Symmetrical gaits of Cebus apella: implications for the functional significance of diagonal sequence gait in primates

Quadrupedal locomotion of primates is distinguished from the quadrupedalism of many other mammals by several features, including a diagonal sequence (DS) footfall used in symmetrical gaits. This presumably unique feature of primate locomotion has been attributed to an ancestral adaptation for cautious arboreal quadrupedalism on thin, flexible branches. However, the functional significance of DS gait remains largely hypothetical. The study presented here tests hypotheses about the functional significance of DS gait by analyzing the gait mechanics of a primate that alternates between DS and lateral sequence (LS) gaits, Cebus apella. Kinematic and kinetic data were gathered from two subjects as they moved across both terrestrial and simulated arboreal substrates. These data were used to test four hypotheses: (1) locomotion on arboreal supports is associated with increased use of DS gait, (2) DS gait is associated with lower peak vertical substrate reaction forces than LS gait, (3) DS gait is associated with greater forelimb/hind limb differentiation in force magnitudes, and (4) DS gait offers increased stability. Our results indicate that animals preferred DS gait on the arboreal substrate, and LS gait while on the ground. Peak vertical substrate reaction forces showed a tendency to be lower in DS gait, but not consistently so. Pole ("arboreal") forces were lower than ground forces in DS gait, but not in LS gait. The preferred symmetrical gait on both substrates was a grounded run or amble, with the body supported by only one limb throughout most of the stride. During periods of bilateral support, the DS gait had predominantly diagonal support couplets. This benefit for stability on an arboreal substrate is potentially outweighed by overstriding, its associated ipsilateral limb interference in DS gait and hind foot positioning in front of the hand on untested territory. DS gait also did not result in an optimal anchoring position of the hind foot under the center of mass of the body at forelimb touchdown. In sum, the results are mixed regarding the superiority of DS gait in an arboreal setting. Consequently, the notion that DS gait is an ancestral adaptation of primates, conditioned by the selection demands of an arboreal environment, remains largely hypothetical.     

Lateral sequence walking in infant Papio cynocephalus: implications for the evolution of diagonal sequence walking in primates

One of the most distinctive aspects of primate quadrupedal walking is the use of diagonal sequence footfalls in combination with diagonal-couplets interlimb timing. Numerous hypotheses have been offered to explain why primates might have evolved this type of gait, yet this important question remains unresolved. Because infant primates use a wider variety of quadrupedal gaits than do adults, they provide a natural experiment with which to test hypotheses about the evolution of unique aspects of primate quadrupedalism. In this study, we present kinematic data on two infant baboons (Papio cynocephalus) in order to test the recent hypothesis that diagonal sequence, diagonal couplets walking might have evolved in primates because their limb positioning provides stability in a small branch environment (Cartmill et al. [2002] Zool J Linn Soc 136:401-420). To assess hindlimb position at the moment of forelimb touchdown, we measured hindlimb angular excursion and ankle position for 84 walking strides, across three different types of gaits (diagonal sequence, diagonal couplets (DSDC); lateral sequence lateral couplets (LSLC); and lateral sequence diagonal couplets (LSDC)). Results indicate that if a forelimb were to contact an unstable substrate, LSLC walking provides as much, and perhaps more, stability when compared to DSDC walking. Therefore, it appears that this moment in a stride was unlikely to be a particularly important selective factor in the evolution of DSDC walking. Further insight into this issue will likely be gained by observations of primate quadrupedalism in natural environments, where the use of lateral sequence gaits might be more common than currently known.     

Stability, limb coordination and substrate type: the ecorelevance of gait sequence pattern in primates

The coordination of limb movements during mammalian locomotion has been well documented in the literature. Most mammals use lateral sequence (LS) gaits, in which a forelimb follows an ipsilateral hind limb during the stride cycle. Primates, however, tend to utilize diagonal sequence (DS) gaits, whereby a contralateral forelimb follows a given hind limb during the stride cycle. A number of scenarios have been offered to explain why primates favor DS gaits, most of them relating to the use of the arboreal habitat and, in particular, the exploitation of a terminal branch niche. Yet to date, there is surprisingly little evidence to support the advantage of DS gaits for negotiating different aspects of the terminal branch environment. Nonetheless, it is apparent that primates possess unique morphologies and a higher than typically recognized degree of flexibility in gait sequence pattern, both of which likely offer advantages for moving upon discontinuous and unstable terminal branches. This paper reviews potential explanations for the use of DS gaits in primates and considers mechanisms by which gait sequence may be altered during different types of arboreal challenges.     

