Introducing Fecal Stable Isotope Analysis in Primate Weaning Studies LAURIE J

American Journal of Primatology 74:926–939 (2012)

RESEARCH ARTICLE Introducing Fecal Stable Isotope Analysis in Primate Weaning Studies LAURIE J. REITSEMA1,2 1Department of Anthropology, University of Georgia, Athens, Georgia 2Department of Anthropology, The Ohio State University, Columbus, Ohio This research investigates the potential of a new, noninvasive method for determining age of weaning among primates using stable carbon and nitrogen isotope ratios in feces. Analysis of stable isotope ratios in body tissues is a well-established method in archeology and ecology for reconstructing diet. This is the first study to investigate weaning in primates using fecal stable isotope ratios. Diets of asingleFrançois’ langur (Trachypithecus francoisi) mother–infant pair at the Toledo Zoo are recon- structed using this technique to track changes in infant suckling behavior over the weaning period. Stable isotope ratios in feces are sampled instead of more traditional samples such as bone or hair to enable daily, noninvasive snapshots of weaning status. Isotopic assessments of weaning status are compared to visual assessments to identify any discordance between the two. Three measurements documented the transition from breast milk to solid foods: stable carbon isotope ratios (δ13C), stable nitrogen isotope ratios (δ15N), and nitrogen content of feces (%N). It appears that solid foods were introduced at approximately 2 months of infant age, but that nursing continued into the 12th month, when sample collection ceased. Stable isotope data exposed a much longer weaning period than what was expected based on previously published data for captive langurs, and clarified visual estimates of weaning status. This reflects the method’s sensitivity to suckling at night and ability to distinguish actual nursing from comfort nursing. After testing this method with zoo animals, it can readily be applied among wild populations. An isotopic approach to weaning provides a new, accurate, and biologically meaningful assessment of interbirth intervals, and facilitates a better understanding of mother–infant interactions. Both of these outcomes are critical for developing successful conservation strategies for captive and wild primates. Am. J. Primatol. 74:926–939, 2012. C 2012 Wiley Periodicals, Inc.

Key words: nursing; lactation; carbon; nitrogen; Franc¸ois’ langur

INTRODUCTION guishing infants from juveniles in groups are some Identifying the age at which primate infants are of these. Because interbirth intervals are considered fully weaned is an obstacle for field primatologists when developing population viability analyses, pre- relying solely on behavioral observations [e.g., Jay, cise assessments of weaning are vital for managing 1965; Lee, 1996; Rajpurohit & Mohnot, 1990]. It is effective conservation strategies [Caro, 1999]. difficult to observe weaning among arboreal or poorly Weaning plays a critical role in primate group habituated groups, and because researchers are lim- interactions and reproductive strategies [Charnov, ited to diurnal observations they are unable to detect 1991]. Because a mother resumes menstruation as suckling at night [Lee, 1996], and cannot account for weaning progresses, age at weaning determines the the actual rate of milk transfer [Martin, 1984]. Ad- length of interbirth intervals and the number of off- ditionally, regardless of how long an infant appears spring a female can bear in her lifetime [Lee, 1987]. to suckle, the behavior may be “comfort nursing” in Weaning represents a genetic conflict of interests which an infant makes contact with its mother’s nip- ple months after weaning takes place as a means of Contract grant sponsor: American Society of Primatologists. reassurance rather than sustenance [Jay, 1965; Lee, *Correspondence to: Laurie J. Reitsema, Department of An- 1987]. Although comfort nursing is physiologically thropology, The Ohio State University, 4034 Smith Lab- meaningless, actual nursing has critical physiologi- oratory, 174 W. 18th Avenue, Columbus, OH 43210. E-mail: [email protected] cal, behavioral, and methodological ramifications in Received 18 November 2011; revised 13 May 2012; revision ac- primate studies. Delaying the mother’s return to sex- cepted 14 May 2012 ual receptivity [Charnov, 1991; Lee, 1996; Pagel & DOI 10.1002/ajp.22045 Harvey, 2002], affecting an infant’s vulnerability to Published online 21 June 2012 in Wiley Online Library infanticide [Crockett & Sekulic, 1984], and distin- (wileyonlinelibrary.com).

