula- species. These results indicate that different develop- tion model (cf. McNulty et al., 2006). Instead of mental models produce estimates of the adult kipunji simulating a discrete adult configuration, the SKAs de- morphology that are no more different than members of velopmental vector was assumed to be infinite and affin- a single species. The exception is the simulation gener- ity was determined by the species mean with the ated from the vector of M. sphinx, whose larger Pro- smallest perpendicular Procrustes distance to this vector crustes distance (see footnote to Table 3) rules it out as at any point along its trajectory. a reasonable estimate of the adult kipunji morphology. On principal component plots of actual and simulated RESULTS adults (Fig. 4a), most SKAs cluster tightly, implying that our estimates of adult morphology are precise, and do Comparison of Developmental Vectors not overlap the distributions of other papionins. Simula- Table 2 shows pairwise angular differences between tions based upon the Chlorocebus, Papio, and Mandril- species developmental vectors. The gelada vector was lus vectors fall away from the core group in the most dissimilar, with interspecific angles in the range of directions of their respective model taxa. Only the latter 41–47, followed closely by Mandrillus sphinx (37–44), is substantially removed from the kipunji cluster, per- and the Kinda baboon (24–41). Angular differences haps reflecting the extended development of male man- between developmental vectors of small-bodied papionins drills (Setchell et al., 2001; Setchell and Wickings, 2004; ranged from 14 to 29. None of these differences were Setchell et al., 2006). On PC1, the M. sphinx simulation significant, however, and even the Theropithecus gelada falls outside the core papionin group in the direction of angles are relatively small given the large number of mandrills and drills. On PC3, the remaining SKAs fall landmarks used in this study. The relative uniformity of closest to, but well separated from, Cercocebus and papionin developmental trajectories, irrespective of phy- Lophocebus, again suggesting that the kipunji adults are logenetic affinity, is apparent in the dendrogram of more different from these genera than either is from the angular differences (Fig. 3a). The pairing of the cercopi- other. Consistent with this finding, the average SKA thecins, Allenopithecus nigroviridis and Chlorocebus clusters as the sister group to other small-bodied papio- aethiops, is the only indication of phylogenetic signal, nins (see Fig. 4b) suggesting that it is phenetically dis- whereas the papionin vectors (Mandrillus and Theropi- tinct from other mangabeys and from macaques. thecus excluded) comprise an unstructured cluster. Procrustes distances between the SKAs and other adult papionins indicate a strong affinity between R. kipunji and Lophocebus aterrimus but also confirm that Morphometric Affinities of Simulated Adults the kipunji is a distinct phenon. Under the nearest The uniformity of papionin developmental vectors is neighbor criterion (see supplemental online material Ta- also reflected in the morphometric similarity of the ble S1), all SKAs but one are closest to L. aterrimus; the simulated kipunji adults derived from them. The pheno- C. agilis–based simulation (K_Cga) falls closest to its gram of simulated and actual papionin adults clusters vector species, C. agilis. When sorted according to the the majority of the SKAs to the exclusion of other small- nearest species mean (SOM, Table S2), all but three bodied cercopithecines (Fig. 3b). Within the SKA cluster, SKAs are closest to L. aterrimus. The K_Ctt and K_Msp the pairings of the A. nigroviridis and Ch. aethiops sim- simulations fall closest to C. torquatus, whereas K_Cta ulations and the C. torquatus simulation and C. torqua- falls nearest to C. atys. Comparisons based on nondis- tus, respectively, show some influence of taxonomic crete developmental simulations (SOM, Table S3) yield vector differences on estimated adult morphologies, but essentially the same results, with every developmental taxonomic patterning is otherwise absent. Only the vector passing closest to the mean of L. aterrimus except Mandrillus sphinx simulation shows a strong, exclusive that of the K_Ctt simulation, which runs closer to C. tor- affinity to its developmental species. quatus. When compared with the intraspecific 394 SINGLETON ET AL. TABLE 3. Descriptive statistics for within- species Procrustes distances to adult-male species means N Mean Std. Dev. Max. A. nigroviridis 8 0.0500 0.0073 0.0596 Ch. aethiops 8 0.0561 0.0117 0.0813 M. mulatta 5 0.0566 0.0082 0.0699 C. agilis 4 0.0442 0.0015 0.0463 C. atys 10 0.0495 0.0038 0.0556 C. torquatus 5 0.0444 0.0040 0.0474 M. leucophaeus 4 0.0491 0.0019 0.0514 M. sphinx 7 0.0735 0.0170 0.1037 T. gelada 7 0.0616 0.0298 0.1273 L. albigena 12 0.0519 0.0056 0.0611 L. aterrimus 10 0.0511 0.0072 0.0658 P. h. kindae 4 0.0554 0.0026 0.0578 Simulated 9 0.0474 0.0080 0.0567 kipunji adultsa aThe simulation based on M. sphinx is excluded from these calculations because its Procrustes distance to the mean (0.1423) indicates a much greater magnitude of developmental shape change, clearly ruling it out as an appropriate model.

