American Journal of Primatology
RESEARCH ARTICLE Oxygen Isotope Ratios in Primate Bone Carbonate Reflect Amount of Leaves and Vertical Stratification in the Diet MELINDA L. CARTER* AND MICHAEL W. BRADBURY Alabama College of Osteopathic Medicine, Dothan, Alabama
The stable isotopic biogeochemistry of free-ranging primates is a unique tool to assess dietary and ecological adaptions among sympatric populations. The present study tested the hypothesis that oxygen isotopes in the bone carbonate of five primate and four ungulate species that live in Kibale National Park, Uganda, would show minimal variability since the species obtain water from a single water source. Bones were analyzed for stable carbon (d13C) and oxygen (d18O) isotope ratios. Results for apatite d13C are consistent with all species feeding in a closed forest habitat and do not exhibit niche partitioning. d18O values, in contrast, cluster by species and correlate positively with the relative contribution of leaves to the whole diet are likely also modified by vertical niche partitioning between taxa within the forest canopy. These results show that biochemical markers from naturally deceased primate remains can aid our understanding of how living animals exploit available resources. Am. J. Primatol. © 2015 Wiley Periodicals, Inc. Key words:
Kibale National Park; Uganda; oxygen isotopes; bone carbonate; folivory; primate isotope ecology
INTRODUCTION The application of stable isotope analysis of primate tissues is now well established to better understand both the ecology of living species as well as nutritional adaptations in fossil primates [Crowley, 2012; Sandberg et al., 2012; Sponheimer et al., 2009]. The significance of oxygen isotope ratios from bone carbonate and enamel continues to be investigated as a means to evaluate local climatic and biogeochemical influences on body water composition and as a tool to differentiate herbivorous animals with dietary specializations [Levin et al., 2006]. This is particularly useful among sympatric forest-dwelling animals whose C3-dominated plant diets are relatively indistinguishable through comparison of their d13C tissue values [Cerling et al., 2004; Crowley, 2014; Krigbaum et al., 2013; Nelson, 2013]. These recent studies demonstrate that relative stable isotope ratios in primate tissues can be analyzed directly to test and quantify assumptions made about dietary preferences and niche partitioning by primatologists through years of field observation. The accuracy of results and strength of the analysis can only be made robust by identifying similar trends in diverse species across habitats and continents. Despite evidence that forest-floor and low-canopy plant tissues are depleted in 13C relative to those in upper canopy strata [Farquhar et al., 1989],
© 2015 Wiley Periodicals, Inc.
carbon isotope ratios in bone collagen and carbonate have yet to distinguish niche separation between forest-dwelling animals that feed almost exclusively on vegetation that synthesizes its tissues using the C3-photosynthetic pathway [Carter, 2001; Cerling et al., 2004; Crowley et al., 2010; Krigbaum et al., 2013; Krigbaum, 2000; van der Merwe and Medina, 1991]. Based on similar environmental variables that alter isotope concentration in leaves, oxygen isotope ratios in animal tissues are proving to be a better measure of niche separation between rainforest taxa. The 18O/16O ratio in local water in any given climate is sensitive to mean annual temperature [Dansgaard, 1964]. Bone phosphate d18O values from animals that obtain most of their ingested water
Contract grant sponsor: Office of the President; contract grant sponsor: National Council for Science and Technology; contract grant sponsor: Wenner-Gren Grant #6574 Conflicts of interest: None.
Correspondence to: Melinda L. Carter, Alabama College of Osteopathic Medicine, 445 Health Sciences Boulevard, Dothan, AL 36303. E-mail: [email protected] Received 19 December 2014; revised 19 April 2015; revision accepted 6 May 2015 DOI: 10.1002/ajp.22432 Published online XX Month Year in Wiley Online Library (wileyonlinelibrary.com).
2 / Carter and Bradbury
from leaves reflect relative humidity of the local climate [Ayliffe and Chivas, 1990; Cormie et al., 1994]. The water in leaves that grow in dry habitats are positively enriched in 18O relative to leaves from moist climates [Gat, 1980]. In the study of prehistoric human diets, d18O data from tooth enamel correlate with latitude [Fricke et al., 1995]. The 18O/16O ratio of animal tissues is ultimately influenced by that of drinking water (meteoric and surface water) as well as atmospheric O2, modulated by local temperature and humidity. To maintain balance, oxygen is excreted in the urine and feces, evaporated in sweat, and respired as carbon dioxide [Koch et al., 1989, 1994; Schmidt-Nielsen, 1964, 1997]. The d18O signatures of bone phosphate and carbonate, both recovered from the hydroxyapatite or mineral phase of bone, are assumed to be in equilibrium with that of blood plasma and, therefore, is derived mostly from water in the diet [Kolodny et al., 1983; Longinelli, 1984; Luz et al., 1984, 1990; Luz and Kolodny, 1985]. Oxygen isotope ratios in tropical moist forest vegetation are further altered by evapotranspiration, so leaves have high d18O relative to other parts of the same plant, including fruits [Barbour, 2007; Marshall et al., 2007; Sternberg et al., 1989]. There is a vertical stratification or “canopy effect” in increasing leaf d18O values from forest floor to middle canopy to high canopy due to variations in humidity in local microhabitats [Sternberg et al., 1989]. In an individual plant, cellulose in the leaves has higher d18O values than cellulose from its roots [Epstein et al., 1977; Sternberg et al., 1984, 1986; Sternberg, 1989; Yakir, 1992]. Krigbaum et al. [2013] propose that the proportion of leaves and fruit in the diet will influence the d18O of consumer tissues of folivores and frugivores, respectively, because leaves have a high surface area/low volume ratio in contrast to the low surface area/high volume of fruits, resulting in an enrichment of 18O in leaves because of evapotranspiration. Actual d18O values of leaves and fruit pulp from the same tree have not been published. Research supports the assumption that trends in plant d18O signatures will be preserved in diverse tissue of animals feeding on different types of vegetation. Early studies found that d18O values in herbivore tooth enamel were more positive in browsers (70–95% C3 leaves, shoots, and young twigs) and mixed feeders (>30% C4 grass and >30% C3 browse) than in grazing herbivores (>70% C4 grass) [Cerling et al., 1997, 2003; Kohn et al., 1996]. Bocherens et al. [1996] had opposite results in that 18O was depleted in the enamel of a browser (black rhinoceroses) and mixed-feeder (African elephants), but this may be caused by either differences in body size and metabolic rate or aridity of their respective local habitats [Levin et al., 2006]. Studies on different species of well-studied forestdwelling nonhuman primates present compelling evidence that d18O data in bone and enamel reflect
Am. J. Primatol.