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between mother and infant who are both attempt- isotope ratios are commonly used by archaeologists ing to maximize their evolutionary fitness [Martin, to investigate weaning in past human populations 1984; Trivers, 1974]. Mothers are 50% related to all using bone and teeth [Herring et al., 1998; Richards their infants, whereas infants are 100% related to et al., 2002], and have been used to monitor the tran- themselves and, excepting the case of monozygotic sition among living humans using hair and finger- multiple births, 50% or 25% related to their siblings. nails [Fogel et al., 1989; Fuller et al., 2006]. Some Because of this, it is in the infant’s best interest to research has used strontium stable isotopes in hard prolong nursing, while mothers should be motivated tissues (teeth) to study weaning stress in primates to wean their young as early as possible in the in- [Dirks et al., 2010]. Outside anthropology, ecologists terest of reproducing again. This idea remains con- have used stable isotope signatures to study weaning troversial [Godfrey, 1995; Maestripieri, 2002]. One in mammals, including isotopic analyses of bone col- reason why explaining parent–offspring conflict is lagen to track the durations of lactation and weaning difficult is the lack of a precise measurement of in- among seals [Newsome et al., 2006], of hair to infant weaning status [Martin, 1984]. Proxy indicators vestigate lactation schedules and breeding tactics of of weaning such as time spent on the mother’s ven- meerkats [Dalerum et al., 2007], and of blood compo- tral surface, the transition from riding to walking, nents to identify a weaning signal in 14 other species mother–infant conflicts, and amount of solid foods of mammals [Habran et al., 2010; Jenkins et al., in the infant’s diet provide the nearest visual es- 2001; Kurle, 2002; Polischuk et al., 2001]. In these timates of early life history [Rajpurohit & Mohnot, studies, researchers observe differences in stable iso- 1990; Rhine et al., 1984; Zhao et al., 2008]. This study tope ratios between the mother and infant when the uses a new technique—assessment of stable carbon infant is suckling (trophic-level effects), differences and nitrogen isotope ratios in feces—to determine that decrease throughout or after weaning until the daily snapshots of weaning status among François’ stable isotope ratios of mother and infant are indis- langurs (Trachypithecus francoisi) at the Toledo Zoo tinguishable and the pair consumes the same diet in Ohio. [but see exceptions in Jenkins et al., 2001]. Isotopes are atoms of the same element with Thisisthefirststudytousestableisotopera- the same number of protons, but different numbers tios in feces to track weaning in mammals. Samples of neutrons. Stable isotope analysis in anthropology used in previous isotopic studies of weaning—bone, and ecology is based on the principle “you are what hair, and blood—are difficult or unethical to collect you eat”: stable isotope ratios of foods are reflected in from free-ranging primates, many of which are en- tissues of their consumers. Stable carbon and nitro- dangered, and collection can be stressful even for gen isotopes show trophic-level enrichment patterns, captive animals. Furthermore, these tissue types are revealing relative reliance on plant vs. animal foods formed over periods of weeks to years, representing [Chisholm, 1989; DeNiro & Epstein, 1981; Fuller a time-average of diet rather than the precise life- et al., 2004; Minawaga & Wada, 1984; Schoeninger, history events represented by excretion products. Fe- 1989]. ces are noninvasive to collect, and are synthesized Stable isotope analyses using both hair and fe- quickly [Codron et al., 2006; Hwang et al., 2007; ces have been applied to studies that estimate pri- Sponheimer et al., 2003a], allowing weaning to be mate diet [gorilla (Gorilla gorilla): Blumenthal et al., tracked in day-to-day increments. The rate at which 2011; baboon (Papio ursinis): Codron et al., 2006; feces are synthesized from diet and excreted varies ring-tailed lemur (Lemur catta): Loudon et al., between species and depends on digestive physiology 2007; bonobo (Pan paniscus): Oelze et al., 2011; [Sponheimer et al., 2003a]. For example, ruminants Sponheimer et al., 2006; howler monkey (Alouatta store food in their digestive tracts for relatively long palliata), spider monkey (Ateles geoffroyi), muriqi periods of time during which semidigested cuds are (Brachyteles arachnoids), and capuchin (Cebus ca- regurgitated and chewed, meaning that ruminant pucinus): Schoeninger et al., 1997; galago (Galago feces do not represent the most recent meals. Spon- garnettii and G. zanzibaricus) and white-footed heimer et al. [2003a] report that a switch from C3 sportive lemur (Lepilemur leucopus): Schoeninger et to C4 fodder took 200 hr to be fully reflected in fe- al., 1998; chimpanzee (Pan troglodytes): Schoeninger ces of a ruminant alpaca, compared to only 60 hr et al., 1999]. Beyond diet reconstructions, stable iso- reported for a nonruminant horse. Langurs are non- tope ratios have seen limited applications to studying ruminant foregut fermenters with also the capacity primate nutritional stress [bonobo (P. paniscus): De- to ferment digesta in their hindguts [Caton, 1999; schner et al., 2012; human (Homo sapiens): Fuller et Van Soest, 1994, p 57–76]. This digestive anatomy, al., 2004, 2005, 2006], and have untapped potential uncommon among primates, enables langurs to ex- in nonhuman primate weaning studies. tract energy from structural polysaccharides, such Throughout weaning, an infant’s diet gradually as cellulose from leaves. In a study of feces reten- shifts from 100% mother’s milk to 100% solid foods. tion times between two langur species, Trachyp- The transition can be monitored with stable carbon ithecus obscurus and Trachypithecus cristatus,the and nitrogen isotope ratios of body tissues. Stable mean retention time of food in the digestive tract

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was 2.5 days [Caton, 1999]. This is slow compared to Studies of small mammals demonstrate that fe- cercopithecines, whose mean retention time is just cal δ15N is enriched by approximately +2.5‰ com- 1.5 days. Slower digestion in colobines reflects their pared to δ15N of diet [Hwang et al., 2007]. This diet– unique dual-chamber fermenting anatomy. Diet in- feces enrichment is similar in llamas (+3.0‰) [Spon- formation from a colobine fecal sample is therefore heimer et al., 2003c]. Feces of nursing primate in- more blurred than that from other primate species. fants are therefore expected to exhibit δ15Nvalues Nevertheless, a 60-hr snapshot of weaning status is approximately 2.5‰ higher than δ15N of their moth- drastically more precise than what is possible using ers’ milk (milk having an expected value of 5–6‰). other methods. That is, feces from a suckling infant should exhibit δ15N ratios of 7.5–8.5‰. Feces of the mother should be 2.5‰ higher than that of her available plant foods Predicted Values (having values near 2–3‰), or 4.5–5.5‰. As wean- To monitor the weaning process, three measure- ing progresses, it is predicted that this 3‰ difference ments are considered: stable carbon isotope ratios between feces of infant and mother will gradually (δ13C), stable nitrogen isotope ratios (δ15N), and ni- decline, and finally disappear as the pair eventually trogen content of feces (%N). Stable isotope values consume the same foods. are expressed as delta ratios (δ) of the heavier to the Stable carbon isotope ratios also differ system- lighter isotope in the sample in relation to the ratio atically between diet and consumer. Between plants of the heavier to the lighter isotope in a standard and consumer bone collagen, there is a positive 13C substance (Vienna Pee Dee Belemnite for δ13Cand enrichment of approximately 5‰ duetometabolic AIR for δ15N) using permil (‰) values. fractionation [Schoeninger & DeNiro, 1984]. Be- δ13C ratios are useful for measuring the vegetal cause nearly all foods provided to langurs at the 13 component of diet, including the amount and type Toledo Zoo are C3 plants exhibiting δ Cvaluesof (e.g., grasses; leaves) of plants consumed. In this around −26.0‰ [Nardoto, 2006; Schoeller et al., study, δ13C data are used to monitor (1) the mother’s 1986; Schoeninger et al., 1998], δ13C values of the largely vegetarian diet, and (2) the introduction of mother’s collagen are predicted to be approximately carbon-rich plant foods to the infant’s diet, distin- −21.0‰. Collagen and milk protein have similar guishing between mouthing of solid foods and ac- δ13C values. Fuller [2003] reports that milk pro- tual consumption of those foods. δ15N ratios detect tein δ13C is approximately 1–2‰ lower than finger- an animal’s trophic position (i.e., a suckling vs. a nail δ13C from the same mother. Similarly, collagen weaned infant), and are of primary interest here. δ13C is approximately 1.4‰ lower than fingernail %N measures crude protein content and also moni- δ13C [O’Connell et al., 2001]. Thus, the suckling in- tors the reduction of breast milk in the diet. Stable fant’s diet has an expected δ13Cvaluesimilartoits carbon and nitrogen isotopes in feces represent the mother’s predicted collagen value of −21.0‰. same isotope pool that is retained in the body for tis- Most controlled experiments investigating the sue formation after excretion of urea, meaning feces relationship between diet and feces δ13C have been should show trophic-level enrichment patterns sim- conducted on large-bodied foregut fermenters such ilar to hair, collagen, and nails [Codron et al., 2005]. as llama. Their fecal δ13Cvaluestendtobeap- Stable nitrogen isotope ratios in animal tissues proximately 0.3–1.3‰ lower than diet [Sponheimer reflect trophic position in the local food web. This is et al., 2003a]. Controlled feeding experiments sug- because, as nitrogen from food sources is ingested gest the diet–feces space is smaller for C3 diets and incorporated into consumer tissues, the lighter (−0.6‰)thanforC4 diets (−1.0‰) [Sponheimer et isotope 14N breaks down more readily than the heav- al., 2003a]. Larger diet–feces differences of up to ier isotope 15N, and is excreted with urea [Steele & −5.9‰ are reported for smaller mammals that use Daniel, 1978]. As a consequence, urea is isotopically hindgut fermentation, such as chipmunk and vole “light,” while remaining δ15N ratios of consumer tis- [Hwang et al., 2007]. Neither large foregut rumi- sues such as bone, hair and milk are approximately nants nor small hindgut fermenters present an ideal 3‰ higher than those of food sources [Minawaga & model for langurs, which are medium-bodied, non- Wada, 1984]. Because most plants exhibit δ15N ratios ruminant foregut fermenters [Caton, 1999]. For this near 2–3‰ [Nardoto, 2006], it is predicted that the study, the δ13C diet–feces space of −3.1‰ will be langur mother investigated in this study will have used because it was the mean value determined ex- body tissues with a δ15N ratio of approximately 5– perimentally by Hwang et al. [2007] to be appro- 6‰. No reports of stable isotope values of nonhuman priate for small-bodied animals fed mixed C3/C4 di- primate milk could be found in previously published ets, which the langurs at the Toledo Zoo consume. literature, but among humans, it has previously been With this −3.1‰ diet–feces spacing value in mind, reported that the δ15N value of human breast milk feces of the mother are predicted to exhibit δ13C protein (the milk component primarily used for in- values around −29.1‰. Feces of the nursing infant tissue-building) is similar to that of body tissues fant are expected to exhibit δ13Cvalues3.1‰ lighter [Fuller, 2003]. than breast milk, or −24.1‰. The expected 5‰ δ13C