Comparisons of Estimated Adult Morphology Consistent with the PCA and cluster results, the visualizations of the simulated adult crania are extremely similar to each other and to their average shape (Fig. 5). Where individual simulations deviate from the average, they do so in ways that are consistent with the morphology of the vector species. Thus, the Lophocebus simulations (K_Laa and K_Laj) have more anteriorly positioned zygomatic roots, deeper suborbital fossae, and lower neurocrania. The simulation based on the vector of the Kinda baboon (K_Phk) has a higher frontal bone, more prominent glabellar region, more globular neurocranium, and greater facial prognathism. The C. torquatus simulation (K_Ctt) is also relatively prognathic, with shallower suborbital fossae and somewhat inflated maxillary ridges. The SKAs’ mutual similarities, however, clearly outweigh their differences, and this consistency recommends the average SKA as a robust estimate of the kipunji’s adult cranial morphology (Fig. 5, K_Mean). Comparisons between the average SKA and the mean Fig. 3. Cluster analyses. (a) Dendrogram of angular differences adult-male shapes of actual papionins reveal cranial between species developmental vectors. Absence of taxonomic patterning reflects relative uniformity of developmental vectors in small- shape differences consistent with those described by Sin- bodied papionins. (b) Phenogram of simulated kipunji adults (SKAs) gleton (2009) for the juvenile cranium (Figs. 6 and 7). In and adult male means. Simulated adults are identified by ‘‘K_’’ plus comparison with most other papionins—including L. the taxon abbreviation of the species whose vector was used to cre- aterrimus, the taxon to which it is most similar—the ate it, e.g., K_Laa is the adult simulation based on the vector of adult kipunji (average SKA) exhibits a taller, broader Lophocebus aterrimus. Taxon abbreviations follow Table 1. neurocranium; broader face and suborbital fossa; more concave anteorbital profile; shorter nasal bones; and a more dorsally rotated palate. In contrast with L. aterrimus, in particular, the adult kipunji exhibits broader, Procrustes-distance distributions (Table 3), none of the less restricted suborbital fossae, and its zygomatic simulated kipunji adults falls within the adult-male arches are less sinuous and more laterally positioned. In range of variation of any species examined. Procrustes contrast with the juvenile specimen (Singleton, 2009), distances between adult-male papionins and the average the simulated adult neurocranial length is relatively estimated kipunji adult also equal or exceed distances greater than that of L. aterrimus but similar to L. albi- between members of different genera (Table 4). This is gena and, interestingly, Papio hamadryas kindae. The further evidence that the kipunji cannot be accommo- adult kipunji’s cranial base is most similar to that of dated within any papionin taxon sampled and corrobo- L. aterrimus, whereas its frontal bone shape most resem- rates Singleton’s (2009) finding that the kipunji bles C. agilis. The kipunji’s neurocranial height and over- represents a distinct genus. all shape are most similar to that of P. h. kindae, whereas DEVELOPMENTAL SIMULATION OF Rungwecebus 395