subsistence strategies adapted to different average feeding heights in the forest canopy [Crowley et al., 2014; Krigbaum et al., 2013; Nelson, 2013]. Cerling et al. [2004] suggest that the d18O values from folivorous colobines are enriched relative to frugivores because their water source is predominantly leaves. Kibale National Park (795 km2) is a mid-altitude tropical evergreen forest in southwestern Uganda (0° ́ ́ N and 30°19–30°32 ́ ́ E) where primate ecolo13–0°41 gy and conservation have been studied for over 40 years [Bryer et al., 2013; Chapman and Lambert, 2000; Struhsaker, 1997]. Mean annual rainfall is 1712 mm (1990–2004) and occurs across two rainy seasons [Chapman et al., 2002a; Rode et al., 2006]. The primates and ungulates in the present study were collected from three ecologically distinct camps: Ngogo, Kanyawara, and Kanyanchu. Much of Kibale National Park is classified as Parinari forest [Skorupa, 1988]. Differences in altitude, however, result in differences in climate and vegetation. The climate at Ngogo is warmer and drier than at Kanyawara and the vegetation is quite different [Butynski, 1990]. The region of forest near Kanyanchu that has been documented is called the Dura River site, located roughly 15 km south of Kanyawara and is a combination of moist evergreen forest and lowland tropical forest [Chapman and Chapman, 1997]. At this time, small sample sizes of mammalian species from Kibale National Park preclude the investigation of the influence of local habitat on stable isotope ratios. Species presence and population density are known to vary for most primate and ungulate species at these study sites. Bone carbonate d18O and d13C data from five well-studied East African primates are presented here. Data were generated from the analysis of skeletal remains of sympatric primates living in Kibale National Park prior to 1995 and represent a subset of data from a multi-isotopic study that tested various hypotheses derived from both stable isotopic biogeochemistry and primate ecology [Carter, 2001]. d13C (bone collagen and apatite) and d15N (bone collagen) data from these same specimens are published and discussed elsewhere [Crowley et al., 2010; Smith et al., 2010]. The present study tested the hypothesis that sympatric primates that obtain water from local sources will have similar d18O values. Differences in d18O values are considered meaningful and related to diet. METHODS Bone samples were collected from individuals representing five primate and four ungulate species from Kibale National Park (KNP), Uganda, in September, 1995 (Fig. 1). Permission to conduct research in Uganda was obtained from the Office of the President and related agencies upon agreement
Primate Bone Isotopes Reflect Folivory / 3
Fig. 1. Location of Kibale National Park, Uganda. Skeletal remains were collected from designated study sites: Kanyawara, Ngogo, and Kanyanchu.
to adhere to legal requirements. Permission to export biological samples was obtained from the Uganda Wildlife Authority and Kenya Wildlife Authority and, following authorization from US Fish and Wildlife Service, samples were imported to the United States for analysis. The acquisition and handling of materials followed the Principles for the Ethical Treatment of Nonhuman Primates established by the American Society of Primatologists. The skeletal remains studied here were from naturally deceased animals that had been collected from various sites throughout the forest. Certain specimens had been previously identified [Struhsaker and Leakey, 1990]. A history of the Kibale faunal collection and means of recovery are discussed
in Carter [2001]. Details of the acquisition of the chimpanzee remains is described elsewhere [Carter et al., 2008]. Between 2–10 grams of bone were collected from each specimen, primarily from long bones, ribs, or the cranial base (Table I). When available, long bone cortex was preferred to control for potential taphonomic contamination. Each of the following species was chosen for this study based on availability of remains, dietary specialization (e.g., folivory or frugivory), and vertical niche stratification (documented differences among average heights at which they forage for food within the forest canopy) (Table I): Procolobus rufomitratus spp. tephrosceles (red colobus; total sample numbers: N ¼ 37; P. tephrosceles, N ¼ 28; Colobus spp., N ¼ 9), Lophocebus albigena johnstoni (grey-cheeked
Am. J. Primatol.
Am. J. Primatol.
N6/TTSK 71
KFB 56
N15/TTSK 50
N7/TTSK 55 KFB 107 KFB 17 KFB 93 KFB 105 KFB 106 KFB 18 KFB 20 KFB 1 KFB 4 KFB 3
K57B
K41B
K63B
K56B K1B K2B K3B K4B K5B K6B K7B K55B K59B K60B
KFB 111
KFB 109
K15B
K16B
K14B
No KFB # No KFB # KFB 125 KFB 124 No KFB # KFB 30 N18/TTSK 61 KFB 113
K24B K27B K25B K28B K29B K30B K62B
80 63 62 16 64 29
KFB 127
K42B
KFB KFB KFB KFB KFB KFB
KFB 126
Primates K40B
K34B K35B K36B K37B K38B K39B
Kibale ID
Lab No.
anubis anubis anubis anubis anubis anubis
Procolobus rufomitratus Procolobus rufomitratus Procolobus
Colobus? Colobus? Colobus sp. Colobus sp. Colobus sp. Colobus sp. Colobus sp.
