Versus Fast-Moving Primates

J Mammal Evol (2016) 23:353–368 DOI 10.1007/s10914-016-9323-3

ORIGINAL PAPER

Different Level of Intraspecific Variation of the Bony Labyrinth Morphology in Slow- Versus Fast-Moving Primates Alexandre Perier 1,2

&

Renaud Lebrun 1

&

Laurent Marivaux 1

Published online: 9 March 2016 # Springer Science+Business Media New York 2016

Abstract The vestibular system of the inner ear detects the motions of the head and is involved in maintaining balance. For this reason, this organ has been deeply studied and several scientists have tried to link its morphology with the locomotor behavior of an animal. Via high-resolution computed microtomography and geometric morphometric methods, we analyzed the intraspecific variation of the 3D morphology of the bony labyrinth (inner ear) in four species of primates differing in their locomotor adaptations: two being slow-moving taxa (Nycticebus and Perodicticus), and two being fastmoving taxa (Callithrix and Microcebus). Basically, there are very few analyses of the inter-individual variation of this organ in mammals in general, and this approach has never been attempted in primates thus far. Our results show that variation of the bony labyrinth morphology is expressed by the same ways in the different species (e.g., differences in the size, shape, and orientation of the semicircular canals, and in the width and height of the cochlea), but that slow-moving taxa exhibit a higher amount of intraspecific variation than Electronic supplementary material The online version of this article (doi:10.1007/s10914-016-9323-3) contains supplementary material, which is available to authorized users. * Alexandre Perier [email protected] * Laurent Marivaux [email protected]

1

Laboratoire de Paléontologie, Institut des Sciences de l’Évolution de Montpellier (ISE-M, UMR 5554, CNRS/UM/IRD/EPHE), c.c. 064, Université de Montpellier, place Eugène Bataillon, F-34095 Montpellier Cedex 05, France

2

African Primate Initiative for Ecology and Speciation, Department of Zoology and Entomology, University of Fort Hare, Alice 5700, South Africa

do fast-moving taxa. Our results strengthen support for a previously published hypothesis, according to which a relaxation of the selective pressure applied to the morphology of the bony labyrinth is the likely reason for this higher amount of intraspecific variation in slow-moving taxa, and that it may be related to a reduced functional demand for rapid postural adjustments. Keywords Inner ear . Semicircular canals . Intraspecific variation . Geometric morphometrics . Primates . Locomotion

Introduction During the Cenozoic, placental mammals have colonized several ecological niches using resources differentially. There is now a wide array of morphotypes and adaptations, which are expressed by multiple and various life history traits. The diversity of activities and positional behaviors (postures) related to locomotion highlights the diversity of the possibilities in the access to trophic resources (e.g., Brown and Yalden 1973; Van Valkenburgh 1985). Describing these locomotor adaptations and characterizing them from a morphological point of view based on skeletal elements or soft anatomy have been the subject of numerous studies (e.g., Fleagle 1977; Biewener 1 9 8 3 ; F l e a g l e a n d M e l d r u m 1 9 88 ; D o ra n 1 9 93 ; Panagiotopoulou et al. 2015). For paleontologists, the ecology and biology of living species are otherwise the only references for interpreting these parameters in extinct species. However, characterizing the locomotion of extinct taxa has proven to be somewhat difficult given the fragmentary nature of certain fossil specimens. When postcranial fossil elements (long bones, pelvis, tarsal and carpal bones) are available, it is then possible to reconstruct some aspects of the postural activities and positional behaviors included in their locomotor repertoire

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(e.g., Dagosto 1983, 1988, 1993; Gebo 1987, 1988; Van Valkenburgh 1987; Anemone and Covert 2000). This is more difficult if the sole fossil evidence is the skull. However, some structures of the basicranial region can provide critical information regarding the locomotor adaptations. The petrous bone is dense (Bar-Oz and Dayan 2007) and as such frequently preserves in the fossil record the inner ear (or bony labyrinth) (Fig. 1). This structure is composed of the organ of hearing (cochlea) and the organ of balance (vestibular system). In jawed vertebrates, this latter organ includes three semicircular canals (SCCs) and a vestibule. These are bony structures that enclose membranous organs (the three semicircular ducts, the utricle, and the saccule; Fig. 1). The vestibular system of the bony labyrinth is related to several reflexes (vestibulo-ocular and vestibulo-colic reflexes) and to the detection of angular head accelerations. As such, the vestibular system is responsible for maintaining the balance of the body in relation to the movements performed during locomotion (e.g., Graf and Klam 2006; David et al. 2010). Following this reasoning, interspecific variation in SCC morphology may be attributed to differences in locomotion. Indeed, given that an animal possesses a specific locomotor behavior, it can be expected that the Bmotion sensor^ of the brain exhibits a specialized morphology, which allows it to optimally detect the different types of movements engaged during the walking process. For clinical and therapeutic reasons, the morphology and function of the bony labyrinth are particularly well documented in humans (e.g., Lawrence and McCabe 1959; Lindeman 1969; Schuknecht 1969; Jørgensen et al. 2007; Bradshaw et al. 2010). On the primate model, a few analyses have shown that the morphology of the semicircular ducts is correlated with body mass (see for instance Jones and Spells 1963). It has also been proven that a phylogenetic component is associated with some of the interspecific variation of the 3D morphology of the bony labyrinth in primates (e.g., Lebrun et al. 2010). Several attempts have been made to link the primate SCC morphology (e.g., Spoor and Zonneveld 1998; Spoor et al. 2007) and orientation (Malinzak et al. 2012) to locomotion, in order to reconstruct key aspects of the locomotor adaptations in extinct species (e.g., Spoor et al. 1994; Walker et al. 2008; Silcox et al. 2009; Ryan et al. 2012). Since then, the study of the morphology and function of the inner ear has been extended to other mammals (e.g., Spoor et al. 2002, 2007; Orliac and Ladevèze 2007, 2012; Schmelzle et al. 2007; Orliac et al. 2012; Alloing-Séguier et al. 2013; Benoit et al. 2013, 2015; Berlin et al. 2013; Billet et al. 2013, 2015; Ravel and Orliac 2014; Grohé et al. 2015; Muizon et al. 2015). Most of these studies until recently have relied on an ad hoc hypothesis according to which the intraspecific morphological variation of the mammalian labyrinth is insignificant. However, a recent study has shown the existence of intraspecific variation in this organ (Billet et al. 2012). Billet et al. (2012) focused on