The effects of natural substrate discontinuities on the quadrupedal gait kinematics of free‐ranging Saimiri sciureus

Wild primates encounter complex matrices of substrates that differ in size, orientation, height, and compliance, and often move on multiple, discontinuous substrates within a single bout of locomotion. Our current understanding of primate gait is limited by artificial laboratory settings in which primate quadrupedal gait has primarily been studied. This study analyzes wild Saimiri sciureus (common squirrel monkey) gait on discontinuous substrates to capture the realistic effects of the complex arboreal habitat on walking kinematics. We collected high‐speed video footage at Tiputini Biodiversity Station, Ecuador between August and October 2017. Overall, the squirrel monkeys used more asymmetrical walking gaits than symmetrical gaits, and specifically asymmetrical lateral sequence walking gaits when moving across discontinuous substrates. When individuals used symmetrical gaits, they used diagonal sequence gaits more than lateral sequence gaits. In addition, individuals were more likely to change their footfall sequence during strides on discontinuous substrates. Squirrel monkeys increased the time lag between touchdowns both of ipsilaterally paired limbs (pair lag) and of the paired forelimbs (forelimb lag) when walking across discontinuous substrates compared to continuous substrates. Results indicate that gait flexibility and the ability to alter footfall patterns during quadrupedal walking may be critical for primates to safely move in their complex arboreal habitats. Notably, wild squirrel monkey quadrupedalism is diverse and flexible with high proportions of asymmetrical walking. Studying kinematics in the wild is critical for understanding the complexity of primate quadrupedalism.     

The effect of branch diameter on primate gait sequence pattern

Most mammals use lateral sequence gaits during quadrupedal locomotion, a pattern characterized by the touchdown of a forelimb directly following the ipsilateral hind limb in a given stride cycle. Primates, however, tend to use diagonal sequence (DS) gaits, whereby it is the touchdown of a contralateral forelimb that follows that of a given hind limb most closely in time. A number of scenarios have been offered to explain why primates favor DS gaits, most of them relating to the use of the arboreal habitat and, in particular, the exploitation of a narrow branch niche. This experimental study explores the potential explanation for the use of DS gaits by examining the relationship between branch diameter and gait patterns in 360 strides collected from six species of quadrupedal strepsirrhine primates on broad and narrow diameter supports. Gait sequence is quantified using limb phase, or the percentage of time during a stride cycle that a forelimb touchdown follows an ipsilateral hind limb touchdown. Although Loris, Nycticebus and Eulemur rubriventer individuals in this study did exhibit significantly lower locomotor velocities on narrower supports (P<0.01 in all three species), analyses of covariance showed no significant differences in limb phase values between broad and narrow diameter supports. Hence, results indicate surprisingly little evidence to suggest that alterations in gait sequence pattern provide a specific advantage for negotiating narrow supports.     

Gait selection and the ontogeny of quadrupedal walking in squirrel monkeys (Saimiri boliviensis)

Locomotor researchers have long known that adult primates employ a unique footfall sequence during walking. Most mammals use lateral sequence (LS) gaits, in which hind foot touchdowns are followed by ipsilateral forefoot touchdowns. In contrast, most quadrupedal primates use diagonal sequence (DS) gaits, in which hind foot touchdowns are followed by contralateral forefoot touchdowns. However, gait selection in immature primates is more variable, with infants and juveniles frequently using LS gaits either exclusively or in addition to DS gaits. I explored the developmental bases for this phenomenon by examining the ontogeny of gait selection in juvenile squirrel monkeys walking on flat and simulated arboreal substrates (i.e., a raised pole). Although DS gaits predominated throughout development, the juvenile squirrel monkeys nonetheless utilized LS gaits in one-third of the ground strides and in one-sixth of pole strides. Multiple logistic regression analyses showed that gait selection within the juvenile squirrel monkey sample was not significantly associated with either age or body mass per se, arguing against the oft-cited argument that general neuromuscular maturation is responsible for ontogenetic changes in preferred footfall sequence. Rather, lower level biomechanical variables, specifically the position of the whole-body center of mass and the potential for interference between ipsilateral fore and hindlimbs, best explained variation in footfall patterns. Overall, results demonstrate the promise of developmental studies of growth and locomotor development to serve as "natural laboratories" in which to explore how variability in morphology is, or is not, associated with variability in locomotor behavior.     