Papio Papio Papio Papio Papio Papio
Lophocebus albigena Pan troglodytes Pan troglodytes Pan troglodytes Pan troglodytes Pan troglodytes Pan troglodytes Pan troglodytes Pan troglodytes Pan troglodytes Pan troglodytes
Cercopithecus ascanius Cercopithecus ascanius Cercopithecus ascanius Cercopithecus ascanius? Cercopithecus ascanius?
Species
folivory
folivory
folivory
folivory folivory folivory folivory folivory folivory folivory
omnivory omnivory omnivory omnivory omnivory omnivory
frugivory frugivory frugivory frugivory frugivory frugivory frugivory frugivory frugivory frugivory frugivory
frugivory
frugivory
frugivory
frugivory
frugivory
Primary diet
radius tibia
80%d
7%c 7%c 7%c 7%c 7%c 7%c
— 10%b 10%b 10%b 10%b 10%b 10%b 10%b 10%b 10%b 10%b
80%d
cranial
21%a
femur
parietal
21%a
80%d
caudal vertebra, ribs
21%a
80%d 80%d 80%d 80%d 80%d 80%d 80%d
caudal vertebra
21%a
Cercopithecus avg. SD ribs rib occipital occipital rib rib femur occipital tibia femur rib Pan avg. SD mandible occipital occipital occipital occipital occipital Papio avg. SD tibia fibula mandibular ramus tibia tibia frontal cranial
multiple bones
Bone sampled
21%a
Leaves in diet average %
0.59 0.82 0.60
15.86 16.78 16.39
0.78 0.46 0.49
16.03 17.06 18.13
0.39 1.29 1.26 1.16 7.32 1.82 0.18 0.72 1.05 0.75 0.48 0.51 0.71 0.10 2.61 1.29 1.74 1.42 1.70 2.46 2.33 1.82 0.48 2.16 1.76 2.51 0.94 1.06 2.22 0.69
1.77
17.18
16.66 0.54 15.48 15.64 12.09 12.65 15.46 15.99 15.61 16.82 15.43 15.72 15.60 15.10 1.50 14.67 17.50 15.90 15.75 15.24 16.97 16.00 1.06 15.45 16.15 16.11 16.06 16.46 15.78 15.88
1.69
d18Ocarbonate (PDB ‰)
17.08
d13Ccarbonate (PDB ‰)
TABLE I. Sample Description and Isotope Data From Bone Carbonate. Differentiation of Colobinae is Discussed in the Text
4 / Carter and Bradbury
KFB 117
KFB 116
KFB 112
KFB 61
KFB 19
KFB 115
KFB 59
KFB 15
KFB 58
KFB 21
N12/TTSK 60 N20/TTSK 53 N9/TTSK 58
K18B
K19B
K20B
K21B
K22B
K23B
K26B
K31B
K32B
K33B
K58B
No KFB # KFB 130
KFB 25 KFB 44 No KFB # KFB 6
Ungulates K43B K50B
K49B K44B K12B K61B
K68B
K67B
K66B
K65B
N27/TTSK 59 N21/TTSK 69 N11/TTSK 57
KFB 41
K17B
K64B
Kibale ID
Lab No.
TABLE I. Continued
Cephalophus harveyi Cephalophus monticola Cephalophus harveyi Cephalophus sp. Cephalophus sp. Cephalophus sp.
rufomitratus Procolobus rufomitratus Procolobus rufomitratus Procolobus rufomitratus Procolobus rufomitratus Procolobus rufomitratus Procolobus rufomitratus Procolobus rufomitratus Procolobus rufomitratus Procolobus rufomitratus Procolobus rufomitratus Procolobus rufomitratus Procolobus rufomitratus Procolobus rufomitratus Procolobus rufomitratus Procolobus rufomitratus Procolobus rufomitratus Procolobus rufomitratus
Species
herbivore/browser herbivore/browser herbivore/browser herbivore/browser
herbivore/browser herbivore/browser
folivory
folivory
folivory
folivory
folivory
folivory
folivory
folivory
folivory
folivory
folivory
folivory
folivory
folivory
folivory
folivory
folivory
Primary diet
femur caudal vertebra, ribs occipital, mandible ribs ribs, metacarpal, phalanx occipital occipital, zygomatic occipital, parietal occipital ulna occipital multiple bones occipital occipital vertebrae
80%d 80%d 80%d 80%d 80%d
80%d 80%d 80%d 80%d 80%d 80%d 80%d 80%d 80%d
rib cranial ulna, ribs occipital phalanx, calcaneus scapula
— — — — — —
Colobine avg. SD
occipital
80%d
80%d
ribs
Bone sampled
80%d
Leaves in diet average %
1.05 2.25 1.32 0.80 1.15 0.76 0.37 0.85 0.19 0.74 0.43 1.26 0.44 2.11 1.50
17.00 15.81 16.58 15.28 17.03 16.47 16.13 17.08 16.56 15.86 15.46 16.05 15.81 16.11 15.44
16.44 15.34 15.85 15.84
16.45 14.53
2.15 1.78 2.17 2.88
2.42 1.72
0.80 1.02
1.04
17.19
16.27 0.66
0.06
d18Ocarbonate (PDB ‰)
16.20
d13Ccarbonate (PDB ‰)
Primate Bone Isotopes Reflect Folivory / 5
Am. J. Primatol.