Fig. 4. Phenetic afﬁnities of simulated kipunji adults (SKAs). (a) (b) Phenogram of adult male means. The average simulated kipunji Principal components analysis of SKAs and actual adult males. With (Rkn) clusters as a sister group to small-bodied papionins, indicating the exception of the simulation based on M. sphinx, the SKAs cluster its phenetic distinctness from other mangabeys and from macaques. tightly in morphospace and do not overlap the distributions of other Taxon abbreviations follow Table 1. species. As a group, the SKAs fall closest to Lophocebus aterrimus. its facial proﬁle is closest to that of Macaca mulatta. The et al., 2005; Chakraborty et al., 2007), are Red Listed as short palate of the simulated adult is likely an artifact of soon as they are described (Davenport and Jones, 2008; the abbreviated premaxillary alveolar process of the juve- Kumar et al., 2008). Ethologists, morphologists, and nile specimen, whose incisors are not fully erupted. molecular phylogeneticists must race poachers and habitat destruction to characterize and understand newly discovered primates while they still exist in DISCUSSION their natural environments. Conservation-mandated The curious case of the kipunji is a pointed reminder scarcity of physical voucher specimens and the steady that modern primatologists work in a very different mi- attrition of irreplaceable comparative collections require lieu from that of their predecessors. Truly new primate that bioanthropologists embrace new approaches to the species, much less genera, are exceedingly rare and of- study of primate morphological diversity. Increasingly, ten, like the kipunji and Arunachal macaque (Sinha we will need to adopt minimally destructive methods 396 SINGLETON ET AL. TABLE 4. Procrustes distances among cercopithecine adult-male means Rkna Ann Cax Cga Cta Ctt Laa Laj Mmx Msp Phk Tgn R. kipunjia 0 0.0990 0.1218 0.0888 0.0791 0.0866 0.0770 0.0925 0.0862 0.2571 0.1200 0.1681 A. nigroviridis 0.0990 0 0.0737 0.0785 0.0768 0.0920 0.0783 0.0842 0.0773 0.2619 0.1186 0.1787 Ch. aethiops 0.1218 0.0737 0 0.1004 0.1030 0.1269 0.1018 0.1016 0.0972 0.3017 0.1451 0.2168 C. agilis 0.0888 0.0785 0.1004 0 0.0420 0.0785 0.0536 0.0678 0.0629 0.2627 0.0910 0.1717 C. torquatus 0.0791 0.0768 0.1030 0.0420 0 0.0628 0.0534 0.0721 0.0552 0.2497 0.0886 0.1595 C. atys 0.0866 0.0920 0.1269 0.0785 0.0628 0 0.0761 0.0972 0.0755 0.2207 0.0988 0.1442 L. aterrimus 0.0770 0.0783 0.1018 0.0536 0.0534 0.0761 0 0.0506 0.0602 0.2596 0.0984 0.1770 L. albigena 0.0925 0.0842 0.1016 0.0678 0.0721 0.0972 0.0506 0 0.0815 0.2647 0.1177 0.1983 M. mulatta 0.0862 0.0773 0.0972 0.0629 0.0552 0.0755 0.0602 0.0815 0 0.2595 0.0923 0.1617 M. sphinx 0.2571 0.2619 0.3017 0.2627 0.2497 0.2207 0.2596 0.2647 0.2595 0 0.2258 0.2055 P. h. kindae 0.1200 0.1186 0.1451 0.0910 0.0886 0.0988 0.0984 0.1177 0.0923 0.2258 0 0.1383 T. gelada 0.1681 0.1787 0.2168 0.1717 0.1595 0.1442 0.1770 0.1983 0.1617 0.2055 0.1383 0 aAverage estimated adult kipunji. Abbreviations follow Table 1.