Substrate determines asymmetrical gait dynamics in marmosets (Callithrix jacchus) and squirrel monkeys (Saimiri boliviensis)

Studies of skeletal pathology indicate that injury from falling accounts for most long bone trauma in free‐ranging primates, suggesting that primates should be under strong selection to manifest morphological and behavioral mechanisms that increase stability on arboreal substrates. Although previous studies have identified several kinematic and kinetic features of primate symmetrical gaits that serve to increase arboreal stability, very little work has focused on the dynamics of primate asymmetrical gaits. Nevertheless, asymmetrical gaits typify the rapid locomotion of most primates, particularly in smaller bodied taxa. This study investigated asymmetrical gait dynamics in growing marmosets and squirrel monkeys moving on terrestrial and simulated arboreal supports (i.e., an elevated pole). Results showed that monkeys used several kinematic and kinetic adjustments to increase stability on the pole, including reducing peak vertical forces, limiting center of mass movements, increasing substrate contact durations, and using shorter and more frequent strides (thus limiting disruptive whole‐body aerial phases). Marmosets generally showed greater adjustment to pole locomotion than did squirrel monkeys, perhaps as a result of their reduced grasping abilities and retreat from the fine‐branch niche. Ontogenetic increases in body size had relatively little independent influence on asymmetrical gait dynamics during pole locomotion, despite biomechanical theory suggesting that arboreal instability is exacerbated as body size increases relative to substrate diameter. Overall, this study shows that 1) symmetrical gaits are not the only stable way to travel arboreally and 2) small‐bodied primates utilize specific kinematic and kinetic adjustments to increase stability when using asymmetrical gaits on arboreal substrates. Am J Phys Anthropol, 2009. © 2008 Wiley‐Liss, Inc.     

Positional behavior of free-ranging Japanese macaques (<Emphasis Type="Italic">Macaca fuscata</Emphasis>)

Positional behavior was quantitatively studied in identified free-ranging Japanese macaques (Macaca fuscata). Five male and 11 female adults were observed in a forested mountain habitat. Data were analyzed for proportion of bout distance, number and time of each locomotion and postural type. Japanese macaques are semiterrestrial, and mainly walk and run quadrupedally. This supports the notion that Macaca are generally quadrupeds. Sex differences in positional behavior were found in the preference of substrate and types of positional behavior. Males and females tend to be terrestrial and arboreal, respectively. Males leap more frequently and longer in distance than do females when they are feeding in trees. These sex differences are considered to be related to differences in morphology, food choice, social activity, and the nursing of infants. Frequencies of leaping and the distance covered by leaping in Japanese macaques are more than those of long-tailed macaques which are arboreal quadrupeds. However, Japanese macaques leap shorter distances at a time than do long-tailed macaques, which indicates that body size may be related to leaping distance more than the frequency of leaping and the distance covered by leaping. Japanese macaques are not as specialized for terrestrial locomotion as pig-tailed macaques. They use both terrestrial and arboreal supports, and are considered to be semi-terrestrial quadrupeds, somewhere between the arboreal long-tailed macaque and the terrestrial pig-tailed macaque. Electronic Publication     

Body mass distribution and gait mechanics in fat-tailed dwarf lemurs (Cheirogaleus medius) and patas monkeys (Erythrocebus patas)

Most quadrupeds walk with lateral sequence (LS) gaits, where hind limb touchdowns are followed by ipsilateral forelimb touchdowns. Primates, however, typically walk with diagonal sequence (DS) gaits, where hind limb touchdowns are followed by contralateral forelimb touchdowns. Because the use of DS gaits is nearly ubiquitous among primates, understanding gait selection in primates is critical to understanding primate locomotor evolution. The Support Polygon Model [Tomita, M., 1967. A study on the movement pattern of four limbs in walking. J. Anthropol. Soc. Nippon 75, 120-146; Rollinson, J., Martin, R.D., 1981. Comparative aspects of primate locomotion, with special reference to arboreal cercopithecines. Symp. Zool. Soc. Lond. 48, 377-427] argues that primates' use of DS gaits stems from a more caudal position of the whole-body center of mass (COM) relative to other mammals. We tested the predictions of the Support Polygon Model by examining the effects of natural and experimental variations in COM position on gait mechanics in two distantly related primates: fat-tailed dwarf lemurs (Cheirogaleus medius) and patas monkeys (Erythrocebus patas). Dwarf lemur experiments compared individuals with and without a greatly enlarged tail (a feature associated with torpor that can be expected to shift the COM caudally). During patas monkey experiments, we experimentally shifted the COM cranially with the use of a weighted belt (7-12% of body mass) positioned above the scapulae. Examination of limb kinematics revealed changes consistent with systematic deviations in COM position. Nevertheless, footfall patterns changed in a direction contrary to the predictions of the Support Polygon Model in the dwarf lemurs and did not change at all in the patas monkey. These results suggest that body mass distribution is unlikely to be the sole determinant of footfall pattern in primates and other mammals.     