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difference between feces of mother and infant should cally the reliability of observational data in assess- gradually decline through weaning as mother and ing weaning age of the François langur infant. The infant come to consume the same foods. extent of any incongruence between observational δ15N primarily reflects the major calorie sources and chemical methods can be considered toward a in diet. If an infant meets 100% of its caloric needs more precise assessment of weaning. By investigat- from nursing, its δ15N ratio will not necessarily re- ing François’ langurs, which are highly endangered, flect minor amounts of nitrogen-poor plant foods this study provides a new perspective on weaning be- added on top of milk consumption. Plant foods, which havior and interbirth intervals for this species that are carbon-rich, have a stronger effect in diluting the will help improve species survival plans and popu- carbon pool available to the infant, making δ13Cmore lation viability analyses. Based on the above discus- sensitive to the introduction of plant foods in diet sions, the following hypotheses are tested: [Fuller et al., 2006]. Although δ15N ratios might show δ13 little change with the introduction of solid foods, δ13C (1) Feces of the mother will exhibit Cvaluesof − ratios may show an earlier and more rapid decline, approximately 29.1‰. δ13 as has been documented among fingernails of hu- (2) Feces of the suckling infant will exhibit Cval- − man infants [Fuller et al., 2006]. The addition to ues of approximately 24.1‰ during the first solid foods is significant as it exposes the infant to month after birth. δ13 a new pathogen environment, and reduces the lac- (3) The expected C difference between feces of the tation stress on a female who may then return to mother and of the suckling infant will decrease estrus. δ13C will be used as a secondary source of and disappear as weaning proceeds. δ15 information on weaning. (4) Feces of the mother will exhibit Nvaluesof Nitrogen content (%N) in feces is another use- 4.5–5.5‰. δ15 ful measure that represents crude protein content (5) Feces of the infant will exhibit Nvaluesof of diet. In previous studies of primates, nitrogen 7.5–8.5‰ during the first month of age. δ15 content was used to assess diet and habitat qual- (6) The expected N difference between feces of the ity [Chapman et al., 2005; Codron et al., 2006]. %N mother and of the suckling infant will decrease values are produced in the process of stable isotope and disappear as weaning proceeds. δ13 analyses and in the present study will be a third (7) C values of infant feces will decline earlier and δ15 measurement used to detect consumption of breast more rapidly than N values. milk, which among Old World monkeys (baboons, talapoin monkeys, rhesus macaques) contains approxi- METHODS mately 16 mg/ml protein [Buss, 1968; Buss & Cooper, 1970; Kunz & Lonnerdal,¨ 1993]. It is expected that Materials the infant will exhibit higher %N than its mother The protocol for this study adheres to the legal while subsisting off breast milk and that %N will requirements of the United States and the specific gradually decline as the infant is weaned onto less principles of the American Society of Primatologists nitrogen-rich plant foods. Nutritional status of moth- for the ethical treatment of animals, and met the ers is an important factor influencing the weaning approval of the Toledo Zoo Animal Care and Man- process [Lee, 1987]. In this study, %N data of the agement Committee and the Executive Director. mother while she is pregnant, lactating, and nonlac- In this pilot study, fecal stable isotope analysis is tating are investigated to examine the crude protein applied to a captive mother and infant François’ lan- content of her diet and subsequently her nutritional gur dyad at the Toledo Zoo and Aquarium. François’ status throughout these phases. langurs are endangered primates that occupy sub- The first goal of this research is to investigate tropical limestone hill regions of China and Viet- the isotopic relationships between feces and diet in nam [Li et al., 2007]. Historically their range was a captive mother–infant François’ langur dyad. Data much larger but due to habitat destruction and hunt- on the diet–feces relationship used as an interpretive ing, in the last few decades numbers have dramati- baseline for this study are from small mammal stud- cally declined [Hu & Wei, 2002; Li et al., 2007; Zhou ies including primarily rodents; there is currently no et al., 2007]. In Guangxi Province, China, which in primate-specific experimental stable isotope spacing the 1980s contained the largest global population of data relating diet to feces. Once this relationship is François’ langurs (4,000–5,000 individuals), the total assessed among captive animals with semicontrolled number estimated in 2002–2003 was just 307 indi- diets, the method can be applied to other mother– viduals [Li et al., 2007]. Conservation measures will infant pairs in both captive and wild groups, and will be critical for the survival of this species in the wild require fewer fecal samples to track weaning. The but have only recently been implemented in Guangxi second goal of this research is to evaluate the differ- Province [Hu & Wei, 2002; Li et al., 2007]. Wild ent types of information provided by stable carbon François’ langurs feed primarily on leaves, along and nitrogen isotopes in relation to the weaning pro- with fruit and seeds [Hu, 2011]. François’ langurs cess. A final goal of this study is to evaluate isotopi- are relatively understudied, partially owing to their

