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Int J Primatol DOI 10.

1007/s10764-012-9582-7

Stable Isotope Techniques and Applications for Primatologists


Brooke E. Crowley

Received: 18 February 2011 / Accepted: 18 October 2011 # Springer Science+Business Media, LLC 2012

Abstract Stable isotope biogeochemistry is useful for quantifying the feeding ecology of modern and extinct primates. Over the past three decades, substantial advances have been made in our knowledge of the physiological causes of isotopic patterns as well as effective methodology to prepare samples for isotopic analysis. Despite these advances, the potential of stable isotope biogeochemistry has yet to be fully exploited by primate researchers, perhaps due to the prolific and somewhat daunting nature of the isotopic literature. I here aim to present a cogent overview of stable isotope applications to nonhuman primate feeding ecology. I review the factors that influence ecological patterns in carbon, nitrogen, and oxygen stable isotopes. I present methods for collecting and preparing samples of tooth enamel and bone mineral hydroxyapatite, bone collagen, fur and hair keratin, blood, feces, and urine for isotope analysis. I discuss both the existing and potential applications of these isotopic patterns to primate feeding ecology. Lastly, I point out some of the pitfalls to avoid when interpreting and comparing isotopic results. Keywords 13C . 15N . 18O . Hydroxyapatite . Proteinaceous tissues . Stable isotope . Trophic discrimination

Introduction Stable isotope biogeochemistry is a powerful tool for enhancing our understanding of the diets and niche partitioning of extant species as well as reconstructing the diets and habitats of extinct species. Our current knowledge of nonhuman primate dietary needs derives largely from field observations, dental wear, and morphology. Al-

Electronic supplementary material The online version of this article (doi:10.1007/s10764-012-9582-7) contains supplementary material, which is available to authorized users. B. E. Crowley (*) Departments of Geology and Anthropology, University of Cincinnati, Cincinnati, OH 45221, USA e-mail: brooke.crowley@uc.edu

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though each of these methods has its merits, each is also inherently incomplete (Sponheimer et al. 2009). Observed and actual feeding rates may differ because many species are cryptic and difficult to study. Dental wear may be biased toward gritty or acidic foods and may only reflect an animals most recent meals (Grine 1986). Morphology clearly reflects dietary adaptations, but such adaptations might be more influenced by the consumption of fallback foods (Lambert et al. 2004; Marshall and Wrangham 2007). As a result, actual feeding ecology may be much broader than morphological adaptations would suggest. Isotopic data, providing a quantitative record of an animals feeding ecology, can validate observational and morphological data sets. The application of stable isotope biogeochemistry to ecological research has been increasing steadily over the last three decades. A wealth of papers on topics including habitat characterization, plant and animal physiology, and food web dynamics is now available. However, this information is not always accessible to those unfamiliar with the isotopic literature. Here I provide background information on carbon, nitrogen, and oxygen isotopes, the three stable isotope systems that are most commonly used in feeding ecology studies. I review collection and sample preparation methods for the tissues and body products most relevant to primate feeding ecology (tooth enamel and bone hydroxyapatite, bone collagen, fur and hair keratin, blood, feces and urine), and present some examples of stable isotope applications in primate foraging ecology. Stable isotope biogeochemistry has been widely applied in archaeological studies. Whereas summarizing this literature is beyond the scope of this article, I do refer to human-based studies when examples for nonhuman primates are lacking. A number of excellent recent reviews cover the fundamentals of isotope biogeochemistry in plants (Barbour 2007; Handley et al. 1999; Heaton 1999; Kohn 2010; Marshall et al. 2007) and animals (Gannes et al. 1998; Koch 2007; Kohn and Cerling 2002; Lee-Thorp et al. 2003; Martnez del Rio et al. 2009; Schoeninger 2010; Sponheimer et al. 2009). I refer readers to these sources for a more in-depth discussion of the material presented here. Isotopic analysis of additional tissues, e.g., tooth dentine, and additional isotopes, e.g., hydrogen, strontium, and sulfur, may also have applications to primate foraging ecology (Gannes et al. 1998; Koch 2007). However, discussion of these topics is beyond the scope of this article.