Effects of support size and orientation on symmetric gaits in free-ranging tamarins of Amazonian Peru: implications for the functional significance of primate gait sequence patterns

The adoption of a specific gait sequence pattern during symmetrical locomotion has been proposed to have been a key advantage for the exploitation of the fine branch niche in early primates. Diverse aspects of primate locomotion have been extensively studied in technically equipped laboratory settings, but evolutionary conclusions derived from these investigations have rarely been verified in wild primates. Bridging the gap from the lab to the field, we conducted an actual performance determination of symmetrical gaits in two free-ranging tamarin species (Saguinus mystax and Saguinus fuscicollis) of Amazonian Peru by analyzing high-speed video recordings of naturally occurring locomotor bouts. Tamarins arguably represent viable models for aspects of early primate locomotion. We tested three specific hypotheses derived from laboratory studies to test for the influence of support size and orientation and to gain further insight into the functional significance of primate gait sequence patterns: (1) The tamarins utilize symmetrical gaits at a higher rate on small supports than on larger ones. (2) During symmetrical locomotion on small supports, diagonal sequences are utilized at a higher rate than on larger supports. (3) On inclines, diagonal sequences are predominantly used and on declines, lateral sequences are predominantly used. Our results corroborated hypotheses 1 and 3. We found no clear support for hypothesis 2. In conclusion, our results add to the notion that primate gait plasticity, rather than uniform adoption of diagonal sequence gaits, enabled early primates to accommodate different support types and effectively exploit the small branch niche.     

Palmar and Plantar Pressure While Walking on a Horizontal Ladder and Single Pole in <Emphasis Type="Italic">Macaca fuscata</Emphasis>

To investigate biomechanical function in the hand and foot during quadrupedal locomotion in nonhuman primates, physical anthropologists and primatologists measure the pressure under them. We collected hand and foot pressure data while a Japanese macaque (Macaca fuscata), a semiterrestrial anthropoid, walked on 2 different simulated arboreal substrates, a horizontal ladder and a single pole, to explore differences in hand and foot use between the 2 substrates. The ladder rungs were perpendicular to the craniocaudal axis of the subject, and the pole was parallel to the subject’s craniocaudal axis. We tested the hypothesis that the pole was a more challenging substrate for the macaque than the ladder. Focusing on a diagonal sequence, diagonal couplets gait, we calculated gait characteristics and computed mean peak-pressure images of the hand and foot for each substrate from individual peak images via translation registration. We found several substrate differences that supported the hypothesis. The Japanese macaque walked at significantly slower speeds when traveling on the pole than on the ladder. Slower travel speed on the pole suggests that the Japanese macaque needed a wider support base to maintain balance on this substrate. Mean peak-pressure images suggest that the ladder invoked a more stepping-like behavior, but the pole invoked a more grasping-like behavior, especially of the foot. We show that the hand and foot use of the Japanese macaque would be adaptable to biomechanical challenges posed by different substrates.     

Muscle dimensions in the Japanese macaque hand

Macaques have been used as an important paradigm for understanding the neural control mechanisms of human precision grip capabilities. Therefore, we dissected the forearms and hands of two male Japanese macaques to systematically record the muscle mass, fascicle length and physiological cross-sectional area (PCSA). Comparisons of the mass fractions and PCSA fractions of the hand musculature among the Japanese macaque, chimpanzee, and human demonstrated that the sizes of the thenar and hypothenar eminence muscle groups are more balanced in the macaque and chimpanzee, but those of the thenar eminence group are much larger in the human, indicating that the capacity to generate force at the tip of the thumb is more restricted in macaques, despite their high manual dexterity. In the macaque, however, the extrinsic flexor muscles are much larger, possibly to facilitate weight bearing by the forelimbs in pronograde quadrupedal locomotion and forceful grasping of arboreal supports in gap-crossing movements such as leaping. Taking such anatomical differences imposed on the hand musculoskeletal system into consideration seems to be an important method of clarifying the mechanisms of precision grip in macaques.     