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inaccessible rocky limestone forest habitat. Recent baseline. During Phase 2, when the infant is exclu- captive breeding successes at zoos (e.g., the Taronga sively nursing, samples were to be collected twice Zoo in Australia and US zoos in Toledo, Ohio, per week from both mother and infant. This phase Evansville, Indiana and Omaha, Nebraska) are pro- lasted from late August through November, when viding valuable new information on care and repro- the infant appeared to obtain virtually all its calo- ductive management among this species. ries from breast milk (see also Zhao et al. [2008] for In the wild, the weaning period of Francois’ wild animal data). However, it was not until Septem- langurs has been estimated at 19–21 months [Zhao ber 14, 2010 (3 weeks of infant age) that the first et al., 2008]. According to a publication by Y.M. Lai infant sample could be collected. Prior to this, defe- in 1987 [as cited by Zhao et al., 2008], the duration cation occurred while clinging to the mother, which of nursing is shorter in captivity, on the order of just the mother would clean. Phase 3 began in December 6 months. The difference, if accurate, is likely due when the infant was observed eating considerably to higher nutritional quality of captive animal diets, more solid foods and spending more time apart from which are based on vegetables and fruits. In the wild, the mother. During Phase 3 samples were collected Hanuman langur infants (Trachypithecus entellus) twice weekly from the mother and daily from the in- are completely dependent on their mothers for at fant. Phase 3 was initially expected to last through 6 least 1 month after parturition [Jay, 1965]. Solid months of infant age, as this was the weaning period foods are part of the wild Hanuman langur infant expected for captive langurs, based on a 1987 report diet around 3 months of age [Rajpurohit & Mohnot, by Y.M. Lai [as cited by Zhao et al., 2008]. 1990]. Between 4 and 10 months, weaning begins Diets of the Toledo Zoo langurs comprise (by food as the mother begins to reject her offspring [Jay, weight) approximately 45% vegetables, 45% leafy 1965; Rajpurohit & Mohnot, 1990]. This intensifies greens, and 10% primate biscuit. Periodic enrich- at 11 months of age until weaning is complete by 15 ment items include fruit, peanuts, browse, and pasta, months [Jay, 1965; Rajpurohit & Mohnot, 1990]. A which are offered once or twice a week. Such a shorter timeline is expected as the framework of the diet is estimated to have a δ13C value of approxi- present study, which involves captive langurs. mately −26‰ and a δ15N value of 2–3‰.Tocalcu- During the sample collection period, the late a dietary baseline, a number of foods were ana- François’ langur group at the Toledo Zoo comprised lyzed, including tomato, green pepper, carrot, broc- four individuals: one adult female, one adult male, coli, zucchini, green beans, eggplant, turnip, turnip one juvenile male, and one infant. The infant, a male, greens, and a primate “Leafeater” biscuit. These food was born on August 25, 2010. The infant’s birth was samples were taken by zookeepers from among the anticipated because the Toledo Zoo conducts routine foods offered to the animals during several days in ultrasounds, which detected pregnancy at an early August, September, and October 2011. Food samples stage in March 2010. Gestation lasted an estimated were frozen, freeze-dried, and homogenized in a mor- 6 months. Following birth, the mother received tar/pestle. Depo-Provera® shots as a method of birth control Fecal sample preparation followed previously es- and a melengestrol acetate implant was inserted in tablished protocol [Codron et al., 2006]. Fecal sam- mid-February, neither of which disrupted lactation. ples were stored frozen at the zoo in plastic collection bags until they could be transported The Ohio State University and oven-dried for several days at 60– Sample Collection, Preparation, and Analysis 80◦C. Dried feces were homogenized with a mortar Feces were collected by zookeepers from the ex- and pestle so that the powdered sample could pass hibit area shortly following deposition during routine through a 0.5-mm mesh sieve. An effort was made to cleanings while the animals were not present. The remove undigested food particles and hair from the mother was fed oatmeal cookies containing green sample while grinding. food dye so that her feces could be distinguished from Subsamples weighing between 1.3 and 1.5 mg for the rest of the group. Food dye is not expected to feces and 2.4 and 2.5 mg for foods were weighed into significantly alter bulk fecal stable isotope values, tin capsules at The Ohio State University Stable Iso- and as a constant ingredient in the mother’s diet tope Biogeochemistry Laboratory. Subsamples were throughout the study, should not affect the tran- analyzed on a Costech Elemental Analyzer coupled sitions of interest here. Feces of the infant were to a Finnigan Delta IV Plus stable isotope ratio easily identifiable throughout the collection period, mass spectrometer under continuous flow using a being smaller and often pelleted, and no labeling CONFLO III interface. Stable carbon (δ13C = per- was required. Samples were collected by zookeep- mil deviation of the ratio of 13C:12C relative to ers at different frequencies over the course of three the Vienna Peedee Belmenite Limestone standard) research phases between July 2010 and September and stable nitrogen (δ15N = permil deviation of 2011. During Phase 1, commencing 1 month before 15N:14N relative to air) isotope measurements were expected parturition, feces of the mother were sam- made where the average standard deviation of re- pled eight times to establish a preparturition dietary peated measurements of the USGS24, IAEA-N1, and