Am. J. Primatol.
KFB 78 KFB 75 KFB 76 KFB 77 No KFB #
No KFB # KFB 82
No KFB # KFB 46 KFB 129 KFB 45 KFB 128 KFB 50
K8B K10B K11B K52B K54B
K13B K9B
K46B K47B K48B K51B K53B K45B
c
b
Chapman et al. [2002a]. Potts et al. [2011]. Johnson et al. [2012]. d Chapman et al. [2002b].
a
Kibale ID
Lab No.
TABLE I. Continued
porcus porcus porcus porcus porcus
Tragelaphus scriptus Tragelaphus scriptus Tragelaphus scriptus Tragelaphus scriptus Tragelaphus scriptus Tragelaphus scriptus?
Syncerus caffer Syncerus caffer?
Potamochoerus Potamochoerus Potamochoerus Potamochoerus Potamochoerus
Species
herbivore/browser herbivore/browser herbivore/browser herbivore/browser herbivore/browser herbivore/browser
herbivore/grazer herbivore/grazer
omnivory omnivory omnivory omnivory omnivory
Primary diet
— — — — — —
— —
— — — — —
Leaves in diet average % Cephalophus avg. SD mandible mandible mandible mandibular ramus rib Potamochoerus avg. SD occipital mandible Syncerus avg. SD tibia ribs calcaneus rib rib, occipital, C1 cranial Tragelaphus avg. SD
Bone sampled
0.56 3.54 2.05 2.11 19.65 19.23 20.62 16.72 19.60 19.47 19.21 1.31
15.74 0.73 14.40 9.63 14.81 13.76 16.91 13.90 2.66
d13Ccarbonate (PDB ‰)
1.22 1.62 1.42 0.28 1.60 2.09 3.32 1.29 1.62 1.09 1.84 0.80
2.19 0.43 2.87 3.29 3.08 1.68 3.25 2.83 0.67
d18Ocarbonate (PDB ‰)
6 / Carter and Bradbury
Primate Bone Isotopes Reflect Folivory / 7
mangabey; N ¼ 2), Cercopithecus ascanius schmidti (redtail guenon; N ¼ 7), Papio hamadryas anubis (olive baboon; N ¼ 6), and Pan troglodytes schweinfurthii (chimpanzee; N ¼ 12). Bones in this study listed under “Procolobus” were predominantly identified as red colobus; specimens listed as “Colobus spp” or “Colobus?” could not be assigned to species with confidence and may represent Colobus guereza (black-and-white colobus). Both species are specialized folivores. For comparison to non-primate herbivores, bone samples were also collected from the following ungulates: bushbuck (Tragelaphus scriptus; N ¼ 6), bush pig (Potomochoerus porcus; N ¼ 5), duiker (two species: Cephalophus harveyi and C. monticola; N ¼ 5), and African buffalo (Synerus caffer; N ¼ 2). As with the primate remains, certain ungulate specimens could not be identified to species level with confidence. Other than chimpanzee remains, the age and sex of most animal remains could not be determined. Most specimens were adults, based on dental maturation and epiphyseal closure. Leaves comprise at least 73–87% of diet of red colobus and 77–88% of that of black-and-white colobus, categorizing the colobines as the most folivorous of the Kibale primates [Chapman and Chapman, 1999; Chapman et al., 2002b; Struhsaker, 1975; Struhsaker and Oates, 1975] (Table I). Colobinae have a multi-chambered stomach that allows for a ruminant digestive physiology [Baranga, 1982; McKey et al., 1981; Ohwaki et al., 1974]. Frugivorous primates from Kibale include grey-cheeked mangabeys and redtail guenons, though both species supplement their diets substantially with arthropods [Struhsaker, 1978, 1980; Waser, 1975, 1977]. Kibale baboons were thought to be omnivorous and opportunistic predators [Rowell, 1966; Struhsaker, 1997], though recent study documents significant frugivory and limited predation [Johnson et al., 2012]. Kibale chimpanzees are well studied and are frugivorous omnivores, with fruit comprising as much as 80% of their annual diet [Potts et al., 2009, 2011; Wrangham et al., 1992, 1993a, 1993b]. While research on acquisition and metabolism of dietary water in primates is limited, the assumption is that they obtain water indirectly by consuming succulent plant foods (that contain meteoric water) instead of drinking directly from surface waters [Kempf, 2009; Rothman et al., 2012]. As for the nonprimates in this study, the bushbuck is a forest bovid that browses on shrubs and leguminous herbs [Haschick and Kerley, 1997]. Bush pigs have an omnivorous diet, similar to that of Kibale baboons, that includes low-canopy/forest-floor items such as fruits, berries, insects, and small animals [Ghiglieri et al., 1982; Harris and Cerling, 2002; Kingdon, 1979; Nummelin, 1990]. A suid, the bush pig is a water-dependent browser and the only non-ruminant among the ungulates in this study
[Harris and Cerling, 2002]. Red (Cephalophus harveyi) and blue (Cephalophus moniticola) duikers, both found in Kibale National Park, mainly eat fruits that drop from forest trees and shrubs, but may supplement with floral parts and leaves [Lwanga, 2006; Nummelin, 1990]. The African buffalo is a strict grazer that is less water-dependent than the other Kibale ungulates and is seen foraging only in Kibale grasslands, located predominantly in central and southern regions of the park [Levin et al., 2006; Struhsaker, 1997; Wanyama et al., 2010]. They may consume low-canopy browse in forest margins [Field, 1976]. Bone samples were cleaned by removing the outer cortex with a rotary sander. Large pieces were fragmented and sonicated in distilled water with multiple rinses. When dry, bone fragments were powdered using a Wiley mill. The smallest fraction (
RESEARCH ARTICLE Oxygen Isotope Ratios in Primate Bone Carbonate Reflect Amount of Leaves and Vertical Stratification in the Diet MELINDA L. CARTER* AND MICHAEL W. BRADBURY Alabama College of Osteopathic Medicine, Dothan, Alabama
The stable isotopic biogeochemistry of free-ranging primates is a unique tool to assess dietary and ecological adaptions among sympatric populations. The present study tested the hypothesis that oxygen isotopes in the bone carbonate of five primate and four ungulate species that live in Kibale National Park, Uganda, would show minimal variability since the species obtain water from a single water source. Bones were analyzed for stable carbon (d13C) and oxygen (d18O) isotope ratios. Results for apatite d13C are consistent with all species feeding in a closed forest habitat and do not exhibit niche partitioning. d18O values, in contrast, cluster by species and correlate positively with the relative contribution of leaves to the whole diet are likely also modified by vertical niche partitioning between taxa within the forest canopy. These results show that biochemical markers from naturally deceased primate remains can aid our understanding of how living animals exploit available resources. Am. J. Primatol. © 2015 Wiley Periodicals, Inc. Key words:
Kibale National Park; Uganda; oxygen isotopes; bone carbonate; folivory; primate isotope ecology
INTRODUCTION The application of stable isotope analysis of primate tissues is now well established to better understand both the ecology of living species as well as nutritional adaptations in fossil primates [Crowley, 2012; Sandberg et al., 2012; Sponheimer et al., 2009]. The significance of oxygen isotope ratios from bone carbonate and enamel continues to be investigated as a means to evaluate local climatic and biogeochemical influences on body water composition and as a tool to differentiate herbivorous animals with dietary specializations [Levin et al., 2006]. This is particularly useful among sympatric forest-dwelling animals whose C3-dominated plant diets are relatively indistinguishable through comparison of their d13C tissue values [Cerling et al., 2004; Crowley, 2014; Krigbaum et al., 2013; Nelson, 2013]. These recent studies demonstrate that relative stable isotope ratios in primate tissues can be analyzed directly to test and quantify assumptions made about dietary preferences and niche partitioning by primatologists through years of field observation. The accuracy of results and strength of the analysis can only be made robust by identifying similar trends in diverse species across habitats and continents. Despite evidence that forest-floor and low-canopy plant tissues are depleted in 13C relative to those in upper canopy strata [Farquhar et al., 1989],
© 2015 Wiley Periodicals, Inc.
carbon isotope ratios in bone collagen and carbonate have yet to distinguish niche separation between forest-dwelling animals that feed almost exclusively on vegetation that synthesizes its tissues using the C3-photosynthetic pathway [Carter, 2001; Cerling et al., 2004; Crowley et al., 2010; Krigbaum et al., 2013; Krigbaum, 2000; van der Merwe and Medina, 1991]. Based on similar environmental variables that alter isotope concentration in leaves, oxygen isotope ratios in animal tissues are proving to be a better measure of niche separation between rainforest taxa. The 18O/16O ratio in local water in any given climate is sensitive to mean annual temperature [Dansgaard, 1964]. Bone phosphate d18O values from animals that obtain most of their ingested water
Contract grant sponsor: Office of the President; contract grant sponsor: National Council for Science and Technology; contract grant sponsor: Wenner-Gren Grant #6574 Conflicts of interest: None.
Correspondence to: Melinda L. Carter, Alabama College of Osteopathic Medicine, 445 Health Sciences Boulevard, Dothan, AL 36303. E-mail: [email protected] Received 19 December 2014; revised 19 April 2015; revision accepted 6 May 2015 DOI: 10.1002/ajp.22432 Published online XX Month Year in Wiley Online Library (wileyonlinelibrary.com).
2 / Carter and Bradbury
from leaves reflect relative humidity of the local climate [Ayliffe and Chivas, 1990; Cormie et al., 1994]. The water in leaves that grow in dry habitats are positively enriched in 18O relative to leaves from moist climates [Gat, 1980]. In the study of prehistoric human diets, d18O data from tooth enamel correlate with latitude [Fricke et al., 1995]. The 18O/16O ratio of animal tissues is ultimately influenced by that of drinking water (meteoric and surface water) as well as atmospheric O2, modulated by local temperature and humidity. To maintain balance, oxygen is excreted in the urine and feces, evaporated in sweat, and respired as carbon dioxide [Koch et al., 1989, 1994; Schmidt-Nielsen, 1964, 1997]. The d18O signatures of bone phosphate and carbonate, both recovered from the hydroxyapatite or mineral phase of bone, are assumed to be in equilibrium with that of blood plasma and, therefore, is derived mostly from water in the diet [Kolodny et al., 1983; Longinelli, 1984; Luz et al., 1984, 1990; Luz and Kolodny, 1985]. Oxygen isotope ratios in tropical moist forest vegetation are further altered by evapotranspiration, so leaves have high d18O relative to other parts of the same plant, including fruits [Barbour, 2007; Marshall et al., 2007; Sternberg et al., 1989]. There is a vertical stratification or “canopy effect” in increasing leaf d18O values from forest floor to middle canopy to high canopy due to variations in humidity in local microhabitats [Sternberg et al., 1989]. In an individual plant, cellulose in the leaves has higher d18O values than cellulose from its roots [Epstein et al., 1977; Sternberg et al., 1984, 1986; Sternberg, 1989; Yakir, 1992]. Krigbaum et al. [2013] propose that the proportion of leaves and fruit in the diet will influence the d18O of consumer tissues of folivores and frugivores, respectively, because leaves have a high surface area/low volume ratio in contrast to the low surface area/high volume of fruits, resulting in an enrichment of 18O in leaves because of evapotranspiration. Actual d18O values of leaves and fruit pulp from the same tree have not been published. Research supports the assumption that trends in plant d18O signatures will be preserved in diverse tissue of animals feeding on different types of vegetation. Early studies found that d18O values in herbivore tooth enamel were more positive in browsers (70–95% C3 leaves, shoots, and young twigs) and mixed feeders (>30% C4 grass and >30% C3 browse) than in grazing herbivores (>70% C4 grass) [Cerling et al., 1997, 2003; Kohn et al., 1996]. Bocherens et al. [1996] had opposite results in that 18O was depleted in the enamel of a browser (black rhinoceroses) and mixed-feeder (African elephants), but this may be caused by either differences in body size and metabolic rate or aridity of their respective local habitats [Levin et al., 2006]. Studies on different species of well-studied forestdwelling nonhuman primates present compelling evidence that d18O data in bone and enamel reflect
Am. J. Primatol.