Background on Stable Isotope Biogeochemistry Stable isotope ratios in animal tissues are informative because they provide a record of the food or water actually ingested and assimilated into an animals body tissues. These ratios are predicted to vary as a function of habitat, physiology, and dietary proclivity (Koch 2007; Schoeninger 2010). Accordingly, stable isotopes have been used to investigate foraging ecology and niche partitioning among both living and extinct nonhuman primates (Codron et al. 2006; Crowley et al. 2011a; Dammhahn and Kappeler 2010; Fourie et al. 2008; Lee-Thorp et al. 2003; Loudon et al. 2007; Schoeninger et al. 1997). Stable isotope ratios are ideal for these purposes because the difference in mass between isotopes of an element is measurable. Isotopes with more neutrons, e.g., 13C, 15 N, and 18O, have heavier masses than isotopes with fewer neutrons, e.g., 12C, 14N,

Stable Isotope Techniques and Applications

and 16O. These mass differences result in slightly different thermodynamic and kinetic properties among isotopes of the same element (Hoefs 1997; Urey 1947). They also lead to partitioning, or fractionation, of lighter and heavier isotopes between different materials during physical and chemical reactions (Hoefs 1997). As a result of these fractionation processes, different materials have discrete isotope ratios, reflecting the relative amounts of lighter and heavier isotopes. Because the absolute ratio of heavy to light isotope tends to be very small, isotopic abundance is generally referred to as parts per thousand (per mil, ) in reference to an international standard using a standardized notation, e.g., 13C, where d Rsample =Rstandard 1 1000, and R is the ratio of heavy to light isotope (13C/12C, 15N/14N or 18O/16O). Positive and negative values indicate that the sample has more or less of the heavy isotope relative to the standard, respectively (Hoefs 1997). Whereas nitrogen and carbon isotopes have only one international standard, oxygen has two: 15N is reported relative to atmospheric nitrogen (AIR); 13C is reported relative to PeeDee Belemnite (PDB), an extinct cephalopod; and 18O is reported relative to both PDB and standard mean ocean water (SMOW) (Koch 2007). As a result, the scales for values from different elements are not directly comparable. Nevertheless, trends in 13C, 15N, and 18O values are the same. If a material has relatively less of the heavy isotope (less 13C, 15N, or 18O), then its value is lower. Conversely, if a substance has relatively more of the heavy isotope (more 13C, 15N, or 18O), its value is higher. Isotope data are typically graphed as bivariate scatter plots. Carbon is conventionally placed on the x-axis, and nitrogen or oxygen on the y-axis.

Interpreting Stable Isotope Ratios in Primates Stable isotope values in a primates tissues can only distinguish isotopically distinct dietary items. They usually cannot be used to determine the relative contribution of a single plant or arthropod species. Nevertheless, stable isotope values can differentiate consumption of plant and animal matter as well as determine the relative contribution of certain plant parts, e.g., leaves versus fruits, or plant functional types, e.g., C4 grasses, succulents, trees with N-fixing symbionts. Dietary Inputs Because isotope values in primates reflect diet, it is important to understand the factors underlying isotopic variation in plants, which comprise the majority of most primate diets. Carbon isotope values mainly reflect plant physiology. Plants use three main types of photosynthetic pathways: C3, C4, and crassulacean acid metabolism (CAM). Most trees, shrubs, and grasses from regions with cool growing seasons use the C3 photosynthetic pathway. Grasses that grow in tropical regions or temperate regions with warm growing seasons use the C4 photosynthetic pathway (Koch 2007; Kohn and Cerling 2002). The majority of leaf succulents, and many epiphytic orchids and bromeliads, use CAM (Keeley and Rundel 2003). These three plant types can be distinguished based on their isotope values. The average 13C values for C3 and C4