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IAEA-N2 standards were 0.05‰ for δ13C and 0.13‰ greens; green pepper). The range of δ13Cvaluesis for δ15N. Data are reported to the nearest 0.1‰,ac- smaller, from −30.9‰ (turnip greens) to −22.4‰ cordingly. Approximately 10% of all samples were (“Leafeater” biscuit, containing ground corn, a plant run in duplicate. Both stable carbon and nitrogen enriched in 13C). Three tomatoes and three types of isotopes are reported in relation to the accepted lettuce were assayed and reveal that the stable iso- standards according to the equation δ = ([Rsample − tope values of even a single food type vary widely. Rstandard − 1] × 1,000). In addition to stable isotope ratios, the mass spectrometer measures overall carbon and nitro- Integrity of Data gen content (%C and %N) of the samples. From Duplicate analyses of fecal samples were per- these measurements, carbon to nitrogen (C:N) ra- formed to investigate the extent of within-sample tios were calculated according to the equation C:N variation. One triplicate and five duplicate samples = (%C/12)/(%N/14). The C:N ratios of samples can were analyzed from the mother, and eight duplicate detect inclusion of unwanted substances with %C or samples were analyzed from the infant. In general, %N values that differ from feces, and are used to in- replicability was very high. Other than one sample vestigate sample integrity, and also reflect changes from the mother (the δ15N values of its duplicates in %N with varying protein content of the diet. differing by 0.7‰) all replicate isotope analyses were Descriptive statistics are presented hereafter as within 0.3‰ for δ15N, 0.4‰ for δ13C, and 0.5 for %N. means and standard deviations. Pearson’s correla- The maximum and mean differences between each tions are used to explore any relationships between set of replicate samples are presented in Table II. C:N ratios and stable isotope values. Both r values Possible “contamination” of samples by microbes and associated P values are reported, and P values or undigested foods is investigated by comparing C:N are considered significant at the 0.05 level. Other in- ratios to stable isotope values of feces. There is no ferential statistics are not used on the basis of the relationship between C:N ratios and either δ13C(r = small sample used for this pilot study. −0.289, df = 223, P < 0.001, N = 225) or δ15N(r = −0.161, df = 223, P = 0.016, N = 225; Fig. 1). Fecal C:N ratios of the mother range from 10.7 to 17.9. RESULTS Those of the infant range from 7.0 to 14.8, with the Foods lowest values occurring during the first 3 months. Stable isotope signatures of foods are highly vari- The integrity of only one sample is suspect on the able, especially for nitrogen (Table I). Most δ15Nval- basis of questionable %C and %N data. The C:N ratio ues are low with the exceptions of a green pepper for this infant sample from October 19, 2011 was ex- and turnip greens. The range of δ15Nvaluesis0.4‰ tremely high at 60.7, indicating that the substance (green bean, a low-15N legume) to 12.0‰ (turnip analyzed was not characteristic of most feces, per- haps containing a disproportionate amount of undigested high-carbon fatty acids from breast milk. This TABLE I. Food Stable Isotope Values sample’s stable isotope values were still within the overall range of variation for the infant, although Food item δ15N(‰) δ13C(‰) the δ13C value was slightly lower than the other val- Broccoli 3.3 −28.3 ues from October 2010. Macroscopically, this sam- Turnip 0.9 −27.9 ple was clearly unlike the others in having a greasy Turnip greens 12.0 −30.9 texture and appearance. This sample appears in Zucchini 6.4 −26.8 Figure 1 but will be excluded from further discus- Eggplant 5.5 −26.8 sion and does not appear in the remaining figures. Green bean 0.4 −28.0 Tomatoes Regular 3.0 −27.8 Fecal Samples − Roma 0.9 27.9 δ15 δ13 Roma 2.0 −28.0 Mean fecal Nand C values and standard Green pepper 12.0 −29.3 deviations for each calendar month are presented in Carrot 1.3 −26.7 Table III. Figure 2 displays both the mother’s and 15 13 Lettuce the infant’s fecal δ N, δ C, and %N values, and Iceberg 4.8 −27.6 represent all 143 days for which samples were an- Romaine 3.4 −27.5 alyzed. Figure 3 displays similar information using Leaf 1.2 −29.4 just one set of data points each, which represent Leafeater biscuit 2.9 −22.4 the difference between the mother’s and the infant’s Mean ± SD 4.0 ± 3.7 −27.7 ± 1.8 δ15N, δ13C, and %N values. These values were calculated by subtracting the mother’s from the infant’s Stable nitrogen (δ15N) and stable carbon (δ13C) isotope values of a sub- sample of foods routinely fed to Toledo Zoo langurs. Each food listed was values, and, unlike in Figure 2, only days when both analyzed once. the mother and infant were sampled are represented

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TABLE II. Replicate Analyses

δ15N(‰) δ13C(‰) N content (%)

Maximum Mean Maximum Mean Maximum Mean difference difference difference difference difference difference

Mother 0.7 0.2 0.3 0.1 0.3 0.1 Infant 0.3 0.2 0.4 0.1 0.5 0.1

Stable nitrogen (δ15N) and stable carbon (δ13C) isotope and nitrogen/protein content (%N) results from replicate analyses of feces from mother and infant, showing both the mean and the maximum differences obtained from repeated analyses.

ment of infant fecal 15N above the mother was 3.5‰ (October 26, 2010). The mean δ13C value of the mother was −26.4 ± 0.5‰. The overall mean δ13C value of the infant was −25.9 ± 0.8‰, but prior to the second month, when the infant was exclusively suckling, the mean δ13Cvaluewas−23.7 ± 1.2‰ (n = 6). For the ﬁrst 3 weeks of sample collection and the ﬁrst 6 weeks after birth, the infant fecal δ13C values are consistently higher than its mother (Figs. 2B and 3B). Excepting one low-13C outlier, which may contain undigested fatty acids, infant δ13C values are on average 3.1‰ higher than mother values. On October 8, 2010 (approximately 6 weeks), there is a change in the infant δ13C: after this date, infant values are lower. Start- ing on December 17, 2010 (approximately 4 months), the δ13C values of infant and mother become similar. The mean fecal %N value of the mother was 3.5 ± 0.4%, and of the infant was 4.5 ± 0.8%. During October and November 2010, the infant’s fecal values are up to 3.4% higher than its mother, but after 3 months of age the infant’s fecal %N drops to within 1% of its mother (either higher or lower; Figs. 2C and 3C).

DISCUSSION Fig. 1. The relationship between the carbon to nitrogen (C:N) Data Concerning Foods and Diet–Feces ratios and stable carbon isotope ratios (δ13C) for all mother and infant samples (A), and the relationship between the carbon to Spacing nitrogen ratios and stable nitrogen isotope ratios (δ15N) for all The stable isotope values of foods in this mother and infant samples (B). study vary widely, which can result from different production strategies (e.g., fertilization techniques (n = 82). This normalizes the data for ﬂuctuations in [Bogaard et al., 2007; Meints et al., 1975]). Con- stable isotope ratios that are merely due to day-to- sequently, daily ﬂuctuations in the mother’s δ15N day variations in the mother’s diet, and emphasizes values can be expected, and attributed to varia- the variation that is due to nursing. These infant– tions in the stable isotope signatures of foods con- 15 15 mother values ( Ninfant–mother) also appear in sumed. Fluctuations in a suckling infant’s δ Nval- Table III. ues should approximately match those of its mother, The mean fecal δ15N value of the mother is but more detached variation can be expected when 5.0 ± 0.9‰ (n = 82) with 90% of her δ15Nvalues the infant is weaning. falling between 4.0‰ and 5.5‰. The mean fecal δ15N Different foods contribute more or less to the value of the infant is 6.4 ± 0.8‰ (n = 143). As shown bulk stable isotope signature of the langur diet by Figures 2A and 3A, many (n = 22) of the in- depending on how much (fresh food weight in kg) fant’s fecal δ15N values are more than 2‰ higher is offered each day. By considering how many kg of than those of the mother. The maximum enrich- each type of food is provided for the animals daily

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TABLE III. Stable Isotope Results for Mother and Infant

15 Mother Infant Ninfant–mother (‰)

δ15N δ13C δ15N δ13C%N Month n (‰)(‰)%Nn (‰)(‰)