Fig. 5. Three-dimensional representations of simulated kipunji size for Lophocebus. Each SKA is identiﬁed by ‘‘K_’’ plus the taxo- adults (SKAs). SKAs based on different species vectors (periphery) are nomic label (following Table 1) of the species whose vector was used scaled to unit size to facilitate shape comparisons. The virtual juvenile to create it. K_Mean is the mean of the other simulations. Differences cranium (center) is scaled to 85% of adult size, the approximate ratio among the SKAs are minor, which suggests that the estimate of adult- of the juvenile cranial centroid size to the adult-male average centroid male kipunji morphology is robust. DEVELOPMENTAL SIMULATION OF Rungwecebus 397

Fig. 6. Superimposition of the average adult kipunji (blue) on the sally rotated palate. Its neurocranial height and overall shape are most adult-male averages of selected papionin species (red). In lateral view, closely matched by Papio (Phk). The simulated adult’s snubbed palate the kipunji is distinguished from most papionins by its relatively tall is an artifact of the kipunji juvenile’s abbreviated premaxillary alveolar neurocranium, short nasal bones, concave anteorbital profile, and dor- process. Taxon abbreviations follow Table 1. that maximize information gleaned from limited samples major papionin clades and, in all cases, insignificant. In and preserve existing comparative material for future the cases of Mandrillus sphinx and Theropithecus researchers, making conservation the watchword in the gelada, species with large angles but small subadult collection as well as in the field. samples, lack of statistical significance may be attribut- In this study, we apply one such method, develop- able to small sample sizes. Our results contrast those mental simulation, to the sole physical specimen of a of Leigh (2007), who found developmental trajectories unique and critically endangered primate, the kipunji. in Papio to diverge strongly from those of other papio- By simulating the kipunji’s adult cranial morphology nins. Leigh associated this divergence with increased using a range of developmental models, we sought to body size in the Papio lineage, so our discrepant gain a fuller understanding of the kipunji’s morphology results may be attributable to our use of P. h. kindae, and taxonomic affinities as well as new insights into the smallest extant baboon. The failure of papionin papionin cranial development. Among the questions vectors to cluster by genus (Fig. 3a) implies that addressed were: How different are papionin develop- small-bodied papionins share a common developmental mental patterns? How much does the choice of develop- pattern distinct from that of cercopithecins. That this mental model affect estimates of adult cranial shape? pattern appears to be present in both Macaca and in What is the contribution of pre-M1 morphogenesis to African forms suggests it is retained from the common adult cranial morphology? and, How do estimated papionin ancestor. kipunji adult crania compare to those of actual papio- In morphometric and visual comparisons, simulated nins? Unlike similar analyses of fossil material, our kipunji adults (SKAs) based on different papionin vec- results are ultimately testable once adult specimens tors are extremely similar. The phenogram of actual and become available. simulated adults (Fig. 3b) clusters simulated kipunjis to the exclusion of other small-bodied papionins. The general failure of simulated kipunji adults to cluster accord- Developmental Simulation ing to vector source further corroborates the uniformity Differences between species’ developmental vectors of papionin developmental patterns. That said, the pair- (Table 2) were remarkably uniform across the three ing of simulations based on Cercocebus torquatus and 398 SINGLETON ET AL.

Fig. 7. Superimposition of the average adult kipunji (blue) on the adult-male averages of selected papionin species (red). In frontal view, the kipunji is distinguished from all papionins by its greater neurocranial height and midfacial breadth, less restricted suborbital fossae, and shorter nasal bones. Taxon abbreviations follow Table 1.