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the bony labyrinth morphology in the South American xenarthrans, and showed that Bradypus variegatus (the three-toed sloth) exhibits a surprisingly high degree of intraspecific variation, notably on the SCCs. This arboreal xenarthran of the monogeneric family Bradypodidae is characterized by very slow locomotion, with slow and nonstereotypical movements. Thus, would the intraspecific variation of the morphology of the bony labyrinth be more important in slow-moving taxa? Billet et al. (2012) suggested the existence of a release of selective pressure on the SCCs in three-toed sloths for explaining their morphological variation. Besides, they hypothesized a link between this release of selective pressure and the rapidity of locomotion of Bradypus: BThe most plausible reason for such a released selective pressure on their SC[SCCs] morphology lies in their reduced activity pattern^ (Billet et al. 2012: 3937). These two hypotheses will serve as a working base for the present study. Among placental mammals, primates exhibit a wide range of activities and positional behaviors, which are associated with various types of locomotion (quadrupeds [arboreals, terrestrials], leapers, climbers, etc.; e.g., Ashton and Oxnard 1964; Napier and Napier 1967; Rollinson and Martin 1981; Martin 1990; Hunt et al. 1996). All primates perform different types of locomotion during their daily routine. As such, a given primate species cannot be described by a single locomotion category, but rather by a whole repertoire widely overlapping with that of many other species. If most primates are active and fast, some others, like lorisids, move more slowly with deliberate and non-stereotypical movements. As such, it is tantalizing to compare the degrees of the labyrinthine morphological variation in so-called slow and fast primate taxa. Spoor et al (2007) have proposed an agility scale. This scale ranks animals according to the rapidity of their movements, ranging from slow to fast. For simplification purposes, we have chosen to use their scale. In this paper, we assessed the existence of different levels of intraspecific variation between fast and slow (sensu Spoor et al. 2007) primate species using a twofold approach: we analyzed (i) the bony labyrinth 3D shape and size variability using geometric morphometric methods (GMM), and (ii) the variation in orientation between the SCCs. These approaches were applied on digital model of labyrinths obtained by X-ray microcomputed tomography (μCT) surface reconstruction. We analyzed the inner ears of several individuals among four extant primate species differing in their locomotor adaptations: two being considered as active and middle-fast to fast-moving taxa (Microcebus murinus and Callithrix jacchus) according to Spoor et al. (2007), and two being considered as slow-moving taxa (Perodicticus potto edwardsi and Nycticebus coucang). With such a species selection, which includes three distinct taxonomic groups (callitrichine anthropoid, cheirogaleid and lorisid strepsirhines) and distinct locomotor adaptations, we aimed at assessing the amount of

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Fig. 1 Anatomical position, anatomy and orientation of the bony labyrinth in the skull of a specimen of Microcebus murinus (UMC 547, UM). 1: cochlea; 2: ampullae of the semicircular canals; 3: semicircular canals; 4: utricle; 5: saccule; 6: anterior semicircular canal; 7: common crus; 8: posterior semicircular canal; 9: lateral semicircular canal

inter- and intraspecific variation of the 3D morphology of the bony labyrinth in primates, and their relationships to the patterns of locomotion (fast versus slow behaviors).

Material and Methods Selected Taxa The specimens analyzed in this study come from the collections of the Anthropological Institute and Museum (AIM) of Zurich, the University Museum of Zoology (UMZC) of Cambridge, the Université de Montpellier (UM), the Museum für Naturkunde (MN) of Berlin, and of the Muséum National d’Histoire Naturelle (MNHN) in Paris (see Online Resource). We sampled specimens from the following species: Callithrix jacchus Linnaeus, 1758 (Platyrrhini, Cebidae, Callitrichinae): this taxon, also dubbed the common marmoset can be found throughout northwestern Brazil, where it lives in the bush, swamp, or tree plantations (e.g., Rowe et al. 1996). This marmoset is diurnal and arboreal. It is active throughout the day but with a peak of activity early in the morning and late in the evening. It generally makes a break at mid-day for

about 1 h during which it takes a nap or cleans itself (e.g., Stevenson and Poole 1976). Its diet consists primarily of fruit, animal prey (insects, small birds, and lizards), and gum (15 % of total diet) (Rowe et al. 1996). Its locomotion is fast and of two types (following Stevenson and Poole 1976). The first type is called Bnormal^ and includes walking, running, climbing, and jumping between two objects. In all of these activities, the animal gait is relaxed and its tail is extended. The second type contains a Bbouncing^ approach, which consists of a displacement with exaggerated movements of leaps and bounds from objects on a frequent basis. In the wild, their fastest type of locomotion is the Bgallop,^ while in captivity, they show 11.8 % of Bcanter^ (49.4 % if they move on a pole; Young 2009). Microcebus murinus Miller, 1777 (Strepsirhini, Lemuriformes, Cheirogaleidae) is the well-known Malagasy gray mouse lemur, which inhabits the drier forests of the south, west, and north coasts of the island (Mittermeier et al. 1994), which make it the primate with the smallest geographic distribution of our sample. It is nocturnal and arboreal, and its activity declines during the dry season when it enters into Btorpor^ (Ortmann et al. 1997). Its diet consists of fruit, flowers, gums, nectar, and animal matter (beetles, spiders, occasionally frogs and chameleons, and secretions of

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Hemiptera larvae; e.g., Rowe et al. 1996). Regarding its locomotion, this species is engaged in a form of fast arboreal quadrupedalism, including primarily vertical clinging and leaping on vertical support (e.g., Gebo 2011) and climbing with quick and agile movements. However, it can also be engaged in slow movements (Martin 1972). In a study conducted by Gebo (1987), M. murinus proved to be the most frequent leaper among the cheirogaleids observed (the other species were Cheirogaleus medius, Ch. major, and Mirza coquereli). Although it moves mainly in trees at night, this species occasionally is on the ground to dig and search for insects (e.g., Némoz-Bertholet and Aujard 2003). To catch insects while in the trees, it can perform a cantilevering movement. On the ground, it moves or jump with its four members. It can also bridge between different supports (stretching over) and especially with the branches above itself (Gebo 1987). Nycticebus coucang Boddaert, 1785 (Strepsirhini, Lorisiformes, Lorisidae), also known as the slow loris, is found in Southeast Asia. Its geographic distribution roughly equals that of C. jacchus in term of the area covered. It is a nocturnal and arboreal animal that lives in evergreen forests, preferring forest edges where supports and prey (insects) are abundant. Its diet consists of 50 % fruit, 30 % animal prey, 10 % gum, bird eggs, and cocoa beans (Rowe et al. 1996). The most frequent movements included in its locomotion are suspension and slow-moving quadrupedalism (Walker 1974), followed by bridging and slow climbing. Each of these movements rank between 20 and 30 % of its locomotor behavior (Gebo 1987). However, the suspension movements are attributed more to the postural behavior of the animal than to its locomotor behavior (Curtis 1995). It can move occasionally on the ground and in a serpentine style of quadrupedalism. However, its foot movements remain in a stereotypic sequence (Gebo 1987). Perodicticus potto edwardsi Müller, 1766 (Strepsirhini, Lorisiformes, Lorisidae), dubbed the potto, inhabits equatorial rainforests of Africa. There are four subspecies of Perodicticus potto and we only selected specimens of the subspecies Perodicticus potto edwardsi, which could be considered as a distinct species (Butynski and Jong 2007). This subspecies has a widespread West-East distribution, which extends from Nigeria to Angola and the eastern Congo Basin, and possibly to the Democratic Republic of Congo (Butynski and Jong 2007), which make it the primate with the widest geographic distribution of our sample. This species lives in the canopy of primary and secondary forests with dense vegetation and eats fruit (65 %), gums (21 %), and animal prey (10 %) (Rowe et al. 1996). It is a cryptic nocturnal animal that moves slowly (Forbes 1894) though it can run at times (Walker 1969). In the study of Gebo (1987), P. potto moved quadrupedally and climbed more frequently than did N. coucang, but it was less suspensory. It can also bridge between supports in the small branch periphery (Gebo 1987) but this