July 2010 2 4.5 ± 0.1 −27.4 ± 0.0 2.9 ± 0.0 0 August 2010 3 5.4 ± 0.6 −26.9 ± 0.4 3.1 ± 0.2 0 September 2010 1 5.0 −26.3 3.3 4 6.7 ± 0.6 −23.9 ± 1.6 6.2 ± 1.6 1.7 October 2010 5 5.2 ± 0.7 −26.6 ± 0.2 3.3 ± 0.3 8 6.8 ± 0.8 −24.8 ± 1.0 5.4 ± 1.0 1.6 November 2010 6 5.4 ± 1.1 −26.6 ± 0.4 3.3 ± 0.3 11 7.2 ± 0.7 −25.0 ± 0.4 4.4 ± 0.9 1.8 December 2010 7 4.4 ± 0.5 −26.3 ± 0.4 4.0 ± 0.2 22 6.6 ± 0.6 −25.7 ± 0.3 4.6 ± 0.4 2.1 January 2011 8 4.9 ± 0.7 −26.2 ± 0.5 3.8 ± 0.3 23 7.1 ± 0.6 −25.9 ± 0.3 4.4 ± 0.6 2.2 February 2011 13 4.8 ± 0.5 −26.2 ± 0.3 3.7 ± 0.3 19 6.6 ± 0.6 −26.0 ± 0.4 4.9 ± 0.3 1.7 March 2011 9 4.8 ± 0.5 −26.4 ± 6.0 3.3 ± 0.3 13 6.0 ± 0.7 −26.5 ± 0.4 3.8 ± 1.0 1.2 April 2011 7 4.8 ± 0.9 −26.0 ± 0.3 3.3 ± 0.4 15 5.7 ± 0.8 −26.4 ± 0.3 4.0 ± 0.7 0.9 May 2011 8 4.4 ± 0.9 −26.6 ± 0.6 3.5 ± 0.3 13 5.6 ± 0.6 −26.7 ± 0.3 4.4 ± 0.5 1.3 June 2011 2 6.6 ± 3.3 −26.8 ± 0.3 3.3 ± 0.1 2 5.5 ± 0.4 −26.9 ± 0.0 4.0 ± 0.1 1.1 July 2011 3 5.9 ± 0.9 −26.9 ± 0.5 3.6 ± 0.3 3 6.3 ± 0.5 −26.8 ± 0.7 4.4 ± 0.0 0.4 August 2011 1 7.3 −26.1 2.9 2 5.4 ± 0.3 −26.0 ± 0.4 4.0 ± 0.7 1.9

Stable nitrogen (δ15N) and stable carbon (δ13C) isotope values and nitrogen/protein content (%N) of feces from the mother and her infant. Values are grouped by month; sample sizes are shown for each month. The average monthly difference between mother and infant δ15N fecal values are shown in the right-most column (15N). and “weighting” each item’s isotopic contribution to than that of the calculated diet, which is a lower diet– the bulk diet accordingly, it was possible to calculate feces spacing than previously reported for either ranges of bulk diet values for six hypothetical diets rodents or large-bodied herbivores [Hwang et al., containing various combinations of foods. The δ13C 2007; Sponheimer et al., 2003c]. As previously men- range for hypothetical diets was −23.5‰ to −24.6‰ tioned, this is possibly due to the high-15N green pep- and the δ15N range was 3.8–4.3‰.Bothrangesare per, which raises calculated diet values somewhat slightly above what was expected based on values thereby reducing the diet–feces spacing. of modern foods [Nardoto, 2006]. Higher δ13Cval- ues of the diet explain why mother fecal δ13Cvalues are approximately 3‰ higher than expected. Above- Relationship Between Mother and Infant expected dietary δ15N may be because of the impact δ15 of the high-15N green pepper analyzed here on the N and the persistence of suckling calculations,aspeppersarefedtothelangursev- It appeared to zookeepers that by the fifth month ery day and this high value consequently contributes (January 2011) the infant was suckling very little. δ15 to every one of the calculated diets. In light of the Indeed, the first day that the infant’s fecal Nvalue wide δ15N variation of tomatoes and lettuce reported is nearly identical to its mother’s is January 8, 2011. here, it is entirely possible that other green peppers Yet according to the stable isotope data, a consider- might exhibit lower δ15N values, which would lower able amount of the infant’s calories were still derived the bulk average of actual diets by 1–2‰.Forδ13C, from suckling on many days in January 2011: some δ15 higher than expected values calculated for bulk di- of the infant’s fecal N values are still nearly 2‰ etsareduetotheprimatebiscuitthatconstitutes higher than its mother, consistent with one trophic approximately 10-12% by weight of the daily diet position. Either what appeared to be comfort nurs- and has a relatively high δ13C ratio (−22.4‰)at- ing after January 2011 was in fact actual nursing, or tributable to corn as an ingredient. Wild animal diets nursing continued for a prolonged period at night (or would likely more consistently reflect the predicted both). The infant continued to suckle throughout the δ15Nandδ13C values, although wild plants also vary intended 6-month sample collection period, exhibit- δ15 in their stable isotope values and should be mea- ing fecal N values 1–3‰ higher than its mother. sured [Schoeninger, 2010]. Because of this, the sample schedule was extended in In terms of the diet–feces spacing relationships, an effort to capture the cessation of suckling. Begin- based on the mother’s fecal stable isotope values and ning in April 2011 (month 8), values of the infant be- using the calculated diet values, δ13C of feces was gin to drop below those of the mother (Figs. 2A; 3A), approximately 2–3‰ lower than δ13Cofdiet,more an indication of nutritional independence for some δ15 consistent with the values reported by Hwang et al. days. Two-thirds of the infant’s fecal Nvaluesare [2007] for small rodents than those reported by Spon- still higher than the mother’s after the initial drop in heimer et al. [2003a] for large-bodied animals. Fecal April 2011, and even 1 year after birth, the infant’s δ15 δ15N of the mother was approximately 1‰ higher N values are elevated, indicating the persistence

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Fig. 2. Comparing stable nitrogen isotope values (δ15N) (A), stable carbon isotope ratios (δ13C) (B), and percent nitrogen content in feces (%N) (C) of all mother and infant fecal samples through time. of suckling. The infant could be seen suckling as late variable and easier to interpret. Zoo animals were as October 2011 (14 months after birth). Stable iso- chosen for this pilot study in part because of their tope data suggest this was for physiological reasons controlled diets, yet although a precise “menu” of and not comfort nursing. foods can be known, the isotopic variability of these Numerous fluctuations in fecal δ15Nofthe commercially produced foods is more uncertain than mother throughout the sample period are not sur- what may be expected for wild diets. prising, considering feces are synthesized quickly A final observation regarding δ15Nisthatatthe and fecal stable isotope values are barely “smoothed end of the sample period (June–August 2011), the out” by time averaging. Similar day-to-day variation mother’s values are unexpectedly high (as much as has been reported among bonobos using stable iso- 3.2‰ higher than the infant). The only change to an- tope values in urine [Deschner et al., 2012]. Although imals’ diets during this time was addition of more physiology likely plays a role in this, the fluctua- browse (mulberry, willow, and grapevine), of which tions are probably primarily due to differences in langurs will eat the leaves and bark. Within plants, the isotopic signatures of foods offered each day. An- leaf δ15N can indeed be higher than δ15Nofother imals may have consumed high-15N turnip greens roots and stems due to patterns of nitrogen assim- one day and low-15N green beans another. As ev- ilation from nitrate [Evans, 2001]. Yet browse was idenced by the variable stable isotope signatures of also available during the previous summer and no multiple tomatoes and lettuce in this study, even one pattern of δ15N enrichment in the mothers’ feces is food type can produce a considerable range of sta- observed during this time (Fig. 2A). Also, the infant’s ble isotope variation among animals. The fact that δ15N values are not as high as the mother’s during feces represent a snapshot of approximately 60 hr this time though they had access to the same foods, helps smooth some of this variation. Because diets cautioning against a dietary explanation for the en- of wild primates are less heterogeneous day-to-day richment. It is nevertheless possible that mother and than foods produced using modern agricultural tech- infant simply chose to eat different foods (different niques, their stable isotope signatures would be less amounts of browse or high-15N vegetables).