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plants are 28.5 (range, 20 to 37) and 14 (range, 12 to 16), respectively (Kohn 2010; Marshall et al. 2007). Most CAM plants can switch between full CAM photosynthesis (night and day cycles) and full C 3 photosynthesis (stomata and fixation both occurring during the day). As a result, CAM plants tend to have 13C values that range between those typical of C3 and C4 plants, depending on the degree of succulence, temperature, and water stress (Marshall et al. 2007). Nitrogen isotope values in plants reflect nutrient availability, plant physiology, and microbial associations (Handley et al. 1999; Marshall et al. 2007; Werner and Schmidt 2002). In the majority of terrestrial systems, plants obtain their nitrogen directly from soil nitrate and ammonium, and their 15N values are greater than AIR (ca. 0). In some systems, particularly moist forests, plant 15N values can be <0 (Handley et al. 1999). Certain plant taxa, including most legumes, have symbiotic bacteria that provide nitrogen fixed directly from the atmosphere. These plants tend to have 15N values close to 0 (Handley et al. 1999). Importantly, plants with Nfixing bacteria do not always fix nitrogen (Codron et al. 2005; Muzuka 1999). Depending on the research question, it is crucial to verify if legumes or other potentially N-fixing plants in the study area do indeed have 15N values close to 0. There may be small 15N differences between sympatric C3, CAM, and C4 plants, as well as between plant parts (Codron et al. 2005, 2006; Werner and Schmidt 2002). For example, CAM plants can have significantly higher 15N values than sympatric C3 or C4 plants (Codron et al. 2006; Crowley et al. 2011b; Muzuka 1999). Oxygen isotope values in plant tissues mirror the 18O of the plants water source. Water sources include surface water fed by sporadic local precipitation as well as more stable groundwater. Although most plant tissues are not enriched in 18O relative to their source water, evapotranspiration can increase 18O values in leaf tissue (Barbour 2007), particularly in arid localities. Oxygen isotope values may be used to distinguish plants that use C3 and CAM/C4 photosynthesis (Marshall et al. 2007); C3 plants are more affected by evapotranspiration than either CAM or C4 plants. Oxygen isotopes combined with 15N and 13C values may be particularly effective at distinguishing CAM consumption, at least in arid localities. This combination of isotopes may also be useful for distinguishing consumption of hemiparasitic parasites, such as mistletoes from the family Loranthaceae, which tend to have lower 13C, 15N, and 18O values than their host plants (Cernusak et al. 2004; Schulze et al. 1991). Because leaf 18O is increased during evapotranspiration, oxygen isotope values may also be able to discriminate, folivores, frugivores, and root-consuming animals (Carter 2001; Cerling et al. 2004; Lee-Thorp et al. 2003). Isotopic Differences Between Primate Tissues and Diet Carbon and nitrogen isotope values in herbivores are higher than those in plants, and 13C and 15N values in faunivores are higher than those in herbivores. Such isotopic differences are referred to as trophic discrimination (Martnez del Rio et al. 2009). More than three decades of research has been dedicated to understanding trophic discrimination. Pioneering work in the 1970s and 1980s determined that trophic discrimination between consumer protein and diet for carbon is ca. 35 for herbivores and 02 for more faunivorous consumers (DeNiro and Epstein 1978;

Stable Isotope Techniques and Applications

Lee-Thorp et al. 1989a; Schoeninger and DeNiro 1984). Trophic discrimination for nitrogen is ca. 3 for both herbivores and faunivores (DeNiro and Epstein 1981; Schoeninger and DeNiro 1984). Finally, trophic discrimination between the 13C values of bone carbonate and diet was determined to be 910 for nonungulate herbivores (DeNiro and Epstein 1978). Researchers have subsequently examined inter- and intraindividual isotopic variability in discrimination factors for captive taxa feeding at similar trophic levels (Hare et al. 1991; Howland et al. 2003; Jim et al. 2004; Passey et al. 2005; Sponheimer et al. 2003b). In general, trophic discrimination estimates generated from these studies resemble those calculated in earlier work. However, they also indicate that there can be substantial variability among taxa and tissues. For example, in captive herbivores, discrimination between tooth enamel 13C and diet ranges from 11.3 to 14.6 (Passey et al. 2005), and discrimination between fur keratin and diet ranges from 2.7 to 3.5 and 3.0 to 6.5 for 13C and 15N, respectively (Sponheimer et al. 2003a,b). This variability most likely results from differences in digestive physiology and the manufactured nature of provisioned diets, e.g., protein content and C3- versus C4-derived foods. A small but valuable data set on trophic discrimination factors exists for wild animals. Estimated discrimination between tooth enamel and plant 13C values for large-bodied ruminants (ca. 14.1) is similar to measured values in captive herbivore taxa (Cerling and Harris 1999); however, this group differs from most primates in both diet and physiology. Probable trophic discrimination factors for small Malaysian mammals also agree with those calculated for captive animals: 13C and 15N values for clipped toes and fur keratin from herbivores are ca. 2.4 and 2.1 higher than isotope values in plants (their presumed diet), and 13C and 15N values for faunivores exceed those for herbivores by ca. 2.0 and 2.5, respectively (Hyodo et al. 2010). Using these values, one can predict trophic discrimination factors for primate tissues such as keratin (Table I). Measured keratinplant discrimination factors for nitrogen agree well with these predictions: Nitrogen isotope values for herbivorous Propithecus edwardsi and omnivorous Microcebus spp. are ca. 3.6 and 5.9 higher than those in plants, respectively. However, keratinplant discrimination factors for carbon are unexpectedly higher for Propithecus edwardsi (ca. 7.2) than Microcebus spp. (ca. 5.0) (Crowley et al. 2011b; McGee and Vaughn 2003). These results illustrate the need for more isotope data to estimate trophic discrimination factors accurately for primates. In addition to diet, 15N values in primates can reflect nutritional stress. The 15N values of an animals tissues reflect both nitrogen assimilation and loss. Anabolic tissue building preferentially incorporates 15N over 14N. When an animal consumes a protein-rich diet, it does not use all of the dietary amino nitrogen to build body tissue. Excess 15N-depleted amine groups are excreted as urine (Hedges and van Klinken 2000; Koch 2007; Martnez del Rio et al. 2009). On low-protein diets, an animal uses most of its dietary nitrogen to build body tissues. As a result, more 14N is incorporated into body tissues rather than excreted. Finally, if an animal does not consume sufficient nitrogen to meet metabolic requirements, it will begin to break down body tissue proteins and reincorporate liberated amino acids into metabolically expensive tissues (Koch 2007; Martnez del Rio et al. 2009). These liberated amino acids are 15N enriched relative to dietary amino acids. Importantly, nitrogen loss and