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kind of behavior is relatively rare (Jungers 1979). Like N. coucang, it is rare for P. potto to be on the ground, but when it does, it also moves using a serpentine style of quadrupedalism, but with a more fluid motion. A total of 87 individuals (18 Callithrix, 32 Microcebus, 15 Nycticebus, and 22 Perodicticus) were analyzed for the present study. The sample contains both males and females, mostly adults. In order to increase the sample size for each species studied, we also included several juveniles (sub-adults and a few younger individuals having fully erupted first molars or having their first molars about to be erupted). In the human embryo, the bony labyrinth attains an adult equivalent size and shape between 17 and 19 weeks of gestation (Jeffery and Spoor 2004). No study has yet been conducted to investigate the labyrinthine development within the four non-human primate species studied here, but based on the human model, it can be expected that the bony labyrinths of the non-adult specimens sampled here have already achieved their full development (adult labyrinthine size and shape). 3D Data Acquisition The 3D data acquisition was performed on entire skulls or focused on the basicranial region (petrosal bone) using X-ray microtomography. Some specimens already scanned for other specific analyses (e.g., Lebrun et al. 2010, 2012) were also re-used for this study. Most specimens from the AIM of Zurich were scanned on site using a Scanco μCT80 facility, at a resolution ranging from 36 to 74 μm. Some specimens of P. potto edwardsi from the AIM (see Online Resource) were also scanned at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) using the ID17 (45.71 μm) and ID19 (60 μm) beam lines. Specimens from UMZC of Cambridge, MNHN in Paris, MN in Berlin, and UM of Montpellier were scanned using a Skyscan 1076 μ-CT (MRI; ISE-M) with a resolution of 36 μm. The scans were then processed using the software Avizo 7.1 (FEI Visualization Sciences Group), and a virtual segmentation of the left bony labyrinth for each individual was performed. Structure, Orientation and Configuration of the Semicircular Canals (SCCs) For several decades, a canonical model of the configuration of the SCCs has been largely accepted, and expressed in three points that are thought to be respected across vertebrates: (i) the SCCs from the same bony labyrinth are orthogonal one to another; (ii) the bony labyrinth from one side of the skull is the mirror of the bony labyrinth from the other side - implying there is a bilateral symmetry and that the two bony labyrinths have the same morphology (i.e., same shape, size, and orientation); (iii) the corresponding SCCs between the right and left inner ears occupy the same planes. Nonetheless, Berlin et al.

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(2013) have raised serious questions concerning the validity of the canonical model hypothesis: they measured an average deviation from orthogonality of 6° between all canal pairs of the same bony labyrinth for a large sample of mammalian species. Furthermore, the same authors have found an average deviation of 4.3° between the same canal pairs from the inner ear of each side of the skull, while they measured an average deviation from coplanarity of 10.1° between the left and the right inner ear SCCs. Concerning the overall shape of the vestibular system, each SCC is roughly circular. The LSCC is generally closely parallel to the ground during the customary head position of animals (Hullar 2006) (see Fig. 1), but it is not a good indicator for the exact habitual position of the head because variation in the LSCC orientation can be observed (Berlin et al. 2013). Each SCC encloses a semicircular duct (SCD). The complete membranous bony labyrinth is filled with endolymph and each duct possesses an ampulla, in which hair cells are located. When the head moves, the endolymph moves inside the ducts, and the hair cells detect the direction and intensity of the movements, thereby transforming them into nerve impulses. The brain receives and analyzes these nerve impulses, and activates the associated reflexes to keep the balance of the body. Likewise, the walls of the utricle and saccule are filled with hair cells, and each of these organs possesses otoliths (small calcium carbonate particles). During body movements, the otoliths move along the walls of the utricle and saccule, and pass on the hair cells, which produce shear forces. The properties of those forces are transmitted to the brain via nerve impulses (Graf and Klam 2006). The otolith organs are sensitive to gravity and to linear accelerations; the utricle is sensitive to a change in horizontal movements, while the saccule to a change in vertical movements (e.g., inside an elevator). The SCCs detect angular accelerations. For a more detailed description of the bony labyrinth, see Rabbitt et al. (2004), David et al. (2010), and Ekdale (2015). It is worth noting that the endolymph flow inside the ducts is affected by canal shape, which influences the animal’s sensitivity to rotation (Malinzak et al. 2012). There are three main ways canal morphology may vary: by size, shape, and orientation (which comprises the deviation from orthogonality). For instance, animals having more orthogonal canals are assumed to encounter higher angular head velocities (fast head rotation) during locomotion (Malinzak et al. 2012). Differences in size and orientation of the SCCs are linked to differences in the sensitivity of the vestibular system (see Berlin et al. 2013 for more information on this matter). Three Dimensional Morphological Quantification of the Bony Labyrinth Landmarks are points that are useful for defining specific structures (usually hypothesized as homologous) of the

