Introduction

Vietnam contains 10 species of leaf-eating primates representing 3 genera (Nadler et al. 2004). Though dietary data on the primates are limited, researchers have hinted at ecomorphological relationships among diet, feeding behavior, craniodental morphology, and gut physiology in these highly endangered colobines (Caton 1998; Jablonski et al. 1998; Kirkpatrick 1998). Over the past decade data have accumulated that demonstrate a relationship between the dental and cranial morphology of leaf-eaters and the average toughness of their diets in the wild. For example, Wright (2005) showed that the average toughness of foods masticated by howlers exceeds that of 5 other platyrrhine taxa in a primate community in central Guyana. Howlers exhibit well developed crests on large molars that make the comminution of tough foods more effective and time efficient (Lucas 2004; Rosenberger 1992).

Members of the Colobinae exhibit well-developed crests on their teeth and sacculation of the gut, which do not occur in other behavioral folivores, e.g., howlers and gorillas (Caton 1998, 1999; Fleagle 1999; Jablonski et al. 1998). Though researchers have documented a high percentage of leaf consumption in members of the Colobinae, my colleagues and I made initial qualitative observations of the morphology and ecology of leaf-eating monkeys in Vietnam that suggested differences among the primates in the way they process leaves to extract required nutrients. We compare the ingestive and digestive strategies of 2 genera of Vietnam leaf monkeys. We include 2 species of Trachypithecus (limestone langurs) and 2 species of Pygathrix (douc).

Is there more than one way to eat a leaf?

Leaves demand both mechanical and chemical mechanisms for the extraction of water and nutrients (Cheng et al. 1980; Dominy et al. 2001; Lucas et al. 1995). For mammals to gain required nutrients from leaves, they must be exposed to microbes that convert structural and nonstructural carbohydrates initially into monosaccharides and disaccharides and ultimately into volatile fatty acids (Cheng et al. 1980; Van Soest 1994, Waterman and Kool 1994). The effectiveness of the process can be influenced in 3 primary ways: 1) by retaining the leaves in the gut for extended periods, thus permitting longer exposure to gut fauna; 2) by expanding the gut and permitting more food to be digested at any one time; and 3) by increasing the surface area for the microbes to act upon by breaking leaves into smaller pieces via mastication (Lucas et al. in prep). Reptiles exhibit adaptations permitting the first 2 methods (Pafilis et al. 2007: gut retention; O’Grady et al. 2005: gut morphology). Birds also exhibit variation in gut retention times (Fukui 2003) and morphology (Battley and Theunis 2005; Grajal et al. 1989), and some exhibit adaptations of the bill and hyoid bone for food processing (Korzoun et al. 2003). Like birds, primates and other mammals exhibit gut and oral adaptations, particularly dental adaptations, to ingest and to digest foliage (Lucas 2004). But this begs the question: Do folivorous animals, particularly primates, emphasize any of these methods to the exclusion of others, and does the emphasis on any one method differ among closely related species? By holding dietary toughness constant, we sought to identify the degree to which Pygathrix spp. and Trachypithecus spp. are dependent on the mastication of leaves before digestion in the gut. We augment our findings by comparing size-adjusted molar areas between them and by a review of literature on variation in gut size and morphology among them.

Dietary Toughness and its Selective Influence on Primate Morphology and Masticatory Behavior

Toughness is the energy consumed in propagating a crack of a given area and is measured as the area under a force-displacement curve divided by crack area (Ashby 1992; Lucas 2004; Vincent 1992). During mastication, fragmentation of food between the teeth is largely dependent on either toughness or a combination of toughness and stiffness, and can be expressed as fragmentation indices (Agrawal et al. 1997; Lucas et al. 2002; Williams et al. 2005). Food tissues that can withstand high strains before crack propagation are termed displacement limited (Lucas et al. 2000) and demand such adaptations as well-developed crests on the postcanine dentition. Leaves are the quintessential displacement-limited foods in the diets of primates. Toughness alone is a good indicator of the mechanical demands that they place on the masticatory system, particularly among colobines, which are relatively dependent on leaves. Colobine primates exhibit relatively large molars, which increases the degree to which foods are comminuted per chew, and they exhibit crests that assist in driving cracks through tough materials, much the way scissors cut through paper (Lucas 2004).