B.E. Crowley Table I Hypothetical trophic discrimination patterns for primate fur keratin

Carbon and nitrogen isotope values in primates reflect their diet, increasing with increasing trophic level. For illustrative purposes trophic discrimination values are presented for fur keratin from Malaysian small mammals (Hyodo et al. 2010), and 15 N values are based on a legume-derived diet. Carbon isotope values for C3 and C4 plants are based on global averages (Kohn 2010; Marshall et al. 2007) while the 13 C value for CAM plants is based on succulents from southwest Madagascar (Crowley et al. in press)

related increases in body 15N do not happen uniformly in all tissues. During brief periods of food shortage, protein recycling may have an isotopic effect only on excreta or tissues with rapid metabolic turnover, e.g., urine, blood. However, prolonged fasting, i.e., starvation, should result in 15N enrichment of all proteinaceous tissues, including those with slow turnover rates (Gannes et al. 1998; Martnez del Rio et al. 2009). Oxygen isotope values in an animal predominantly reflect the water it drinks, water in the foods that it eats, and metabolically released oxygen (Bryant and Frlich 1995; Fricke and ONeil 1996; Luz and Kolodny 1985). Therefore, dietary signals can be complicated by both environmental conditions and the variable intake of water from food versus drinking (discussed in detail later). Nevertheless, within a particular habitat, 18O values may be able to differentiate primates that feed at different trophic levels; faunivores tend to have lower 18O values than sympatric herbivores, particularly in arid habitats (Sponheimer and Lee-Thorp 2001). Lower 18O values in faunivores may result from the consumption of 18O-depleted animal fats and proteins or from more frequent drinking in faunivores than sympatric herbivores (Sponheimer and Lee-Thorp 2001). Importantly, internal body temperature and metabolic rate can have a substantial effect on animal 18O values (Koch 2007). To interpret 18O values correctly, body temperature should be ca. 37C and metabolic rate should be relatively stable (Bryant and Frlich 1995). Although these conditions are met for most primates >1 kg, they are less likely to hold for species that are <1 kg, particularly those that undergo daily or seasonal torpor. Interpreting Habitat Regional and local abiotic and biotic factors affect the isotope values in plants, and these effects are passed on to animal consumers. Rainfall is the primary regional

Stable Isotope Techniques and Applications

factor that influences the 13C values in C3 plants, but temperature and elevation may have secondary effects (Kohn 2010). Moist, cool habitats at high elevations exhibit lower 13C values than hot, dry habitats at low elevations. Carbon isotope values in C4 plants are not affected by these factors (Swap et al. 2004). However, primates inhabiting coastal localities may have high 13C values if they consume marine foods. Coastal seagrasses tend to have high 13C values, similar to C4 plants (Koch 2007). As a result, primates that eat marine-derived foods (such as coastal invertebrates) could exhibit 13C values resembling those of a C4 consumer. In most cases, it will be easy to dismiss either C4 or marine consumption based on behavioral data or the floral composition of the locality. Lastly, canopy density, relative humidity, light availability, tree height, and soil moisture can all affect the 13C values of C3 plants at a more local scale (Heaton 1999; van der Merwe and Medina 1989). In open settings, plants obtain their carbon from atmospheric carbon dioxide (CO2). In dense forest settings with limited air circulation, a portion of the available carbon comes from 13C-depleted CO2 derived from soil decomposition and microbial respiration (Heaton 1999; van der Merwe and Medina 1989). In addition, moist and shady conditions lead to lower 13C values in understory plants (Heaton 1999). Collectively, these factors can help distinguish C3 plants growing in more open woodlands, grasslands, or exposed forest canopies from those growing in more closed-forest understory environments (Fig. 1). Whereas C3 plants growing in open, dry, sunny habitats have relatively high 13C values (21 to 27), understory plants can exhibit 13C values < 31.5 (Kohn 2010). This isotopic pattern, frequently called the canopy effect, is also apparent for animals living in these respective microhabitats (Schoeninger 2010). Regionally, increasing temperature and decreasing rainfall have been related to increasing 15N in C3 plants (Handley et al. 1999; Swap et al. 2004). However, nitrogen isotope values in C4 plants do not appear to correlate with climatic variables (Swap et al. 2004). Locally, relative humidity; rooting depth; mycorrhizal associations; and soil moisture, chemistry, and age can all affect 15N values in C3

Fig. 1 Foliar carbon, nitrogen, and oxygen isotope values reflect microhabitat. Width of shaded triangles indicates increasing aridity; temperature; and 13C, 15N, and 18O values. Moister, cooler localities have lower 13C, 15N, and 18O values than drier, warmer, more open localities. In forested localities, 13C and 18O values tend to increase with increasing distance from the forest floor.