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anatomy of an individual (bone sutures, foramina, etc.). However, it is difficult to detect homologous points (type 1 landmarks; Bookstein 1991) on the surface of the bony labyrinth, which is a curvy organ, and as such, landmark positions along curves or surfaces cannot be homogenized across different individuals. In contrast, semilandmarks are equidistant points that allow to optimally characterize surfaces or curves (Gunz and Mitteroecker 2013; see also Bookstein 1997 and Gunz et al. 2005). Therefore, we used semilandmarks here to quantify the three dimensional shape and size of the bony labyrinth. Semilandmarks Protocol We used ISE-MeshTools (Lebrun 2014; http://morphomuseum. com/meshtools) for digitizing the semilandmarks. First, we digitized 3D curves to model the shape of the bony labyrinth. We placed a curve along the center of the lumen of the cochlea (anatomical position of the basilar membrane) on the two first turns, as it is the minimal number of coiling turns observed for all sampled individuals. Curves were also placed along the centroid of the lumen of each SCCs (midline curve). Curves defined along the ASCC and PSCC were fused at the common crus (the common crus was considered as an independent curve) and each SCC was kept opened (curves were not passing through the vestibular part of the SCCs; Fig. 2). A total of five curves (one for each SCC, one for the common crus, and one for the cochlea) were digitized. The curves were then re-expressed interactively into equidistant semilandmarks in MorphoTools (Specht et al. 2007; Lebrun 2008). In order to optimally characterize the 3D morphology of the bony labyrinths while keeping as little redundant information as possible, we retained 25 semilandmarks for the LSCC, 20 semilandmarks for the ASCC and PSCC, five semilandmarks for the common crus, and 30 semilandmarks for the cochlea (see Fig. 2 for an illustration). In this context, each SCC was characterized by 25 semilandmarks (as the ASCC and PSCC share the five semilandmarks placed on the common crus). Common Crus Length, SCC Centroid Size, and SCC Orientation A simple inspection at the bony labyrinth sample indicated to us that the length of the common crus may vary widely within the four investigated species. Therefore, we measured the length of the common crus (CCL) for all specimens. We used the centroid size (CS) of the semilandmarks digitized on each SCC as a proxy for SCC canal size. Because the shape of the SCC often departs widely from circularity, we chose to use this metric designed to capture the size of complex geometries rather than the more commonly used SCC radius of curvature. CS and CCL were then normalized for each species (to reach mean (CS) = 1 and mean (CCL) = 1 for all four species), in

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We used the metrics proposed by Zelditch et al. (2004) to define shape variance: X j¼n V¼

Fig. 2 Placement of the equidistant semilandmarks on a bony labyrinth of a specimen of Callithrix jacchus (CA 10168, AIM)

order to allow for comparison between the species-specific variances. Finally, we measured the angle between the three pairs of canals (and angular variance within the four species), in order to quantify potential departure from orthogonality.

j¼1

d2 j

ðn−1Þ

where V is the shape variance (SV) of a species, dj is the Procrustes distance of individual j, and n is the sample size. The Procrustes distance is the distance of an individual specimen from the mean configuration of its population. This distance is calculated as Bthe square root of the sum of the squared deviations of a specimen’s Procrustes coordinates from the grand mean of each landmark coordinate^ (Young 2006: 627). The shape variance value (SV) is identical to the trace of the variance–covariance matrix of the Procrustes data. From the SV value, a Bootstrap statistical resampling protocol (random sampling with replacement) (Webster and Sheets 2010) was used to compare the variances of each species. This resampling was performed 10,000 times for each species. At each draw, a new value of the SV was calculated and confidence limits for this value were obtained. These confidence limits were then compared. SV values were calculated for the whole bony labyrinth, as well as for the SCCs only and for the cochlea. Each statistical procedure was performed with R 3.0.2 (R Core Team 2014).

Geometric Morphometric Analyses The semilandmarks placed along the three semicircular canals, the common crus, and the cochlea were initially placed evenly along the five curves. As a preliminary step, following the recommendations of Gunz et al. (2012), we used a sliding semilandmark algorithm to discard information derived from the arbitrary spacing of the semilandmarks along these curves. Using an iterative procedure, semilandmarks were allowed to slide along the curves so as to minimize the Procrustes distance between each specimen and the mean shape of the entire sample (for more information on this method, see Gunz and Mitteroecker 2013). After this preliminary step, Procrustes superimposition (Gower 1975) was performed on the data set (rotated, translated, and scaled data) using the software MorphoTools, so that the shape of the bony labyrinth can be compared without taking size into account. A Principal Component Analysis (PCA; Pearson 1901) was then performed on the whole sample for visualizing the spatial distribution of shape variations of the different specimens and different species. Additionally, a PCA on the SCCs only and another PCA on the cochlea only were performed to visualize shape variations for each one of those parts of the bony labyrinth. Moreover, four independent species-specific PCA analyses were performed on the whole bony labyrinth in order to characterize the intraspecific variation of this organ within each species.

Results Patterns of Labyrinthine Interspecific Variation in PCA Space The labyrinths of the four species are clearly discriminated in shape space (Fig. 3), with the exception of P. potto edwardsi and N. coucang, which slightly overlap in PC1-PC2 space. The extent of the projection scores for each species relates to the amount of labyrinthine shape variance. Interestingly, we can observe that the scores of P. potto edwardsi are more widespread in PC1-PC2 space than those of the active/fast species (i.e., C. jacchus and M. murinus). The SV values for the whole bony labyrinth (Table 1) indicate that N. coucang and P. potto edwardsi show a higher amount of variance in labyrinthine shape than C. jacchus and M. murinus. It is noteworthy that P. potto edwardsi exhibits the highest SV value. The confidence limits offer a statistical support to this analysis (there is a statistical distinction between two taxa if their confidence limits do not overlap). The confidence limits of the SV values for the whole bony labyrinth (Table 1) statistically distinguish C. jacchus from N. coucang and P. potto edwardsi, and M. murinus from P. potto edwardsi.

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Fig. 3 Principal component analysis of labyrinthine shape: projection scores of the specimens on the first two principal components. Black square: Callithrix jacchus; Red triangle: Microcebus murinus; Green circle: Nycticebus coucang; Blue diamond: Perodicticus potto edwarsi. Greyed-out specimens: non-adult individuals

When analyzing shape variation of the SCCs only (Fig. 4), the delimitation of the projection scores on the first two PCs of the different species are similar to those previously found on the whole bony labyrinth (Fig. 3). The SV values computed for the SCCs (Table 1) also indicate that N. coucang and P. potto edwardsi show a higher amount of SCC shape variance than C. jacchus and M. murinus. The PCA performed on the cochlea only (Fig. 5) shows a clear distinction between the projection scores of the anthropoid C. jacchus and those of the three strepsirhine species, which slightly overlap on the first axis. The second axis seems to distinguish fast taxa and slow taxa. For each taxon, the projection scores on PC1-PC2 vary widely, which relates to a significant amount of intraspecific shape variation, an observation which is supported by the results presented in Table 1. Again, slow taxa express higher SV values than fast taxa.

Table 1 Intraspecific shape variance and confidence interval for the whole bony labyrinth, the SCCs, and the cochlea Taxa Bony labyrinth Callithrix jacchus Microcebus murinus Nycticebus coucang Perodicticus potto edwardsi SCCs Callithrix jacchus Microcebus murinus Nycticebus coucang Perodicticus potto edwardsi Cochlea Callithrix jacchus Microcebus murinus Nycticebus coucang Perodicticus potto edwardsi

Shape variance

Confidence limits

0.00192 0.00215 0.00277 0.00431

0.00151–0.00209 0.00175–0.00247 0.00212–0.00303 0.00359–0.00462

0.00148 0.00169 0.00223 0.00326

0.00114–0.00162 0.00135–0.00194 0.00167–0.00251 0.00273–0.00346

0.000449 0.000465 0.000534 0.001060

0.000319–0.000533 0.000365–0.000542 0.000396–0.000598 0.000790–0.001230

The inclusion of the non-adult individuals (greyed out specimens) in our sample does not seem to magnify the variation expressed by each species on the interspecific PCAs (Figs. 3, 4 and 5). Additionally, SV values were calculated for the adults of our sample (Table 2); the comparison of the SV values reported in Tables 1 and 2 does not reveal relevant differences.