Mandrillus sphinx with actual C. torquatus and Man- studies showing that interspecific differences in primate drillus adults suggests unique species-vector influences cranial shape are well established before the M1 dental on estimated adult shape. This is particularly interest- stage (O’Higgins and Collard, 2002; McNulty et al., ing given the recent conjecture that C. torquatus is more 2006; Singleton, 2009). It appears that early juvenile closely related to Mandrillus than to other species of morphogenesis establishes a cranial plan that subse- Cercocebus (McGraw and Fleagle, 2006). quent development has only limited ability to modify. The effects of different species vectors on estimated adult shape are seen in the taxonomic clustering of these Phenetic Comparisons two SKAs as well as in subtle, taxon-typical deviations of individual simulations from the average estimate (Fig. Singleton (2009) found the juvenile kipunji cranium to 5). Angular differences between developmental vectors, be phenetically most similar to juveniles of Macaca and some of which (e.g., angles for Mandrillus) may prove Cercocebus but morphologically distinct from all papio- statistically significant with larger samples, certainly nins at a level consistent with generic status. In the pres- contribute to these deviations; however, differences in ent study, Procrustes distance-based determinations of vector length—not investigated in this study—may also phenetic affinity (Tables S1–S3) almost unanimously iden- play an important role in establishing adult shape differ- tify Lophocebus aterrimus as being most similar to the ences. Yet even the most divergent simulation—the adult kipunji’s morphology. However, the SKAs’ position adult based on the vector of Mandrillus sphinx—is as outside the main papionin cluster (Figs. 3b and 4b), their similar to other simulated kipunjis as it is to an adult separation from other papionins in PCA space (Fig. 4a), male mandrill. The consistency of estimated adult cra- and their total lack of overlap with extant species’ Pro- nial shapes suggests that the developmental-simulation crustes-distance distributions (Tables 3 and 4) confirm approach employed in this study has produced a reliable that the kipunji represents a distinct morphology. More- estimate of adult kipunji morphology. The similarity of over, the kipunji adult’s average Procrustes distances to the cranial traits present in the original juvenile speci- other mangabey species (Table 4) are comparable with dis- men and simulated adult kipunjis further corroborates tances between the two mangabey genera. By this DEVELOPMENTAL SIMULATION OF Rungwecebus 399 measure, the kipunji is as deserving of generic recognition shared by L. aterrimus and Cercocebus agilis represent as either Cercocebus or Lophocebus,ifnotmoreso. the primitive African papionin morphology. If so, the In visual comparisons, the adult kipunji is distin- kipunji’s resemblance to L. aterrimus in these features is guished from all other papionins by its relatively tall, uninformative concerning its ancestry. Additionally, the broad neurocranium, broad face and suborbital fossae, kipunji cranium offers no evidence of hybrid ancestry. But concave anteorbital profile, and dorsally oriented palate, there is no reason to expect classic hybrid traits (e.g., su- that is, the differences identified by Singleton (2009) as pernumerary teeth; Ackermann et al., 2006) in a lineage distinctive of the juvenile kipunji cranium. As in the pre- that has had thousands of generations to weed out con- vious study, the estimated kipunji adult shares no fea- flicts between its parental genomes, as witnessed by the tures with Lophocebus to the exclusion of other taxa, fact that the kipunji and other such suspected hybrid taxa and it lacks certain features—such as a low cranial have been identified solely on the basis of their DNA (Tosi vault, sinuous zygomatic arch, and laterally restricted et al., 2000; Sinha et al., 2005; Chakraborty et al., 2007; suborbital fossa (see Figs. 6 and 7)—associated with Karanth, 2008; Karanth et al., 2008; Osterholz et al., Lophocebus (Groves, 1978). Rather, the kipunji cranium 2008; Burrell et al., 2009; Zinner et al., 2009). exhibits a generalized, mangabey morphotype as similar Mangabey biogeography is equally unenlightening as to Cercocebus in many particulars as to its closer rela- to the kipunji’s potential forbears. Modern L. aterrimus tive and consistent nearest neighbor, L. aterrimus. The and C. agilis are sympatric throughout much of Central kipunji’s distinctive neurocranial shape does resemble Africa and approximately equidistant from the kipunji’s that of the Kinda baboon, the only hint, to date, of any East African range (Kingdon, 1997). Fossils attributed to cranial similarity between the kipunji and Papio. This Lophocebus from