subsistence strategies adapted to different average feeding heights in the forest canopy [Crowley et al., 2014; Krigbaum et al., 2013; Nelson, 2013]. Cerling et al. [2004] suggest that the d18O values from folivorous colobines are enriched relative to frugivores because their water source is predominantly leaves. Kibale National Park (795 km2) is a mid-altitude tropical evergreen forest in southwestern Uganda (0° ́ ́ N and 30°19–30°32 ́ ́ E) where primate ecolo13–0°41 gy and conservation have been studied for over 40 years [Bryer et al., 2013; Chapman and Lambert, 2000; Struhsaker, 1997]. Mean annual rainfall is 1712 mm (1990–2004) and occurs across two rainy seasons [Chapman et al., 2002a; Rode et al., 2006]. The primates and ungulates in the present study were collected from three ecologically distinct camps: Ngogo, Kanyawara, and Kanyanchu. Much of Kibale National Park is classified as Parinari forest [Skorupa, 1988]. Differences in altitude, however, result in differences in climate and vegetation. The climate at Ngogo is warmer and drier than at Kanyawara and the vegetation is quite different [Butynski, 1990]. The region of forest near Kanyanchu that has been documented is called the Dura River site, located roughly 15 km south of Kanyawara and is a combination of moist evergreen forest and lowland tropical forest [Chapman and Chapman, 1997]. At this time, small sample sizes of mammalian species from Kibale National Park preclude the investigation of the influence of local habitat on stable isotope ratios. Species presence and population density are known to vary for most primate and ungulate species at these study sites. Bone carbonate d18O and d13C data from five well-studied East African primates are presented here. Data were generated from the analysis of skeletal remains of sympatric primates living in Kibale National Park prior to 1995 and represent a subset of data from a multi-isotopic study that tested various hypotheses derived from both stable isotopic biogeochemistry and primate ecology [Carter, 2001]. d13C (bone collagen and apatite) and d15N (bone collagen) data from these same specimens are published and discussed elsewhere [Crowley et al., 2010; Smith et al., 2010]. The present study tested the hypothesis that sympatric primates that obtain water from local sources will have similar d18O values. Differences in d18O values are considered meaningful and related to diet. METHODS Bone samples were collected from individuals representing five primate and four ungulate species from Kibale National Park (KNP), Uganda, in September, 1995 (Fig. 1). Permission to conduct research in Uganda was obtained from the Office of the President and related agencies upon agreement
Primate Bone Isotopes Reflect Folivory / 3
Fig. 1. Location of Kibale National Park, Uganda. Skeletal remains were collected from designated study sites: Kanyawara, Ngogo, and Kanyanchu.
to adhere to legal requirements. Permission to export biological samples was obtained from the Uganda Wildlife Authority and Kenya Wildlife Authority and, following authorization from US Fish and Wildlife Service, samples were imported to the United States for analysis. The acquisition and handling of materials followed the Principles for the Ethical Treatment of Nonhuman Primates established by the American Society of Primatologists. The skeletal remains studied here were from naturally deceased animals that had been collected from various sites throughout the forest. Certain specimens had been previously identified [Struhsaker and Leakey, 1990]. A history of the Kibale faunal collection and means of recovery are discussed
in Carter [2001]. Details of the acquisition of the chimpanzee remains is described elsewhere [Carter et al., 2008]. Between 2–10 grams of bone were collected from each specimen, primarily from long bones, ribs, or the cranial base (Table I). When available, long bone cortex was preferred to control for potential taphonomic contamination. Each of the following species was chosen for this study based on availability of remains, dietary specialization (e.g., folivory or frugivory), and vertical niche stratification (documented differences among average heights at which they forage for food within the forest canopy) (Table I): Procolobus rufomitratus spp. tephrosceles (red colobus; total sample numbers: N ¼ 37; P. tephrosceles, N ¼ 28; Colobus spp., N ¼ 9), Lophocebus albigena johnstoni (grey-cheeked
Am. J. Primatol.
Am. J. Primatol.
N6/TTSK 71
KFB 56
N15/TTSK 50
N7/TTSK 55 KFB 107 KFB 17 KFB 93 KFB 105 KFB 106 KFB 18 KFB 20 KFB 1 KFB 4 KFB 3
K57B
K41B
K63B
K56B K1B K2B K3B K4B K5B K6B K7B K55B K59B K60B
KFB 111
KFB 109
K15B
K16B
K14B
No KFB # No KFB # KFB 125 KFB 124 No KFB # KFB 30 N18/TTSK 61 KFB 113
K24B K27B K25B K28B K29B K30B K62B
80 63 62 16 64 29
KFB 127
K42B
KFB KFB KFB KFB KFB KFB
KFB 126
Primates K40B
K34B K35B K36B K37B K38B K39B
Kibale ID
Lab No.
anubis anubis anubis anubis anubis anubis
Procolobus rufomitratus Procolobus rufomitratus Procolobus
Colobus? Colobus? Colobus sp. Colobus sp. Colobus sp. Colobus sp. Colobus sp.