Patterns of Labyrinthine Interspecific Variation in Physical Space The distinction between C. jacchus and M. murinus, and between N. coucang and P. potto edwardsi in PCA space (Fig. 3), can be described in physical space (see Fig. 6). The height and width definition used in this study have been taken from Spoor and Zonneveld (1995). Callithrix jacchus and M. murinus have a shorter ASCC, which is also tilted medially. However, the width of the ASCC is conserved from one species to another. Within fast taxa, the common crus is shorter and more inclined towards the ASCC. Their PSCC is higher and wider but tilted anteriorly. The LSCCs of N. coucang and P. potto edwardsi are smaller (width and height) and more horizontal. Indeed, the LSCCs of C. jacchus and M. murinus have an oblique orientation (the posterior side of the LSCC is more tilted dorsally than the anterior side). Moreover, in fast taxa, the cochleae are broader than those of slow taxa. In Fig. 5, C. jacchus is distinguished from the other taxa by more constrained turns (the last turns are small) of its cochlea. Microcebus murinus is distinguished from slow taxa by closer turns (cochlea less wide) of its cochlea. If substantial differences in the cochlea are observed between slow and fast taxa, it must be underscored that differences exist also within fast taxa (C. jacchus and M. murinus) and within slow taxa (P. potto edwardsi and N. coucang). For example, the cochlea of C. jacchus is wider than the cochlea of M. murinus. Concerning the SCCs, C. jacchus also exhibits a more oblique LSSC and a wider PSCC. Likewise, P. potto edwardsi exhibits

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Fig. 4 Principal component analysis of semicircular canal shape: projection scores of the specimens on the first two principal components. Black square: Callithrix jacchus; Red triangle: Microcebus murinus; Green circle: Nycticebus coucang; Blue diamond: Perodicticus potto edwarsi. Greyed-out specimens: non-adult individuals

wider LSCC and PSCC than N. coucang, but the latter exhibits a wider ASCC. Patterns of Labyrinthine Intraspecific Shape Variation Among the different specimens of C. jacchus, the first axis of the intraspecific PCA (%var: 23.13; Fig. 7) distinguishes the specimens having more prominent cochleae but shorter SCCs (negative values) from the other specimens (positive values). In contrast, the second axis of the intraspecific PCA (%var: 14.73) distinguishes the specimens having a cochlea less tilted dorsally, a longer common crus, and a less oblique LSCC (negative values) from the other specimens (positive values). Concerning M. murinus, the first axis of the intraspecific PCA (%var: 28.02; Fig. 7) distinguishes the specimens having a smaller cochlea, a wider ASCC and LSCC, and a wider and higher PSCC (positive values) from the other specimens (negative values). The second axis (%var: 12.20) distinguishes the specimens having a higher ASCC, a shorter PSCC, and a LSCC more tilted dorsally (negative values) from the other specimens (positive values). On the first axis of the intraspecific PCA (%var: 27.35; Fig. 7), the specimens of N. coucang, which exhibit a shorter ASCC, a longer common crus, a wider and higher PSCC, a Fig. 5 Principal component analysis of cochlear shape: projection scores of the specimens on the first two principal components. Black square: Callithrix jacchus; Red triangle: Microcebus murinus; Green circle: Nycticebus coucang; Blue diamond: Perodicticus potto edwarsi. Greyed-out specimens: non-adult individuals

LSCC less tilted dorsally, and a wider cochlea (positive values), are distinguished from the specimens situated at negative values. On the second axis (%var: 16.28), specimens situated at negative values possess a shorter and less wide ASCC, a longer common crus, a wider and higher PSCC, which is tilted anteriorly, a LSCC less tilted dorsally, and a wider cochlea. Among the specimens of P. potto edwardsi, the first axis of the intraspecific PCA (%var: 22.29; Fig. 7) distinguishes the specimens having a wider SCC, a shorter common crus, a LSCC less tilted dorsally, and a cochlea more tilted posteriorly from the other specimens (negative values). On the second axis (%var: 14.47), specimens situated at negative values are distinguished from specimens situated at positive values by a higher ASCC, a shorter common crus and a more uplifted PSCC. Angle Between SCCs, Centroid Size, and Common Crus Length Variation For the three pairs of semicircular canals, slow taxa express almost always at least twice as much angle variance than do fast taxa (see Table 3). Fast taxa exhibit a wider mean angle between the lateral SCC and posterior SCC (Lat_Post) than

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Table 2 Intraspecific shape variance and confidence interval for the whole bony labyrinth, the SCCs and the cochlea of the adult specimens only Taxa

Shape variance

Confidence limits

Callithrix jacchus

0.00186

0.00134–0.00203

Microcebus murinus Nycticebus coucang

0.00220 0.00295

0.00168–0.00260 0.00205–0.00324

Perodicticus potto edwardsi

0.00416

0.00313–0.00451

Bony labyrinth

SCCs Callithrix jacchus

0.00142

0.00101–0.00157

Microcebus murinus Nycticebus coucang

0.00178 0.00242

0.00136–0.00212 0.00160–0.00271


0.00323

0.00244–0.00348

Callithrix jacchus Microcebus murinus Nycticebus coucang

0.000460 0.000452 0.000538

0.000302–0.000562 0.000339–0.000535 0.000342–0.000610


0.000987

0.000665–0.001150

slow taxa. This is not the case for the other angles. The angles between the different SCCs do not seem to deviate in average from orthogonality more in slow taxa than in fast taxa. Furthermore, a fast moving species, C. jacchus, exhibits the highest deviation from orthogonality of the sample for a pair of SCC: the mean angle between the ASCC and PSCC is greater than 105° for this species. Slow taxa exhibit the highest amount of variance for both CS and CCL, for each SCC (see Table 4). Specimens of M. murinus display the smallest mean CS of the SCCs, and also exhibit the smallest mean CCL. Slow taxa display the highest mean CS for the ASCC and PSCC. Specimens of C. jacchus display the longest mean length of the common crus.