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plants (Handley et al. 1999; Marshall et al. 2007; Muzuka 1999). The mechanisms behind these patterns are not completely understood. However, they are likely related to changes in the N sources (NO3, NH4+, dissolved organic nitrogen) that are available to plants as well as the rate of processes such as nitrification and ammonia volatilization (Marshall et al. 2007; Muzuka 1999). This habitat-driven variability in plant 15N values is reflected in the primates that eat them. As a result, primates consuming plants from hot, dry habitats exhibit higher 15N values than primates consuming plants from cool, moist habitats (Crowley et al. 2011b; Schoeninger et al. 1997). Lastly, plants from marine and coastal localities tend to have very high 15N values (Muzuka 1999; Schoeninger and DeNiro 1984). Primates living in coastal localities consuming marine-derived nutrients, e.g., crab-eating Macaca fascicularis, could, therefore, have exceptionally high 15N values (Koch 2007). Evaporative 18O enrichment related to differences in degree of irradiance, relative humidity, and rooting depth may be able to distinguish between leaves growing in open vs. closed habitats (Barbour 2007). Within a forest, 18O values may be higher in canopy than forest floor foliage (Fig. 1; Sternberg et al. 1989). Such patterns can be used to both quantify and locate primate foraging in a heterogeneous habitat. In addition to reflecting diet, the 18O values in an animal are strongly affected by drinking water (Bryant and Frlich 1995; Luz and Kolodny 1985). Herbivores that frequently drink water have lower 18O values than those that obtain the majority of their water from their food (Levin et al. 2006). These differences can be particularly pronounced in arid environments. The isotopic composition of drinking water sources varies with climate and the local topography (Dutton et al. 2005). The 18O values of precipitation reflect both the type of precipitation and where it forms: Rain has higher 18O values than snow (Dansgaard 1964). Cooler regions, e.g., higher latitudes and elevations, tend to have precipitation with lower 18O values than warmer regions, e.g., lower latitudes and elevations, regardless of precipitation type (Dansgaard 1964; Dutton et al. 2005). Moreover, evaporation associated with low relative humidity and high ambient temperature can increase the 18O values of drinking water sources as well as plants (Marshall et al. 2007). Thus, 18O values can be used to discriminate between animals living in dry, hot environments and animals living in cool, moist environments. Isotope Summary Stable carbon, nitrogen, and oxygen isotope values reflect dietary inputs and can distinguish faunivores from herbivores as well as frugivores from folivores. Stable isotope values can also reflect the type of biome inhabited by an individual. A primate from a dry, hot, or open habitat should have higher 13C, 15N, and 18O values than a primate from a moist, cool, or closed habitat, even if the two share a similar diet (Crowley et al. 2011b; OBrien and Wooller 2007; ORegan et al. 2008). Even so, because climate and local biotic factors can have dramatic impacts on plant isotope values, 13C, 15N, and 18O differences among dietary categories are valid only within a particular locality. Unless it is possible to account for baseline isotopic variability among localities, i.e., by analyzing the same animal species or plants from different habitats, avoid using isotope values to infer dietary differences between two habitats or regions.