Papio Papio Papio Papio Papio Papio
Lophocebus albigena Pan troglodytes Pan troglodytes Pan troglodytes Pan troglodytes Pan troglodytes Pan troglodytes Pan troglodytes Pan troglodytes Pan troglodytes Pan troglodytes
Cercopithecus ascanius Cercopithecus ascanius Cercopithecus ascanius Cercopithecus ascanius? Cercopithecus ascanius?
Species
folivory
folivory
folivory
folivory folivory folivory folivory folivory folivory folivory
omnivory omnivory omnivory omnivory omnivory omnivory
frugivory frugivory frugivory frugivory frugivory frugivory frugivory frugivory frugivory frugivory frugivory
frugivory
frugivory
frugivory
frugivory
frugivory
Primary diet
radius tibia
80%d
7%c 7%c 7%c 7%c 7%c 7%c
— 10%b 10%b 10%b 10%b 10%b 10%b 10%b 10%b 10%b 10%b
80%d
cranial
21%a
femur
parietal
21%a
80%d
caudal vertebra, ribs
21%a
80%d 80%d 80%d 80%d 80%d 80%d 80%d
caudal vertebra
21%a
Cercopithecus avg. SD ribs rib occipital occipital rib rib femur occipital tibia femur rib Pan avg. SD mandible occipital occipital occipital occipital occipital Papio avg. SD tibia fibula mandibular ramus tibia tibia frontal cranial
multiple bones
Bone sampled
21%a
Leaves in diet average %
0.59 0.82 0.60
15.86 16.78 16.39
0.78 0.46 0.49
16.03 17.06 18.13
0.39 1.29 1.26 1.16 7.32 1.82 0.18 0.72 1.05 0.75 0.48 0.51 0.71 0.10 2.61 1.29 1.74 1.42 1.70 2.46 2.33 1.82 0.48 2.16 1.76 2.51 0.94 1.06 2.22 0.69
1.77
17.18
16.66 0.54 15.48 15.64 12.09 12.65 15.46 15.99 15.61 16.82 15.43 15.72 15.60 15.10 1.50 14.67 17.50 15.90 15.75 15.24 16.97 16.00 1.06 15.45 16.15 16.11 16.06 16.46 15.78 15.88
1.69
d18Ocarbonate (PDB ‰)
17.08
d13Ccarbonate (PDB ‰)
TABLE I. Sample Description and Isotope Data From Bone Carbonate. Differentiation of Colobinae is Discussed in the Text
4 / Carter and Bradbury
KFB 117
KFB 116
KFB 112
KFB 61
KFB 19
KFB 115
KFB 59
KFB 15
KFB 58
KFB 21
N12/TTSK 60 N20/TTSK 53 N9/TTSK 58
K18B
K19B
K20B
K21B
K22B
K23B
K26B
K31B
K32B
K33B
K58B
No KFB # KFB 130
KFB 25 KFB 44 No KFB # KFB 6
Ungulates K43B K50B
K49B K44B K12B K61B
K68B
K67B
K66B
K65B
N27/TTSK 59 N21/TTSK 69 N11/TTSK 57
KFB 41
K17B
K64B
Kibale ID
Lab No.
TABLE I. Continued
Cephalophus harveyi Cephalophus monticola Cephalophus harveyi Cephalophus sp. Cephalophus sp. Cephalophus sp.
rufomitratus Procolobus rufomitratus Procolobus rufomitratus Procolobus rufomitratus Procolobus rufomitratus Procolobus rufomitratus Procolobus rufomitratus Procolobus rufomitratus Procolobus rufomitratus Procolobus rufomitratus Procolobus rufomitratus Procolobus rufomitratus Procolobus rufomitratus Procolobus rufomitratus Procolobus rufomitratus Procolobus rufomitratus Procolobus rufomitratus Procolobus rufomitratus
Species
herbivore/browser herbivore/browser herbivore/browser herbivore/browser
herbivore/browser herbivore/browser
folivory
folivory
folivory
folivory
folivory
folivory
folivory
folivory
folivory
folivory
folivory
folivory
folivory
folivory
folivory
folivory
folivory
Primary diet
femur caudal vertebra, ribs occipital, mandible ribs ribs, metacarpal, phalanx occipital occipital, zygomatic occipital, parietal occipital ulna occipital multiple bones occipital occipital vertebrae
80%d 80%d 80%d 80%d 80%d
80%d 80%d 80%d 80%d 80%d 80%d 80%d 80%d 80%d
rib cranial ulna, ribs occipital phalanx, calcaneus scapula
— — — — — —
Colobine avg. SD
occipital
80%d
80%d
ribs
Bone sampled
80%d
Leaves in diet average %
1.05 2.25 1.32 0.80 1.15 0.76 0.37 0.85 0.19 0.74 0.43 1.26 0.44 2.11 1.50
17.00 15.81 16.58 15.28 17.03 16.47 16.13 17.08 16.56 15.86 15.46 16.05 15.81 16.11 15.44
16.44 15.34 15.85 15.84
16.45 14.53
2.15 1.78 2.17 2.88
2.42 1.72
0.80 1.02
1.04
17.19
16.27 0.66
0.06
d18Ocarbonate (PDB ‰)
16.20
d13Ccarbonate (PDB ‰)
Primate Bone Isotopes Reflect Folivory / 5
Am. J. Primatol.
Am. J. Primatol.