We adapted laboratory equipment and software to field conditions to permit measurement of dietary mechanics. We acquired leaf toughness data via a portable field tester. We collected data on chewing bout lengths and chewing rates via a consumer-grade camera and videotape digitizing software.

Methods

Dietary Toughness

By providing the primates with monospecific bundles of leaves, we ranked the most preferred, moderately preferred, and least preferred foods. The portable universal tester we used was initially designed for testing the mechanical properties of foods processed by primates, and has been refined by Lucas and Darvell along with collaborators at the University of Hong Kong (Darvell et al. 1996; Lucas et al. 2001). The tester (Fig. 1), which is similar to Instron™ machines used in mechanical tests of larger or more mechanically demanding objects, is comprised of 3 essential components: 1) the stainless steel test stand, which is host to 1 of 2 load cells with limits of 10 and 100 Newtons (the load cells are sensitive enough to measure the stress-strain ratio of extremely small deformations); 2) the data integration box, which measures force in compression or tension and also displacement of the stand’s crosshead to which the load cell is attached; and 3) a portable computer with software to read the output from the integration box. Toughness in Joules/m2 is provided after one enters the force in Newtons for a cut of a given area (length and depth) into the computer. Scissor tests control fracture, making them particularly reliable, consistent, and relatively versatile. All reported tests are of toughness derived from scissor tests. One can test toughness in wedging and measure Young’s modulus (stiffness) also by compressing or bending specimens, and obtain the coefficient of friction via a weighted sled device. We conducted 2 sample comparisons via the Mann-Whitney U test between the 2 genera for the toughness of foods in each preference category and for all foods combined (Table I). We chose a nonparametric test statistic owing to the increase in variance that occurs with increasing food toughness (Williams et al. 2005).

Fig. 1
figure 1

Photo of the mechanical test stand, integration box, and computer used for the collection of toughness data. Accessories for a range of different mechanical tests are labeled. Photo courtesy P.W. Lucas.

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Table I Sample sizes for toughness and chewing variables
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Chewing Rates and Bout Lengths

We filmed subjects with a Sony Handycam high-speed consumer-grade camcorder while they were feeding. We imported video clips into Final Cut video software and converted them to Quicktime files (Table I). We did not conduct the video analysis on site, but it is possible given sufficient time. We measured chewing rates from watching lip and jaw movements onscreen, slowing the recordings if necessary to increase accuracy. Timing at any speed was possible because of the onscreen clock, which is synchronized with the video frames no matter the speed. We obtained chewing rate data at 4 different enclosures that housed 1 each of the 4 focal species (Table II). We took chewing rates randomly from various films and could not match them with leaf toughness; however, we paired feeding bouts with leaf toughness for additional analysis. We examined the video frame-by-frame to identify the beginning of feeding bout, end of ingestion phase/beginning of chewing phase, and end of feeding bout. Table I contains the number of bouts for each species and sex. We compared differences in feeding rates and in chewing bout lengths between the species and genera via the nonparametric Mann-Whitney U test statistic (the equivalent of the parametric 2-sample Student’s t statistic).

Table II Number of adult males and females in each study group that were included in the study
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Relative Molar Size and Observations

To augment the findings from the analyses of dietary toughness and feeding behavior, we measured the teeth of all 4 species at the National Museum of Natural History (NMNH) and at the Endangered Primates Rescue Center (EPRC), Vietnam. All 4 focal primates are either endangered (Pygathrix nemaeus and Trachypithecus I. hatinhensis) or highly endangered (P. cinerea and T. delacouri), which together with limited work by Western scientists throughout Southeast Asia through much of the 20th century, has limited the number of museum specimens. Our best represented species for the morphological analysis is Pygathrix nemaeus (n = 11 NMNH, n = 1 EPRC), followed by P. cinerea (n = 6 EPRC), Trachypithecus I. hatinhensis (n = 2, EPRC), and T. delacouri (n = 1 EPRC). We pooled the species in each genus for analysis. We calculated a ratio of molar area (calculated from buccolingual and mesiodistal measurements of M2) divided by cranial vault length (from nasion to opisthocranion) ×100 for each specimen to control for size. We used the Mann-Whitney U test to compare the ratios between the 2 genera. Tooth size scales with body mass, but we found no body mass datum for Pygathrix cinerea, Trachypithecus I. hatinhensis, or T. delacouri.