The Adaptive Significance of Tetrapod Gait Selection

At the slow walk tetrapods avoid lateral couplets gaits to minimizesupport by ipsilateral bipods. Most of them use the lateralsequence because tripods then make larger triangles than forthe diagonal sequence. Of the running symmetrical gaits thesingle foots (in each sequence) permit the smoothest and fastesttravel without suspensions. The trot and pace allow two legsto thrust in unison, the former giving the most stability toanimals not placing the feet well under the body and the latteravoiding interference for long legged runners. The bound andhalf bound are most used by small agile mammals for bursts ofspeed and for maneuvering on rough terrain by a series of leaps.Such animals use the extended suspension. Large cursors on openterrain usually select the shorter more economical, gatheredsuspension. The fastest runners use both suspensions to gainlong strides. At moderate speed the transverse gallop has theadvantages over the rotary gallop that both bipods and tripodsare more stable and that interference may be avoided. At highspeed using both suspensions, none of these advantages pertains.The rotary gallop may then increase maneuverability.     

Origins of primate locomotion: gait mechanics of the woolly opossum

The locomotion of primates differs from that of other mammals in three fundamental ways. During quadrupedal walking, primates use diagonal sequence gaits, protract their arms more at forelimb touchdown, and experience lower vertical substrate reaction forces on their forelimbs relative to their hindlimbs. It is widely held that the unusual walking gaits of primates represent a basal adaptation for movement on thin, flexible branches and reflect a major change in the functional role of the forelimb. However, little data on nonprimate arboreal mammals exist to test this notion. To that end, we examined the gait mechanics of the woolly opossum (Caluromys philander), a marsupial convergent with small-bodied prosimians in ecology, behavior, and morphology. Data on the footfall sequence, relative arm protraction, and peak vertical substrate reaction forces were obtained from videotapes and force records for three adult woolly opossums walking quadrupedally on a wooden runway and a thin pole. For all steps recorded on both substrates, woolly opossums always used diagonal sequence walking gaits, protracted their arms beyond 90 degrees relative to horizontal body axis, and experienced peak vertical substrate reaction forces on forelimbs that were significantly lower than on hindlimbs. The woolly opossum is the first nonprimate mammal to show locomotor mechanics that are identical to those of primates. This case of convergence between primates and a committed fine-branch, arboreal marsupial strongly implies that the earliest primates evolved gait specializations for fine-branch locomotion, which reflect important changes in forelimb function.     

Highly polymorphic STR marker amplified with human DYS389 primers in Japanese macaques (Macaca fuscata)

Amplification products from male and female Japanese macaques were obtained by PCR with human Y-chromosomal DYS389 primers. These products were examined by electrophoresis and sequence analysis. The PCR products from the 12 Japanese macaques tested had different band patterns on an electrophoretogram. Sequence analysis of the products revealed that the high polymorphism originated from variable numbers of repeats of two separate CTAT sequences. The sequences of the Japanese macaque products were similar to those of the reference human DYS389 sequence. However, variable CTGT repeats and a difference in the second forward primer binding site yielded two products in human males, DYS389I and DYS389II, which do not exist in Japanese macaques. Our results suggest that the human DYS389 primers may be a potential tool not only for distinguishing between human and Japanese macaque DNA samples, but also for identifying individual macaques, because of the highly polymorphic alleles.     

Angular momentum and arboreal stability in common marmosets (Callithrix jacchus)

Despite the importance that concepts of arboreal stability have in theories of primate locomotor evolution, we currently lack measures of balance performance during primate locomotion. We provide the first quantitative data on locomotor stability in an arboreal primate, the common marmoset (Callithrix jacchus), predicting that primates should maximize arboreal stability by minimizing side-to-side angular momentum about the support (i.e., L_sup). If net L_sup becomes excessive, the animal will be unable to arrest its angular movement and will fall. Using a novel, highly integrative experimental procedure we directly measured whole-body L_sup in two adult marmosets moving along narrow (2.5 cm diameter) and broad (5 cm diameter) poles. Marmosets showed a strong preference for asymmetrical gaits (e.g., gallops and bounds) over symmetrical gaits (e.g., walks and runs), with asymmetrical gaits representing >90% of all strides. Movement on the narrow support was associated with an increase in more “grounded” gaits (i.e., lacking an aerial phase) and a more even distribution of torque production between the fore- and hind limbs. These adjustments in gait dynamics significantly reduced net L_sup on the narrow support relative to the broad support. Despite their lack of a well-developed grasping apparatus, marmosets proved adept at producing muscular “grasping” torques about the support, particularly with the hind limbs. We contend that asymmetrical gaits permit small-bodied arboreal mammals, including primates, to expand “effective grasp” by gripping the substrate between left and right limbs of a girdle. This model of arboreal stability may hold important implications for understanding primate locomotor evolution. Am J Phys Anthropol 156:565–576, 2015. © 2014 Wiley Periodicals, Inc.     