Stable Isotope Techniques and Applications

Methods for Conducting Isotopic Research Planning a Project Accurate interpretation of isotope values requires the establishment of baseline isotope variability for the habitat(s) and time period(s) of interest. Collecting data from a single study population will be useful only if comparative data already exist. When and what is collected will be dictated by the research question. For example, if the goal is to quantify if the consumption of arthropods changes from season to season, it will be critical to sample a primate tissue that records short-term dietary changes, possibly more than once, i.e., during the seasons of interest. In addition to primate tissues, it will also be crucial to collect representative samples of the main food resources, e.g., both plants and arthropods. If no foods are available, e.g., when dealing with fossils, then the best approach may be to compare data from one or more sympatric mammal species with known or predictable diets, e.g., carnivores, browsing or grazing herbivores, to the primate species of interest. Although this method provides less detailed information about an animals feeding ecology, it still provides important baseline comparative data. This may be the only approach when investigating the isotope ecology of an extinct species (Crowley et al. 2011a; Lee-Thorp et al. 1989b; Nelson 2007). Isotopic applications should be used to complement, rather than replace, other investigative approaches, including observational research. Depending on the question, it may or may not be necessary to include data from more than one isotope. Although analyzing samples for 13C, 15N, and 18O can quickly become expensive, most mass spectrometers allow for measuring both organic carbon and nitrogen isotope ratios from a single sample. Similarly, both carbon and oxygen data can be obtained from the carbonate portion of tooth enamel or bone hydroxyapatite, [Ca10(PO4, CO3)6(OH, CO3)2]. Characterizing Habitat and Diet When investigating the foraging ecology of a modern primate species, it is preferable to collect vegetation samples from all habitats of interest. For example, if the study group inhabits both woodland and grassland, then representative vegetation should be collected from both habitats. If the population inhabits a tropical rain forest, try to sample vegetation from multiple distances above the forest floor, including the height where the individuals forage. Sampling canopy vegetation may require climbing trees or employing extendable loppers (B. E. Crowley, pers. obs.). It may only be possible to sample canopy vegetation from very tall slender trees using a slingshot (N. Dominy, pers. comm.) or a canopy walkway (Herrera et al. 2001). The vegetation sampling scheme should reflect the research question. When investigating short-term seasonal or interannual dietary changes, samples from both animals and vegetation should be collected concurrently. Conversely, when investigating long-term dietary trends, it is less critical that animals and vegetation are sampled at the same time. Collecting samples from multiple seasons or years may be necessary. It is advisable to collect pilot data whenever possible. This will help ascertain how much isotopic variability there is within the system of interest. In

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general, arid and very seasonal localities exhibit more isotopic variability than perennially moist localities. Avoid using vegetation data from other localities. If the goal is simply to characterize habitat, choose several tree species (ideally food species) that are widespread throughout the habitat, and collect leaves from multiple individuals of each species. Collect several leaves from each individual (Codron et al. 2005), selecting only fully mature leaves. When sampling primate foods, you may choose to select representative samples from throughout the study area (Codron et al. 2005; Herrera et al. 2001), or to analyze only partially consumed items that were discarded by the primates (Rothman et al. 2011). Arthropods can be collected using light traps, nets, or by hand-selecting nonvolant individuals (Herrera et al. 2001; Hyodo et al. 2010). Because plants and arthropods can both exhibit substantial interindividual variability (Barbour 2007; Cernusak et al. 2004; Codron et al. 2006; van der Merwe and Medina 1989), it is advisable to collect multiple specimens for each food item. Because most primate research takes place in remote localities that are visited only periodically, a good rule of thumb is to collect more samples than will probably be necessary to address the research question. Pilot analyses will determine if all of the collected samples need to be analyzed. If there is minimal isotopic variability within or among food resources, only a subset of the collected samples will be required. Conversely, if the locality exhibits substantial isotopic variability or some of the samples are lost due to mold or insect damage, it can be very valuable to have additional samples on hand. Plant and arthropod samples can either be air-dried or dried at low heat (<60C) in an oven (Codron et al. 2005; Herrera et al. 2001, 2006; Hyodo et al. 2010). Because mold can alter stable isotope values, it is critical to dry samples completely (Barrow et al. 2008). Further, to maintain dryness, samples collected at humid localities should be stored in airtight containers with desiccant. To measure 18O values in plants, most researchers use either intercellular water or cellulose. Collecting plant water in the field is a difficult task and is beyond the scope of this paper. Details about collecting and extracting plant water are available elsewhere (Cernusak et al. 2004; Peters and Yakir 2008). Fortunately, the 18O values of dried plant material and plant water are correlated (Barbour 2007; Cernusak et al. 2004). It may therefore be possible to use dried plant material to assess 18O differences among food items (Barbour 2007; Cernusak et al. 2004). Water content should be estimated by recording both the fresh wet mass and the dried mass. Homogenize plant and arthropod samples using a mill or an agate mortar and pestle (Herrera et al. 2006; Hyodo et al. 2010). Adding small amounts of liquid nitrogen makes samples brittle and easier to crush. Choosing a Primate Tissue for Analysis Animal tissues vary in the ecological information that they yield because 1) the biological composition of tissues varies, and 2) isotope values in tissues reflect varying lengths of time. First, compositional differences result in isotopic variability among tissues. Consequently, isotope values from these different tissues are not directly comparable. For example, whereas 13C values in bone and tooth enamel carbonate reflect a mix of dietary proteins, lipids, and carbohydrates (Ambrose and Norr 1993; Jim et al. 2004; Passey et al. 2005), feeding and theoretical studies suggest that the 13C values in proteinaceous tissues, e.g., blood, collagen, and