Cochlea

Fig. 6 Comparison of all possible pairs of species-specific bony labyrinth Procrustes mean shapes. The colors were decided by alphabetical order (e.g., Microcebus (blue)/Nycticebus (red); Nycticebus (blue)/ Perodicticus (red))

Discussion The existence of a phylogenetic component in the 3D morphology of the primate bony labyrinth, explaining some of the

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Fig. 7 Patterns of intraspecific labyrinthine shape variation on a: the first axis of the PCA (PC1); b: the second axis of the PCA (PC2). Blue: labyrinthine shape corresponding to the minimal PC score for a given species; red: labyrinthine shape corresponding to the maximal PC score for a given species

interspecific variation, was recently advocated by Lebrun et al. (2010). These authors have shown that Lorisiformes (Lorisidae + Galagidae) exhibit a unique structure of the vestibular system of their inner ear, which allows to clearly distinguish them from other primate groups. Thus, the proximity of the morphological spaces between N. coucang and P. potto edwardsi (both Lorisidae) on the first axis of the PCA performed on the whole labyrinth (Fig. 3), and the distance between slow-moving taxa and the other ones might be related to phylogeny, and to the peculiar morphology of lorisiform bony labyrinths. But the morphology of the inner ear has also been linked to the locomotor behavior of an animal (e.g., Spoor et al. 2002, 2007; Malinzak et al. 2012; Berlin et al. 2013). These studies consisted of interspecific analyses, comparing the morphology of the inner ear between species, but considering only most often only one or two individuals per species. Here, we have investigated the interspecific and intraspecific variation of the 3D morphology of the inner ear with a sample of several specimens per species.

Link Between form and Function

Table 3 Species-specific mean angles (in degrees) and angle variances for each pair of semicircular canals. Lat_Post: angle between the lateral semicircular canal (LSCC) and the posterior semicircular canal (PSCC);

Lat_Ant: angle between the lateral semicircular canal (LSCC) and the anterior semicircular canal (ASCC); Ant_Post: angle between the anterior semicircular canal (ASCC) and the posterior semicircular canal (PSCC)

Mean (Lat_Post) Variance (Lat_Post) Mean (Lat_Ant) Variance (Lat_Ant) Mean (Ant_Post) Variance (Ant_Post)

During head movements, endolymph flows through the membranous semicircular ducts. Thus, different SCC morphologies have an impact on the way the endolymph flows through the ducts, and thus impact the sensitivity to motion of the bony labyrinth (Malinzak et al. 2012). Several authors then tried to find what morphology would allow the best sensitivity to motion considering that fast animals would need a better sensitivity than slow animals. Jones and Spells (1963) observed that the radius of curvature (R) of the SCCs is related to body mass and increases with body size. Larger species have higher values of R. A high value of R is linked to a high afferent sensitivity (Yang and Hullar 2007). Because larger species would have slower head movements, a SCC with a high value of R would be adapted to detect low amplitude angular velocities. Spoor et al. (2007), who were also interested in the radius of curvature, observed that agile (fast) animals had higher R than slower animals of similar size. Contrary to Jones and

Callithrix jacchus

Microcebus murinus

Nycticebus coucang


95.07 3.512 83.96 8.346 105.63 5.518

93.29 5.852 87.06 8.497 95.36 4.566

88.70 13.023 85.90 20.468 91.98 9.257

87.96 22.163 79.60 21.554 96.33 16.260

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Table 4 Species-specific mean centroid sizes (CS), mean common crus length (CCL, in mm), and normalized CS and CCL variances. Speciesspecific normalization was computed in order to achieve mean (CS) = 1, and mean (CCL) = 1 for each of the four involved species. Doing so, the

four variances are comparable across species. Lat_CS: centroid size of the lateral semicircular canal; Ant_CS: centroid size of the anterior semicircular canal; Post_CS: centroid size of the posterior semicircular canal

Callithrix jacchus

Microcebus murinus

Nycticebus coucang


Mean (Lat_CS) Variance (Norm(Lat_CS))

11.623 0.00297

8.859 0.00353

11.227 0.00405

11.667 0.00774

Mean (Ant_CS)

11.569

9.639

15.748

15.438

Variance (Norm(Ant_CS))

0.00268

0.00239

0.00441

0.00490

Mean (Post_CS) Variance (Norm(Post_CS))

11.958 0.00141

8.289 0.00372

12.917 0.00458

13.424 0.00520

Mean (CCL) Variance (Norm(CCL))

1.322 0.0114

0.649 0.0237

1.210 0.0684

1.032 0.0895

Spells (1963), Spoor et al. (2007) thought that high values of R indicate high amplitude angular velocities. Malinzak et al. (2012) rather investigated the orientation of the SCCs than R. They observed that animals with fast head rotations displayed a nearly right angle between each SCC. Animals having an angle between the SCCs that depart from 90° would have slower head rotations. Berlin et al. (2013) studied the bony labyrinths of several mammalian species and observed that the orthogonality between ipsilateral SCCs (from the same bony labyrinth) was never present in their sample instead of what is generally accepted with the canonical model (see Materials and Methods). Moreover, important deviations from orthogonality could sometimes be calculated (31.2° being the largest deviation from orthogonality obtained in their sample). However, they observed that species presenting more orthogonal SCCs present a higher mean vestibular sensitivity to motion. Following Billet et al. (2012), we have focused our analyses on the variation of the morphology of the bony labyrinth at both the interspecific and intraspecific levels because intraspecific variation has been poorly studied before. We found that the labyrinthine morphology of N. coucang and P. potto edwardsi was very distinct from that of the other investigated species, which supports the observation of Lebrun et al. (2010) that Lorisiformes exhibit a unique labyrinthine architecture within primates. More importantly, for each species, we have quantified the intraspecific morphological variation of each part of the bony labyrinth. This variation is expressed on the shape and size of the bony labyrinth, and on the orthogonality of the SCCs, and it is higher in the slowmoving taxa. Thus, the amount of intraspecific variation of the morphology of the inner ear is most probably linked to the need for a certain sensitivity to motion of the vestibular system. It is important to note that our work does not necessarily invalidate the results obtained in preceding studies (e.g., Spoor et al. 2007; Malinzak et al. 2012) taking into account

R or the orthogonality of the SCCs. However, the levels of intraspecific labyrinthine morphological variation we have observed in our sample (and especially in the slow-moving taxa) call for extreme caution regarding the interpretation of measurements based on one or only a few individuals per species. In other words, one should avoid the temptation to infer the locomotor behavior of an extinct species based on the bony labyrinthine morphology of one single individual, but rather, whenever possible, draw conclusions based on the best possible picture of the species wide labyrinthine morphological range. Orthogonality For the four investigated species, we have measured that the SCCs often deviate from orthogonality (Table 3), giving additional support to the stance of Berlin et al. (2013) that the canonical model is an over-simplification of the vertebrate bony labyrinth architecture. Callithrix jacchus and M. murinus, the two fast taxa of our sample, do not always display angles between SCCs, which depart in average the least from orthogonality (for example, see the Lat_Post and the Ant_Post angle measurements). Besides, although C. jacchus is a fast taxon, this species exhibits the highest mean departure from orthogonality for the Ant_Post angle measurement (105.63°). Yet, it exhibits one of the smallest variances for this measurement, and this trend is also observed for two other angles. Fast taxa do not always exhibit angles closer to orthogonality than those of slow taxa, but P. potto and N. coucang always display higher variances for these measurements. Berlin et al. (2013) observed that the angle between the LSCC and the ASCC was always inferior to 90° in their sample. With a larger-sized sample, we observed few individuals exhibiting a Lat_Ant angle superior to 90°, thereby indicating that the Lat_Ant angle inferior to 90° is not a rule.