KFB 78 KFB 75 KFB 76 KFB 77 No KFB #
No KFB # KFB 82
No KFB # KFB 46 KFB 129 KFB 45 KFB 128 KFB 50
K8B K10B K11B K52B K54B
K13B K9B
K46B K47B K48B K51B K53B K45B
c
b
Chapman et al. [2002a]. Potts et al. [2011]. Johnson et al. [2012]. d Chapman et al. [2002b].
a
Kibale ID
Lab No.
TABLE I. Continued
porcus porcus porcus porcus porcus
Tragelaphus scriptus Tragelaphus scriptus Tragelaphus scriptus Tragelaphus scriptus Tragelaphus scriptus Tragelaphus scriptus?
Syncerus caffer Syncerus caffer?
Potamochoerus Potamochoerus Potamochoerus Potamochoerus Potamochoerus
Species
herbivore/browser herbivore/browser herbivore/browser herbivore/browser herbivore/browser herbivore/browser
herbivore/grazer herbivore/grazer
omnivory omnivory omnivory omnivory omnivory
Primary diet
— — — — — —
— —
— — — — —
Leaves in diet average % Cephalophus avg. SD mandible mandible mandible mandibular ramus rib Potamochoerus avg. SD occipital mandible Syncerus avg. SD tibia ribs calcaneus rib rib, occipital, C1 cranial Tragelaphus avg. SD
Bone sampled
0.56 3.54 2.05 2.11 19.65 19.23 20.62 16.72 19.60 19.47 19.21 1.31
15.74 0.73 14.40 9.63 14.81 13.76 16.91 13.90 2.66
d13Ccarbonate (PDB ‰)
1.22 1.62 1.42 0.28 1.60 2.09 3.32 1.29 1.62 1.09 1.84 0.80
2.19 0.43 2.87 3.29 3.08 1.68 3.25 2.83 0.67
d18Ocarbonate (PDB ‰)
6 / Carter and Bradbury
Primate Bone Isotopes Reflect Folivory / 7
mangabey; N ¼ 2), Cercopithecus ascanius schmidti (redtail guenon; N ¼ 7), Papio hamadryas anubis (olive baboon; N ¼ 6), and Pan troglodytes schweinfurthii (chimpanzee; N ¼ 12). Bones in this study listed under “Procolobus” were predominantly identified as red colobus; specimens listed as “Colobus spp” or “Colobus?” could not be assigned to species with confidence and may represent Colobus guereza (black-and-white colobus). Both species are specialized folivores. For comparison to non-primate herbivores, bone samples were also collected from the following ungulates: bushbuck (Tragelaphus scriptus; N ¼ 6), bush pig (Potomochoerus porcus; N ¼ 5), duiker (two species: Cephalophus harveyi and C. monticola; N ¼ 5), and African buffalo (Synerus caffer; N ¼ 2). As with the primate remains, certain ungulate specimens could not be identified to species level with confidence. Other than chimpanzee remains, the age and sex of most animal remains could not be determined. Most specimens were adults, based on dental maturation and epiphyseal closure. Leaves comprise at least 73–87% of diet of red colobus and 77–88% of that of black-and-white colobus, categorizing the colobines as the most folivorous of the Kibale primates [Chapman and Chapman, 1999; Chapman et al., 2002b; Struhsaker, 1975; Struhsaker and Oates, 1975] (Table I). Colobinae have a multi-chambered stomach that allows for a ruminant digestive physiology [Baranga, 1982; McKey et al., 1981; Ohwaki et al., 1974]. Frugivorous primates from Kibale include grey-cheeked mangabeys and redtail guenons, though both species supplement their diets substantially with arthropods [Struhsaker, 1978, 1980; Waser, 1975, 1977]. Kibale baboons were thought to be omnivorous and opportunistic predators [Rowell, 1966; Struhsaker, 1997], though recent study documents significant frugivory and limited predation [Johnson et al., 2012]. Kibale chimpanzees are well studied and are frugivorous omnivores, with fruit comprising as much as 80% of their annual diet [Potts et al., 2009, 2011; Wrangham et al., 1992, 1993a, 1993b]. While research on acquisition and metabolism of dietary water in primates is limited, the assumption is that they obtain water indirectly by consuming succulent plant foods (that contain meteoric water) instead of drinking directly from surface waters [Kempf, 2009; Rothman et al., 2012]. As for the nonprimates in this study, the bushbuck is a forest bovid that browses on shrubs and leguminous herbs [Haschick and Kerley, 1997]. Bush pigs have an omnivorous diet, similar to that of Kibale baboons, that includes low-canopy/forest-floor items such as fruits, berries, insects, and small animals [Ghiglieri et al., 1982; Harris and Cerling, 2002; Kingdon, 1979; Nummelin, 1990]. A suid, the bush pig is a water-dependent browser and the only non-ruminant among the ungulates in this study
[Harris and Cerling, 2002]. Red (Cephalophus harveyi) and blue (Cephalophus moniticola) duikers, both found in Kibale National Park, mainly eat fruits that drop from forest trees and shrubs, but may supplement with floral parts and leaves [Lwanga, 2006; Nummelin, 1990]. The African buffalo is a strict grazer that is less water-dependent than the other Kibale ungulates and is seen foraging only in Kibale grasslands, located predominantly in central and southern regions of the park [Levin et al., 2006; Struhsaker, 1997; Wanyama et al., 2010]. They may consume low-canopy browse in forest margins [Field, 1976]. Bone samples were cleaned by removing the outer cortex with a rotary sander. Large pieces were fragmented and sonicated in distilled water with multiple rinses. When dry, bone fragments were powdered using a Wiley mill. The smallest fraction (