Multivariate comparison of the forelimb between stump-tailed and rhesus macaques.

Rhesus macaques and stump-tailed macaques are sympatric in western Yunnan (China), coexisting or occupying habitats that show little difference. This paper tests hypotheses based on theoretical expectation from the differing biomechanical demands of terrestrial and arboreal quadrupedalism in stump-tailed macaques and rhesus macaques, respectively. Individuals of these two macaque taxa were markedly separated by the first two principal components and discriminant analyses based on 18 variables of the upper limb. The rhesus macaques appear to be more adapted for arboreal quadruped habits because of elongation of the clavicle and forearm, a larger humeral head and greater midshaft sagittal diameters of the radius and ulna.     

Molecular Evidence for Distinct Genotypes of Monkey B Virus (Herpesvirus Simiae) Which Are Related to the Macaque Host Species

Although monkey B virus (herpesvirus simiae; BV) is common in all macaque species, fatal human infections appear to be associated with exposure to rhesus macaques (Macaca mulatta), suggesting that BV isolates from rhesus monkeys may be more lethal to nonmacaques than are BV strains indigenous to other macaque species. To determine if significant differences that would support this supposition exist among BV isolates, we compared multiple BV strains isolated from rhesus, cynomolgus, pigtail, and Japanese macaques. Antigenic analyses indicated that while the isolates were very closely related to one another, there are some antigenic determinants that are specific to BV isolates from different macaque species. Restriction enzyme digest patterns of viral DNA revealed marked similarities between rhesus and Japanese macaque isolates, while pigtail and cynomolgus macaque isolates had distinctive cleavage patterns. To further compare genetic diversity among BV isolates, DNA sequences from two regions of the viral genome containing genes that are conserved (UL27 and US6) and variable (US4 and US5) among primate alphaherpesviruses, as well as from two noncoding intergenic regions, were determined. From these sequence data and a phylogenetic analysis of them it was evident that while all isolates were closely related strains of BV, there were three distinct genotypes. The three BV genotypes were directly related to the macaque species of origin and were composed of (i) isolates from rhesus and Japanese macaques, (ii) cynomolgus monkey isolates, and (iii) isolates from pigtail macaques. This study demonstrates the existence of different BV genotypes which are related to the macaque host species and thus provides a molecular basis for the possible existence of BV isolates which vary in their levels of pathogenicity for nonmacaque species.     

Radiation and phylogeography in the Japanese macaque, Macaca fuscata

The Japanese macaque (Macaca fuscata) presumably differentiated from eastern rhesus macaque (Macaca mulatta) populations during the Pleistocene and the two species are closely related. In order to analyse speciation and subspeciation events in the Japanese macaque and to describe historical and current relationships among their populations, we sequenced and analysed a fragment of 392bp of mitochondrial DNA (mtDNA) control region in 50 individuals belonging to six populations of Japanese macaque and compared these sequences with 89 eastern rhesus macaque control region sequences from GenBank/EMBL database. There were high genetic similarities between both species and only two positions were fixed within each species, which supports the inclusion of the Japanese macaque in a single species with eastern populations of rhesus macaques. Japanese macaque ancestors colonised Japan after the separation of the two species, estimated at between 0.31 and 0.88 million years ago (Mya). The star-like phylogeny, multimodal mismatch distribution, and lack of correlation between geographic and genetic distances are in accordance with a rapid dispersion of macaques throughout the archipelago after the arrival into Japan. The species shows low genetic variation within populations and high levels of genetic differentiation among populations with no mtDNA haplotype shared across populations. Genetic distances between Yakushima macaques (Macaca fuscata yakui) and any other population of Macaca fuscata fuscata subspecies are comparable to the distances between populations of Honshu, Awajishima, and Kyushu, not supporting the classification of Yakushima macaques as a different subspecies.