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It is reasonable to think that a fast-moving individual exhibits fast head rotations, and as such, it probably needs a high sensitivity to motion. Following this idea, and contrary to Malinzak et al. (2012) and Berlin et al. (2013), we do not observe that fast-moving individuals exhibit SCCs closer to orthogonality. These authors stated that the closer to orthogonality is the angle, the higher is the sensitivity to motion. Contrarily, our results rather suggest that sensitivity to motion of the vestibular system is not fully related to orthogonality. We did not measure head rotation on living specimens, but rather calculated 90var (sensu Berlin et al. 2013, as the sum of the absolute deviations from orthogonality of ipsilateral canal pairs, and taking the mean) for each species, and we obtained 8.95° for C. jacchus, 4.10° for M. murinus, 3.59° for N. coucang, and 7.05° for P. potto edwardsi. Should we interpret our measurements of 90var building on the conclusions of Malinzak et al. (2012) and Berlin et al. (2013), we would infer that M. murinus and N. coucang present the fastest head rotations of our sample as they both present in average the smallest deviation from orthogonality (lowest 90var). This would be in contradiction with what is known about the locomotor repertoire of the extant taxa studied here. On the contrary, our results suggest that 90var is not always a good proxy for locomotor behavior, and that studying labyrinthine morphological variability (based on the orientation of the canals, but probably also on the shape and size of the whole labyrinth) might be a much safer approach to attempt to reconstruct the locomotor repertoires of extinct taxa; indeed, we observed here that slow, but also fast taxa, display significant amount of variance for the angle between the different SCC (slow taxa displaying even greater variances than fast taxa). Billet et al. (2012) showed an important intraspecific variation of the morphology of the SCCs in Bradypus variegatus, affecting the width and height of each SCC, but also the angle between them (for instance they report an impressive 44° range of variation for the angle between the anterior and lateral SCCs). Such a difference between two extreme specimens was not observed in our sample (for the same angle, we calculated 10.27° for C. jacchus, 11.47° for M. murinus, 15.26° for N. coucang, and 19.79° for P. potto edwardsi), but like Billet et al. (2012), we have observed that the Lat_Ant angle is always the one presenting the biggest range of variation. And still, the two slow taxa sampled here, present a wider range of variation than the fast taxa. Bradypus variegatus being considered as an extremely-slow taxon (Duarte et al. 2003), it is tempting to conclude that for a given species, the angular range of a given labyrinthine ipsilateral SCC pair, and hence its variability, relates to the sluggishness/rapidity of head movements.

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Global Shape Patterns Vestibular System Building on the results of our geometric morphometric analysis, we must emphasize that all taxa analyzed here exhibit a similar pattern of intraspecific vestibular shape variation. Variations are primarily observed in the height and width of their semicircular canals (SCCs), as well as in the inclination of the LSCC for M. murinus, N. coucang, and P. potto edwardsi. However, the amount of variation is different from one species to another. Indeed, taxa practicing a slow locomotion (P. potto edwardsi and N. coucang) clearly show a higher amount of intraspecific variation in the 3D morphology of the SCC system (cf. Table 1, Fig. 7) than taxa practicing a fast locomotion (C. jacchus and M. murinus), as Billet et al. (2012) first hypothesized. Cochlea Cochlear shape variation patterns can also be observed (Fig. 5; Table 1). At the intraspecific level, the SV values reveal the same trend as that observed for the SCCs. As the same trend can be observed between the vestibular system and the cochlea, analyses on the global shape of the bony labyrinth can be performed (see discussion below). Semicircular Canal Size The shape of the vestibular system has an impact on the flow of the endolymph in the membranous ducts and on the sensitivity to motion of an animal as a result. Orthogonality also influences the sensitivity to motion, but the size of the SCCs may play an important role as well. It is thought that the size of the vertical SCCs reflects requirements for head movements (Hill et al. 2014). On the other hand, Jeffery et al. (2008) considered the LSCC to be the best related to the locomotor agility of an animal. We did not observe a specific trend in the variation of each one of those SCCs. However, for each SCC (and for the common crus), we observed that slow-moving taxa exhibit the highest CS variance (see Table 4). Perodicticus potto edwardsi always shows the highest variance, while C. jacchus the lowest. Those results echo the results obtained with the shape of the bony labyrinth and with those of the orientation of the SCCs. The slower an animal is, the more variable is its bony labyrinth within its species. As the same trends are obtained using the shape, size, and SCC orientation of the vestibular system, these three proxies may be all equally valid when trying to relate labyrinthine morphology and locomotion. Most importantly, whenever possible, our results call for the assessment of intraspecific morphological variability before making assumptions at the interspecific level.

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Cause of the Variability Observed The SCCs detect the angular acceleration of the head and allow posture corrections via vestibular-ocular and vestibulo-colic reflexes (Graf and Klam 2006; Spoor et al. 2007). These reflexes allow to keep the gaze on a target during a movement and to balance the body (Spoor et al. 2007; David et al. 2010). All primates studied here move in forests and climb trees. A loss of the body balance function could then be lethal (i.e., falling). It can therefore be expected that the position and configuration (in the three-dimensional space) of the bony labyrinth in the petrous bone are very constrained from a developmental perspective, in order to ensure an optimum sensitivity and perception of balance (related to fluid flow, i.e., endolymph; see David et al. 2010). Indeed, as Billet et al. (2012: 3936) stated: BMorphological variations in the SC[SCCs] system can dramatically change the animal’s temporal response dynamic (i.e., the behaviour of the cupulae with regard to time during a rotation) and sensitivity to motion.^ A fast-moving primate performs quick and sudden head movements. This primate therefore requires rapid postural adjustments to maintain a balance permanently (i.e., a high sensitivity of the SCCs in order to apprehend the slightest and rapid movements). In contrast, slow-moving primates are expected to have slower head movements. They are therefore subject to lower angular accelerations, and as such, their postural adjustments are lesser and slower. Considering the previous explanations, the greater variation in the 3D morphology of the SCCs of slow taxa should lead to greater differences in relative sensitivity of canals among specimens. The existence of such varying sensitivity patterns suggests that their functional architecture is not associated with a high level of selection on the morphology of the SCCs. A high level of selection would necessarily imply strong selective pressure (Maynard Smith et al. 1985; Arnold 1992). As differences in the morphology of the SCCs would lead to a lower sensitivity to head rotations, high level of selection on the morphology of the inner ear of fastmoving primates is expected to secure the optimum sensitivity of those animals. Our results support the hypothesis of Billet et al. (2012) according to which a release of this selective pressure on the morphology of the SCCs in slow-moving taxa is a plausible explanation for such a different level of intraspecific variation between slow- and fast-moving taxa. However, fast-moving taxa are not statistically distinct from slow-moving taxa (the confidence limits of M. murinus and N. coucang overlap). Microcebus murinus is a fast-moving animal but it performs slow movements from time to time (Martin 1972), while P. potto edwardsi sometimes runs (Walker 1969). This overlapping of the different rapidity of locomotion across taxa may explain why there is not always a statistically significant morphological signal making it possible to distinguish