Stable Isotope Techniques and Applications

keratin, may be biased toward dietary protein rather than bulk diet (Ambrose and Norr 1993; Froehle et al. 2010; Hedges and van Klinken 2000; Jim et al. 2004; Martnez del Rio et al. 2009). Carbon isotope values in tooth enamel and bone mineral can be affected by digestive physiology (Cerling and Harris 1999; Passey et al. 2005). Fortunately, physiological differences among primates do not appear to affect carbon isotope values in bone mineral (Crowley et al. 2010). The 13C values in primate bone collagen tend to be ca. 5.6 lower than those in bone carbonate (Crowley et al. 2010). This difference can be used as a correction factor that can be applied when comparing bone carbonate and collagen 13C values. It is unclear if, or to what degree, 18O might differ between the organic and mineral portions of bones and teeth (G. Bowen, pers. comm.). Differences in 13C and 18O between primate bone and enamel carbonate have not been measured, but it is likely that the two are isotopically similar (Luz and Kolodny 1985; Lee-Thorp and van der Merwe 1991). Small biochemical and metabolic differences can lead to isotopic variability among proteinaceous tissues (Hare et al. 1991), particularly for oxygen (Tuross et al. 2008). For example, 18O values in blood are on average ca. 2 higher than those in keratin and 45 higher than those in collagen for captive pigs. On the other hand, differences in 13C or 15N values among proteinaceous tissues are small (1) and relatively similar among very diverse primate taxa (Crowley et al. 2010). Isotope values in body tissues and excreta reflect the length of time over which they are synthesized. Feces, urine, and blood plasma reflect dietary intake over several days while muscle and whole blood cells integrate several weeks of dietary information. Bone collagen and carbonate synthesis reflects a much longer time period, averaging several years of an animals diet (Barboza and Parker 2006; Gannes et al. 1998; Sponheimer et al. 2003a). Tooth enamel and fur keratin are metabolically inert tissues; once they form, they are not remodeled (Koch 2007). Using serial samples from a metabolically active tissue, such as blood, or an ever-growing incremental tissue, such as keratin, stable isotopes can clarify seasonal dietary or habitat changes as well as individual and population level dietary specializations (Bearhop et al. 2004; Nelson 2007; OBrien and Wooller 2007; Sponheimer et al. 2003b, 2006b). In summary, the choice of tissue depends largely on the specific application of interest. Understanding these patterns is critical for valid research results. Procedures are not yet formally standardized for collecting and preparing samples for isotopic analysis. There are, however, some general requisite guidelines to follow. For example, account for potential environmental variability as much as possible. Given the potential for substantial isotopic variability, multiple individuals (ideally >10) should be sampled (Levin et al. 2006). The number of individuals required depends on the ecology of the species of interest, the research question, and the tissue that is analyzed. Tissues that record only short periods of time, e.g., blood and keratin, tend to be more isotopically variable than tissues that incorporate longer periods of time, e.g., bone collagen and carbonate. A firm understanding of a species dietary proclivities and tissue growth patterns, e.g., continuous hair growth vs. seasonal molting, will help inform sampling strategy. Because temporal resolution cannot be constrained to the same degree in fossil animals, sampling multiple individuals is particularly important. In addition to identifying variability in the species diet or habitat,

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sampling multiple individuals is critical for detecting fossil specimens that may be isotopically altered (Kohn and Cerling 2002). Another good strategy is to minimize the number of variables that might affect isotope values. For example, it is important to account for potential age- and sexrelated differences whenever possible. Depending on the species, males and females may differ in their dietary intake, which can result in isotopic differences (Smith et al. 2010). Infants and juveniles tend to have elevated 15N but lower 13C and 18O values (Koch 2007; Luz and Kolodny 1985; Smith et al. 2010). Unless pursuing questions about age-related isotopic patterns, it is best to sample only adult individuals or tissues that formed post weaning, e.g., permanent molars. In addition, try to collect samples from the same body part, e.g., blood from the brachial vein, fur from the base of the tail, enamel from the third molar tooth. To prevent decay and mold growth, samples should be thoroughly dried or frozen immediately after collection (Barrow et al. 2008). Avoid samples that have been stored in chemical preservatives, such as formalin, because they can alter the isotopic composition of a tissue and are difficult to remove. Many preparation steps do require adding chemicals. Care should be taken to remove these chemicals by thoroughly rinsing samples with ultrapure water, e.g., double distilled or passed through a deionizer. To avoid cross-contamination among samples, always thoroughly wipe clean supplies and equipment using lint-free tissues, e.g., Kimwipes, with a solvent such as methanol, ethanol, or acetone between samples. The sample collection and preparation methods presented in the text that follows are outlined in the electronic supplementary material ([ESM] Tables SI and SII). Initial sample masses should be considered minimum estimates. In addition to standard laboratory apparatus, sample preparation requires items specific to stable isotope analysis (Tables SIII and SIV). Analysis of Tooth Enamel Carbonate (13C and 18O) Tooth enamel is composed mainly of hydroxyapatite, [Ca10(PO4, CO3)6)(OH, CO3)2]. It is highly crystalline, nonporous, and contains only a small percentage of organic material (Kohn and Cerling 2002). Tooth enamel thus preserves its biogenic isotopic signature with high fidelity (Kohn and Cerling 2002; Lee-Thorp and van der Merwe 1991). Once a tooth mineralizes, its 13C and 18O values do not change for the remainder of the animals life. Depending on the species, tooth enamel represents the first few months or years of an individuals life. Carbon isotope values in enamel carbonate reflect a combination of dietary macronutrients (Passey et al. 2005). Sample Collection and Preparation Unless questions about infant diet or weaning are explicitly being addressed, care should be taken to only sample teeth that mineralized after weaning, e.g., permanent molars. Teeth that mineralize before weaning will reflect in vitro or milk-derived nutrients and water sources (Fricke and ONeil 1996). Enamel should be separated from the dentine portion of the tooth and finely powdered, preferably using a dremel equipped with a dental drill bit (Cerling et al. 2004; Fourie et al. 2008). Before beginning sample preparation, contact the laboratory where samples will be analyzed to determine the minimum sample size required for analysis. When dealing with small primate teeth, the entire primate tooth will