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Fig. 3. The difference between mother and infant stable nitrogen isotope values (15N) (A), stable carbon isotope values (13C) (B), and infant nitrogen content in feces (%N) (C) plotted through time. Horizontal lines represent a margin of error which is ± the mean difference obtained from replicate analyses of the same samples (Table II).

If diet differences do not fully explain 15Nen- of the mother could potentially correspond to neg- richment of the mother, there may be physiological ative nitrogen balance, although she was not known explanations. Sponheimer et al. [2003b; 2003c] noted to experience nutritional stress during the sample that herbivores fed high-protein diets in controlled period. Although the reason for the mother’s ele- feeding studies exhibit higher δ15N ratios in hair vated δ15N ratios are not fully understood at present, than those fed lower protein diets. The authors at- these issues underscore the complexity of stable ni- tribute this to the fact that of the two major pathways trogen isotopes in biological systems, and warrant for nitrogen efflux, urine, and feces, urine production further investigation with a greater number of indi- causes fractionation such that urea is isotopically de- viduals. From the elevated δ15N values of the mother pleted, and remaining body pool nitrogen is enriched. in June, July, and August 2011, it at least is apparent When animals have an excess of protein in their di- that the infant is no longer at a consistently higher ets, the excess is lost through urine, not through trophic position compared to its mother from month feces. The result of this is that on high protein di- 11 onwards, although some suckling seems to have ets, relatively more nitrogen is lost through urine, persisted. leading to relatively more isotopic fractionation and δ15 higher N values in body tissues [Sponheimer et δ13C and the addition of solid foods al., 2003b; 2003c]. High protein in the mothers’ diet The stable carbon isotope ratio reflects carbon- (possibly through eating browse), or any other phys- rich plants in the diet and is more sensitive than δ15N iological condition leading to increased bioavailabil- to the addition of solid foods. According to the stable ity of protein, could instigate this metabolic response isotope data, the infant ate very few plant foods dur- 15 and leave mothers’ tissues N-enriched. However, ing the first 6 weeks after birth. Zookeepers noted δ15 %N of the mother does not increase alongside N that in the first month of sample collections the in- (Figs. 2A and C), as would be expected if crude pro- fant was mouthing foods such as broccoli and lettuce, tein in her diet was higher. but the stable isotope data do not suggest these foods Alternatively, during periods of nutritional were actually consumed. After 6 weeks of age (Octo- δ15 stress (negative nitrogen balance), Nvaluesare ber 8, 2010), it appears that solid foods began to con- higher due to the necessary catabolism of an animal’s tribute considerably more to the infant’s diet. By 24 own tissues [Deschner et al., 2012; Fuller et al., 2004, weeks of age, the solid food diets of mother and infant δ15 2005; Hobson et al., 1993]. Increased N ratios are isotopically indistinguishable. Infant δ13Cvalues

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decline more rapidly than do infant δ15Nvaluesas milk values than those animals studied by Jenk- weaning progresses, as has been reported elsewhere ins et al. [2001]. It is worth noting that as lac- for human infants [Fuller et al., 2006]. This supports tation proceeds, crude protein content of milk de- the idea that while δ13C values reflect overall diet, creases while lipid concentration increases [Buss, including dietary energy sources, δ15Nvaluesmore 1968]. Lipids are relatively depleted in 13C [DeNiro closely track the major protein sources in diet. & Epstein, 1977; Fuller, 2003], and potentially, this These data demonstrate a trophic enrichment phenomenon could contribute to the decline in infant in δ13C between mother and infant. The maximum δ13C values as weaning proceeds, independent of the trophic enrichment of infant fecal 13C in the present introduction of solid foods or a trophic-level effect. It study is +3.5‰, somewhat less than the expected would not, however, affect initial trophic enrichment +5‰. It should be borne in mind that during the in the early months. Measuring the isotopic compo- first 3 weeks following birth, no samples could be sition of breast milk throughout lactation would be collected from the infant, and thus the first available very useful for future weaning studies incorporating datum may not represent a pure-suckling diet—that δ13C data. is, solid foods may have been introduced undetected Day-to-day fluctuations represent more or less prior to September 14, 2010. However, no undigested consumption of solid foods, but are also probably ex- solid foods were identified in feces during September aggerated: when consuming solid foods, undigested and October 2010. It may also be the case that langur particles make their way into an animal’s feces [Co- milk protein is slightly depleted in 13Ccomparedto dron et al., 2011]. Many of these are removed in sam- human milk protein which was used to build the pre- ple preparation during sieving, but some particles of diction model [Fuller, 2003], lessening the predicted food are present in the final sample. Whole (albeit mother–infant δ13Cspacing. small) particles are detected in the mass spectrome- Nevertheless, the 3.5‰ mother–infant δ13C ter and are overrepresented compared to the diluted space is greater than what is previously reported signature that would be obtained were the particles for a single trophic position. When δ13Cvaluesfrom fully digested. The particles represent what was not collagen are used in archeology for diet reconstruc- used for nutrition. This is less problematic than it tions, a 13C enrichment of approximately +1‰ has may seem because the stable isotope signatures of been observed between prey and predator (referred undigested parts of foods (e.g., stems) do not differ to as the “carnivore effect”) [Schoeninger & DeNiro, greatly from the digested parts (e.g., leaves) [e.g., Co- 1984]. One explanation for the carnivore effect is that dron & Codron, 2009; Codron et al., 2005] and still δ13C from the flesh of an animal is up to 4‰ lower reflect the ingestion of solid foods. Presence of undi- than δ13C from bone collagen of the same animal, yet gested food particles influences δ13C more strongly the diet–tissue spacing value for δ13Cis+5‰,leav- than δ15N because plant matter has high carbon and ing the carnivore’s collagen 1‰ higher than colla- low nitrogen content. Also for this reason, “contam- gen of its prey [Schoeninger, 1989]. However, Fuller inated” samples potentially could be identified by [2006] observed that fingernails of suckling human examining C:N ratios and removing samples with infants are 1‰ higher than fingernails of mothers, anomalously high values. yet, unlike the case with flesh and collagen, breast milk protein δ13C is just 1–2‰ lower than fingernail %N and the crude protein content in diet δ13C from the same individual [Fuller, 2003]: conse- Fecal %N values of the mother do not vary quently, a fingernail-to-fingernail difference of 3–4‰ widely, reflecting the relatively homogeneous pro- could be expected between mother and infant, yet the tein content of her vegetal diet. Fecal %N values of observed diet–tissue space is lower, evoking the need the infant show broad day-to-day fluctuations, re- for other explanations. The reason why δ13Ctrophic flecting variations in dependence on milk vs. plant enrichment in the present study is greater than pre- foods. The crude protein content of infant feces is viously reported for collagen and fingernails is uncer- high during the first 3 months of age due to breast- tain. At present, few controlled feeding experiments feeding, and drops considerably afterwards, reflect- are available to inform on the δ13C carnivore effect. ing the gradual addition of low-protein plant foods It is likely that more detailed experimental data will in the diet. However, even during the final months make important contributions to understanding this of sample collection, the infant exhibits consistently research area in the future. higher fecal %N than its mother, suggesting a higher It has been reported elsewhere that δ13Cval- protein diet indicative of persistent suckling through ues do not show trophic enrichment in the tissues the 12th month. (blood) of nursing infants because trophic enrichment is “canceled out” by the 13C-depletion of milk when its lipid component is considered [Jenkins et Revisiting the Original Hypotheses and al., 2001]. These results are not replicated here with Future Directions 13 langurs. Interspecific δ C values of breast milk may Fecal δ13C, δ15N, and %N values of the François be at the root of this, with langurs exhibiting higher langurs investigated here are indicative of nursing, a