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fast-moving and slow-moving taxa. However, the overall activity of M. murinus clearly mostly comprises fast movements and the overall activity of slow-moving taxa mostly comprises slow movements. Besides, we expect that the morphology of the bony labyrinth must be best suited for the movements that are performed more frequently. Following Billet et al. (2012), selective pressure must apply on the morphology of the SCCs in fast-moving species, so that the individuals would possess an architecture of the vestibular system that would permit the animal to detect motions optimally. In contradiction to the hypotheses of Malinzak et al. (2012) and Berlin et al. (2013), fast-moving taxa do not exhibit angles that depart less from orthogonality. However, fast taxa exhibit the lowest amount of intraspecific variation regarding the relative orientation of each ipsilateral SCC. A possible interpretation of our result is as follows: 1) all individuals undergo selective pressures that apply on the morphology of the vestibular system to secure the angle at which sensitivity to motion is optimal. But this angle would not necessarily be close to 90° (e.g., the mean Ant_Post angle of 105° displayed by C. jacchus); 2) individuals belonging to fast species undergo greater selective pressures than those belonging to slow species . A difference in sensitivity would surely have a greater impact in fast-moving taxa than in slow-moving taxa. In this context, we can hypothesize that slow- and cautious-moving taxa, such as Perodicticus and Nycticebus, for which the demand in rapid postural adjustments is less, do not present the need for a sensitivity to motion of their SCCs as precise as that displayed by fast-moving taxa. One of our analyses was conducted on the whole bony labyrinth, comprising the organ of hearing (i.e., the cochlea), which is not linked to the balance of an animal. However, variation on the cochlear shape is still observed, and this variation follows the same trends as that observed on the SCCs morphology (Table 1) (i.e., slow-moving taxa exhibit a higher amount of intraspecific variation of the organ of hearing than do fast-moving taxa), which means that the same evolutionary process might be responsible for cochlear morphological variation. It is possible that the effect of a selective pressure applied on the vestibular system would have side-effects on the cochlea as these two bony structures are part of the same anatomical unit (i.e., the bony labyrinth). Thus, a release of the selective pressure on the vestibular system would also impact cochlear morphology, allowing it to be more variable as well. If selective pressure is applied on the bony labyrinth, it may also impact adjacent structures. Hautier et al. (2014) investigated the amount of intraspecific variation of the skull shape compared to the amount of intraspecific variation of the SCCs shape in sloths. They found that sloths depart from usual

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mammalian levels of variation for the SCCs but not for the skull, which means that the variation is primarily concentrated on the SCCs and on the cochlea (Billet et al. 2012).

differentiate slow versus fast-moving taxa via their levels of labyrinthine morphological (shape, size and SCC orientation) variance be confirmed, paleontologists would possess a powerful means to infer the locomotor behavior of extinct species.

Conclusion

Acknowledgments We thank Jacques Cuisin (Muséum National d’Histoire Naturelle [MNHN], Paris), Robert Asher (University of Cambridge), Christopher Zollikofer and Marcia Ponce de León (Anthropological Institute and Museum, Zürich), Loic Costeur (Naturhistorisches Museum Basel, Basel), Nadine Mestre (Laboratoire Vi e i l l i s s e m e n t C é r é b r a l e t P a t h o g e n è s e d e s M a l a d i e s Neurodégénératives, Montpellier [Petter-Rousseaux collection]), Peter Giere (Museum für Naturkunde, Berlin), and Suzanne Jiquel (ISE-M) for access to osteological collections; Guillaume Billet (MNHN, Paris) and Lionel Hautier (ISE-M) for discussions on this study; and Julien Claude (ISE-M) and Nathan Young (University of California, San Fransisco) for their advice regarding some statistical analyses. We are also grateful to the staff of beamlines ID19 and ID17 of the European Synchrotron Radiation Facility [ESRF], Grenoble) and especially to Paul Tafforeau. Moreover, we would like to thank Guillaume Billet (MNHN, Paris) and another anonymous reviewer who provided formal reviews of this manuscript that significantly enhanced the quality of the current version. Finally, many thanks to the Montpellier RIO Imaging (MRI) and the LabEx CeMEB for the access to the μCT-scanning station Skyscan 1076 (ISE-M). This research was supported by the French ANR-ERC PALASIAFRICA Program (ANR-08-JCJC-0017) and the Laboratoire de Paléontologie (ISE-M). This is ISE-M publication n° 2016–028 SUD.

Investigation of interspecific and intraspecific patterns of variations of the morphology of the inner ear of four primate species shows that slow-moving taxa exhibit higher amounts of intraspecific variation of this structure than fast-moving taxa. This variation is primarily expressed by differences in the relative size, shape, and orientation of the semicircular canals, but it is also measurable on the cochlea (width and height). As a whole, our results suggest that patterns of intraspecific labyrinthine morphological variability are linked to the locomotor repertoire of a given species. However, our sample of species and number of specimens per species are rather small and deserve to be increased in order to give additional support to this hypothesis. Sampling Galagidae (closely phylogenetically related to the two slow lorisid species investigated here) specimens, which are fast-moving primates, would allow us to test for potential phylogenetic effects within the Lorisiformes regarding the levels of labyrinthine morphological variation. Besides, although the levels of intraspecific variation differ between slow and fast taxa, this distinction is so far not statistically supported in all cases; increasing the number of specimens sampled per species would probably clarify this issue. Even though our results are still, to some extent, preliminary, they nevertheless strengthen support for the hypothesis of a greater intraspecific variation of the 3D morphology of the bony labyrinth (mostly semicircular canals) in slow taxa, which was recently investigated and supported by Billet et al. (2012). A probable cause for this greater variation recorded in primates having a slow locomotion could be the release of the selective pressure that takes place on the organ of balance during the early stages of development, in relation to a reduced functional demand (there is no need to be as sensitive as fast taxa). If selective pressure actually existed and would be at work, it would most probably act during the development of the individual, when the organ is not yet fully formed. Ontogenetic studies might give us insights into the origin and nature of this selective pressure. Moreover, our results underline the importance of studying intraspecific morphological variation before making assumptions at the interspecific level: because the morphology of the bony labyrinth shows a tremendous amount of intraspecific variation, measurements taken on the organ of one individual might not be sufficient to infer the locomotor behavior of the species to which it belongs. Our analysis was limited only to four species, but these preliminary results are promising. Should the possibility to

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