Results

Leaf Toughness

Mid-ranked leaves were the toughest tissues ingested by the 4 focal species (Table III). The subjects could have dealt with the least preferred items mechanically, but may have avoided doing so because of the presence of secondary chemical compounds. However, there is no significant difference between all pairs of leaf monkey for each preference category and for all leaf specimens pooled (p > 0.05). Thus, it appears that when given the opportunity to exploit the same foods in the absence of interspecific competition, the focal species select foods of comparable toughness (Fig. 2). Thus, we can say with some certainty that toughness was held constant when observing chewing bout lengths and chewing rates.

Fig. 2
figure 2

Box-and-whisker plots of the toughness of ingested leaves for each of the 4 primates species in this study (Mann-Whitney U, p > 0.05 for all comparisons). Vertical line = median, length of box = range within which the central 50% of the values fall, hinges = first and third quartiles.

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Table III Average toughness (J m−2) of high-, middle-, and low-priority foods, eaten by all 4 focal species
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Chewing Bouts

There is a significant difference between Pygathrix cinerea with the shortest bouts and P. nemaeus with the longest bouts (Table IV; Fig. 3) and there is a trend for increasing bout length with an increase in the toughness of ingested leaves (Fig. 4). However, there is no difference between Pygathrix and Trachypithecus for chewing bout length. The bout lengths for the 2 Trachypithecus spp. lie between those for Pygathrix spp.; thus no genus-wide pattern is apparent. Logan and Sanson (2002) found an increase in chewing bout length with an increase in dental wear in koalas. However, King et al. (2005) found that compensatory shearing blades occur up to the age of 18 in Propithecus edwardsi, after which the molars begin to lose their shearing capability. The age of our female Pygathrix cinerea is unknown; however, the age of the male P. cinerea (12 yr) exceeds that of the male P. nemaeus (8 yr) by ca. 4 yr (Table II). It may be that the shearing efficiency of the older male gray-shanked douc actually exceeds that of the younger male red-shanked douc given the findings of King et al. (2005), which may increase the degree of leaf comminution per unit time. However, the difference between the 2 genera is statistically negligible.

Fig. 3
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Log transformed bout length (in seconds) linearly regressed against log transformed toughness (J m−2) for the same food items for each primate species (T. hatinhensis = □, Pygathrix = ○. T. delacouri =△. The pygathrix species were pooled for analysis due to only one specimen of P. nemaeus (the largest value for P. nemaeus at 1371.2 J m−2). Note the general trend for an increase in bout length with an increase in leaf toughness for each primate species.

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Table IV Mann-Whitney U test statistic results for chewing bout length for all species pairs
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Chewing Rates

There are differences in chewing rate between the 2 primate genera and among the primate focal species (Table V; Fig. 5). Trachypithecus spp. (median = 3.01 s−1) ate foods more quickly than Pygathrix spp. did (2.43 s−1; p < 0.01). Among the 4 species, Trachypithecus delacouri (3.18 s−1) chewed the quickest, and the rate is significantly higher than that for the other 3 species (p < 0.01). Trachypithecus I. hatinhensis (2.76 s−1) process foods quicker than either Pygathrix spp. does (P. namaeus = 2.47, P. cinerea = 2.40), while the chewing rates for both Trachypithecus spp. are comparable (p > 0.05). To evaluate if faster chewing by Trachypithecus spp. was due to smaller less efficient molars we compared molar size among the focal species.

Fig. 4
figure 4

Box and whisker plots of chewing bout length in seconds for each of the four primate species in this study (Mann-Whitney U, p > 0.05 between the two represented genera). Vertical line = median, length of box = range within which the central 50% of the values fall, hinges = first and third quartiles. Note the overlap among the species in both of the represented genera.