Am.J.Primatol. Fecal Stable Isotopes Track Weaning / 937

time when the infant is a trophic position higher than the wild may also prove to be underestimations when its mother, and of weaning, the gradual decrease an isotopic approach is applied as an independent in suckling. Hypotheses 1 and 2 predicted that the source of information. mother and suckling infant would exhibit δ13Cval- Application of this method in the wild is straight- ues of −29.1‰ and −24.1‰, respectively. Contrary forward so long as feces can be collected from moth- to these predictions, both the mother and infant ex- ers and infants, and the individuals depositing feces hibited higher values, which may be attributed to can be identified. The primary goal of this study was the higher than expected dietary baseline δ13Cmea- to monitor day-to-day changes in weaning status, sured here. Hypotheses 3 predicted a gradual decline which required a large number of samples. Fewer in infant fecal δ13C to maternal values, and is sup- samples need be collected to apply this method in ported. Together, these results point to the utility of the future, and the number collected will depend on the δ13C ratio in feces to record trophic-level enrich- the questions asked. Because of the substantial daily ment patterns during weaning. fluctuations observed here, it is recommended that Hypotheses 4 and 5, which predict mother and samples be collected in sequences of 3 days at inter- suckling infant fecal δ15N values of 4.5–5–5‰ and vals of interest to the researchers (weekly, monthly, 7.5–8.5‰, respectively, are supported. This indicates seasonally) to determine the extent of an infant’s the utility of fecal δ15N values to record a trophic breastfeeding at a given time. Colobines, with their effect from suckling. Hypothesis 5, which predicted unusual digestive morphology, give fecal samples that δ15N values would decline throughout the course that record foods consumed over the past approxi- of the study, is only partially supported. Judging mately 2.5 days. Feces from virtually all other pri- from the δ15N data, weaning was not complete when mate taxa will yield finer chronological resolutions, sample collection ended: infant fecal δ15Nvaluesdo which may increase apparent daily variations, but not consistently reach maternal values. However, will also refine the method’s precision. A dietary changes in the infant–mother δ15N values through baseline should be estimated in the wild, either by time (particularly before and after March 2011) are measuring stable isotope ratios in available foods, likely indicative of weaning. Hypothesis 7 predicted or by measuring fecal stable isotope ratios of other fecal δ13C values of the infant would decline earlier nonlactating, nonsuckling group members, who in- and more sharply than δ15N values, which is sup- tegrate the signals of the foods consumed and serve ported, indicating these measurements are useful for as a proxy indicator of the overall stable isotope en- recording two different, complementary types of in- vironment [Bump et al., 2007]. In addition to use formation about the weaning process. in the wild, this method has useful applications in These data refine visual estimations because sta- other captive studies; for example, isotopic labeling ble isotope signatures permit detection of suckling of foods to reveal allonursing in groups containing at night, distinguish between actual nursing and more than one lactating female, and monitoring nu- comfort nursing, reflect actual rate of milk transfer, tritional stress and food partitioning during the in- and distinguish between mouthing of solid foods and troduction of new group members. actual consumption of solid foods. Zookeepers wit- nessed mouthing of solid foods prior to 6 weeks, but stable isotope data suggest solid foods may not actu- ACKNOWLEDGMENTS ally have been consumed until after 6 weeks. By 16 I thank Kate Spink and Koral Stoddard with weeks of age, the infant obtained most of its calories the Toledo Zoo for sample collection and providing from solid foods. Through visual observation, nurs- detailed diet information and estimates of weaning ing appeared to be greatly reduced by 20 weeks, but status. I thank Douglas E. Crews with the Human 15 δ N data suggest that the infant still met a signifi- Biology Laboratory, and Andrea´ Grottoli and Yohei cant amount of its dietary needs through nursing at Matsui with the Stable Isotope Biogeochemistry that time, and continued to nurse as late as 1 year Laboratory at The Ohio State University. I thank after birth, which is when sample collection ceased. Katie Daniel, Rachel Kopec, and Dawn Kitchen for The infant was still observed to nurse at 12 months support with sample preparation and interpreta- of age, when sample collection ceased. tion. I am also grateful to anonymous reviewers In this case, the infant’s observed nursing ap- for their constructive feedback on this manuscript pears to have been for dietary purposes and not and to Donald Dunbar for his careful editorial merely comfort nursing, as is frequently suspected. work. It was expected that the weaning period for these captive langurs would be much shorter than 12+ months due to more regular access to highly nutri- REFERENCES tious foods. However, the actual weaning period for Blumenthal SA, Chritz KL, Cerling TE, Rothman JM. 2011. these animals was much longer, and may be com- Stable isotopes in wild gorilla feces document seasonal di- parable to weaning periods reported from the wild etary change and rainfall patterns. Am J Phys Anthropol [e.g., Poirier, 1972]. Reported weaning periods from 144:92–92.

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