Koobi Fora occur as late as 1.38 Ma resemblance is noteworthy given that Papio is character- and establish this genus’s presence in East Africa well ized by accelerated perinatal brain growth and, thus, a into the Pleistocene (Jablonski and Leakey, 2008). But if relatively paedomorphic (for its size) neurocranial shape the East African relict species C. galeritus and C. sanjei (Leigh, 2007). Whether the kipunji’s neurocranial height (the latter also endemic to the Udzungwa mountains) reflects a similar ontogenetic dissociation is a topic wor- are truly descended from a C. agilis–like ancestral popu- thy of future investigation. lation (Grubb, 1978, 1982), Cercocebus ancestry for the kipunji is at least plausible. Whereas DNA, qualitative Kipunji Evolutionary Relationships trait comparisons, and biogeography offer no compelling basis to choose among competing hybridization scenar- and Systematics ios, the evidence presented in this study does. The con- The kipunji adult’s phenetic distance from other species, sistent morphometric affinity of the simulated kipunji lack of unambiguous synapomorphies with other taxa, and adults to L. aterrimus strongly favors Lophocebus as the unique cranial characteristics reaffirm that the kipunji is, paternal progenitor population of Rungwecebus. by present standards, worthy of generic recognition. That If it falls to geneticists to untangle the evolutionary said, many questions concerning the origins and evolution- relationships among African papionins, it is the system- ary history of Rungwecebus remain unresolved. atist’s task to wrestle with the taxonomic and biological The kipunji’s cranial morphology is a mosaic, sharing implications of this increasingly complicated, and per- traits with Lophocebus, Cercocebus, and even Macaca, haps reticulate, phylogeny. Several cercopithecid species although few with Papio. To explain this distribution, are thought to have arisen through intrageneric intro- Singleton (2009) proposed a scenario based on character gression (Tosi et al., 2000; Chakraborty et al., 2007; sorting, with the kipunji retaining more primitive traits Osterholz et al., 2008), and intergeneric hybridization as the lineages of the LPT clade diverged. Recent evi- has been reported in both captivity and (less frequently) dence (Burrell et al., 2009) that kipunji mitochondrial the wild (Markarian et al., 1972; Chiarelli, 1973; Dunbar sequences nest within extant Papio hamadryas cynoce- and Dunbar, 1974; Jolly et al., 1997). Like the kipunji, phalus (Fig. 1) has given rise to two additional scenarios: the golden and capped leaf monkeys (Trachypithecus (1) the kipunji is a yellow-baboon descendant that has geei and T. pileatus) of South Asia are proposed to have convergently evolved a mangabey-like morphology and arisen through intergeneric hybridization between Sem- habitus; (2) the kipunji arose through ancient hybridiza- nopithecus and Trachypithecus (Karanth, 2008; Karanth tion between southeastern yellow baboon females and et al., 2008; Osterholz et al., 2008). These cases suggest male mangabeys. The latter hypothesis is the more plau- that natural hybridization has been more important in sible, but no nuclear sequence links the kipunji to Lopho- Old World monkey evolution than previously thought cebus (or any other taxon) rather than to Papio (Burrell (Arnold and Meyer, 2006) and also raise interesting et al., 2009; Zinner et al., 2009). Citing the juvenile questions for taxonomists. The taxonomic rank of kipunji’s phenetic resemblance to Cercocebus (Singleton, Rungwecebus rests, in part, on the yardstick by which 2009), Burrell et al. (2009) left open the question of this we measure its morphological divergence, namely mor- hypothetical hybrid’s paternity. Zinner et al. (2009), appa- phological distances among other papionin genera. But if rently giving greater credence to nuclear DNA phyloge- members of different cercopithecid genera are capable of nies that link the kipunji to Papio, proposed phylogenetic giving rise to viable hybrid populations, we may be divergence of Rungwecebus and Papio followed by mito- forced to reconsider either our taxonomy or our concep- chondrial introgression, making the kipunji, in a sense, tion of the genus category. its ‘‘own grandpa’’ (Jaffee and Latham, 1948). If molecular methods have yet to resolve the kipunji’s CONCLUSIONS ancestry, what can other lines of evidence tell us? McGraw and Fleagle (2006) hypothesized that the moder- In this study, developmental simulation was used to ately deep suborbital fossae and weak maxillary ridges estimate the adult cranial morphology of Rungwecebus 400 SINGLETON ET AL. kipunji, ‘‘one of the world’s most threatened primates’’ research we thank E. Delson, the late L. Marcus, and (Rovero et al., 2009: p 99). 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