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Fig. 5
figure 5

Box and whisker plots of chewing rates (s −1) for each of the four primate species in this study (Mann-Whitney U, p < 0.01 between the two represented genera). Vertical line = median, length of box = range within which the central 50% of the values fall, hinges = first and third quartiles. Note that the medians for both Trachypithecus species fall above those for both Pygathrix species.

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Fig. 6
figure 6

Box and whisker plots of molar area to cranial vault length ratios for the two primate genera in this study (Mann-Whitney U, p < 0.01). Vertical line = median, length of box = range within which the central 50% of the values fall, hinges = first and third quartiles.

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Table V Mann-Whitney U test statistics for chewing rates (s−1) for all species pairs
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M2 size

Given the small sample size for Trachypithecus delacouri (n = 1) and T. I. hatinhensis (n = 2), we pooled them for analysis of molar size (Fig. 6). The ratio of molar area to vault length for Trachypithecus (median = 56.28) is significantly greater than that for the Pygathrix spp. (median = 47.09). Though the sample sizes are small, the finding suggests that along with increased chewing rate, Trachypithecus increase the degree to which leaves are comminuted per chew.

Discussion

Our study revealed differences among a subset of Southeast Asian colobines in the way they process leaves of comparable toughness. Two distinct strategies emerge. Trachypithecus spp. depend more on high masticatory rates to increase comminution of foods before swallowing. This is predicted to increase the surface area of leaves on which microbes can act. It appears that increased chewing rates in the limestone langurs are combined with larger molars to increase the efficiency of leaf comminution per chew. Pygathrix spp. chew foods for the same amount of time as Trachypithecus spp. do, but they do not chew as quickly. In addition, their molars are smaller. Thus it appears that leaves enter the stomachs of the Pygathrix spp. in larger pieces. However, they may compensate for larger leaf size and less surface area per leaf by increasing stomach size and via the development of a presaccus. The stomach of Pygathrix nemaeus is 20% heavier than that of Presbytis melalophos (Caton 1998). Further, the stomachs of Pygathrix nemaeus, like those of Procolobus, Rhinopithecus, and Nasalis, have a fourth stomach chamber: the presaccus (Caton 1998). The longitudinal muscle coat, squamous epithelial lining, and small size of the presaccus led Caton (1998) to suggest that the structure may be a gastric mill, breaking large ingested food particles into smaller pieces before passing them to the saccus. Though the stomachs of Trachypithecus obscura and T. cristatus exhibit 3 large chambers, they lack a presaccus (Caton 1999). However, they also exhibit an enlarged colic chamber similar to that in cercopithecines and apes, which may act as a secondary fermentation chamber (Caton 1999).

Our findings augment those of Caton (1998, 1999). The presacuss may play the role of additional ingestion and food particle comminution center in Pygathrix spp., thus permitting them to chew more slowly on smaller teeth. Lacking the presaccus, Trachypithecus spp. may rely more on oral comminution of foods, i.e. faster chewing rates and larger teeth, before exposure to microbes in a tripartite stomach.

We are unable to say whether one or the other system is actually more efficient in terms of energy and nutrient return per unit consumed leaf tissue. The lack of an additional colic digestive chamber in Pygathrix may be evidence of a more digestively efficient stomach, while seemingly lower activity levels in the genus suggest a less efficient total digestive system or a less calorically rich diet.

The energetics of positional behavior in the 2 genera differ in ways that demand further inquiry to relate directly to their patterns of food ingestion and digestion. Trachypithecus spp. exhibit higher percentages of leaping and quadrupedal walking than Pygathrix spp. do, while Pygathrix spp. suspend more frequently during foraging and locomotion than Trachypithecus spp. do (Stevens et al. 2008). Brachiation, when done in such a way that collisional energy loss is minimized, may be more energy efficient than initially argued in the primate literature (Bertram 2004), suggesting that the digestive strategy of Pygathrix may be less calorically efficient than the ingestive strategy of Trachypithecus. In addition, Pygathrix spp. are relatively less active than Trachypithecus spp. are (K. Wright et al., in prep).