Stable Isotope Techniques and Applications

probably be needed. It is optimal to powder enough sample to perform at least two analyses. Remember that some sample will be lost during sample preparation. Oxygen isotope values can be measured from either the carbonate or phosphate fraction of hydroxyapatite [Ca10(PO4, CO3)6(OH, CO3)2]. The 18O values of these two fractions are related but not identical (Kohn and Cerling 2002). In general, the phosphate fraction can be analyzed using smaller amounts of sample, but the carbonate fraction is easier to prepare, and carbonate yields both oxygen and carbon isotope data (Kohn and Cerling 2002). Here I discuss the methods used to isolate biogenic carbonate for isotopic analysis. For a discussion of phosphate analysis, see Kohn and Cerling (2002). Laser ablation, releasing CO2 gas directly from the enamel surface, is a promising technique for small or very valuable samples (Sponheimer et al. 2006b). It requires only a minute amount of sample and is considerably less destructive than other sampling methods. Using ablation, seasonal or annual variability can be documented within a single tooth (Sponheimer et al. 2006b). Unfortunately, this method neither allows for the removal of any potential contaminants nor, because of the procedure, can it be employed outside of a few laboratories with the requisite equipment (Kohn and Cerling 2002). Several published methods exist for isolating the carbonate portion of enamel hydroxyapatite for isotopic analysis. I recommend following the carefully tested protocol of Koch et al. (1997). First, powder enamel samples using a dremel equipped with a dental drill bit. Place powdered samples into 1.5- or 20-ml microcentrifuge tubes and record both the mass of the samples and tubes. This is critical for later comparison to final yield. Next, oxidize organic material by adding 23% sodium hypochlorite (NaOCl) to each powdered sample. Follow the ratio of 0.5 ml per 10 mg of enamel. Agitate the samples using a vortex device to ensure thorough mixing, then leave samples for 24 h at room temperature. The samples will release a considerable amount of gas, so ensure that the lids are loose. Centrifuge the samples for 45 min and then remove the NaOCl using a pipette. Thoroughly rinse the samples by adding 11.5 ml of ultrapure water and agitating samples. Now centrifuge samples a second time and remove the water. Repeat the rinse process five times. Next, remove nonlattice-bound carbonates. Add 1 M acetic acid buffered to pH 5 with calcium acetate (following the ratio of 0.5 ml per 10 mg of enamel), agitate, and allow samples to stand at room temperature for 24 h. Centrifuge the samples and remove the buffered acid. Rinse samples five times with ultrapure water following the procedure outlined in the preceding text. After samples have been thoroughly rinsed, they should be lyophilized for 12+ h. Record the new mass of each sample + microcentrifuge tube to determine how much sample was lost during sample preparation. Store samples in a desiccator after they have been dried. Applications Although isotopic patterns in tooth enamel have been applied routinely to populations of humans and our hominin ancestors (Schoeninger 2010), surprisingly little isotopic work has been conducted on tooth enamel in nonhuman primates, possibly because the teeth of most primates are small with relatively thin enamel. Nevertheless, isotopic data gathered to date are promising. For example, enamel 18O values from an historic population of Liberian chimpanzees (Pan troglodytes) suggest

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that the age at weaning was older in males than females (Smith et al. 2010). At Ituri, a rain forest in the Congo, enamel 13C values delineate animals feeding at different levels in the forest canopy, and enamel 18O values distinguish folivorous colobines from more frugivorous taxa (Cerling et al. 2004). Enamel 13C values have mostly been used to discriminate between diets dominated by C3 and C4 plants. Because these two plant groups have such fundamentally different 13C values, a simple mixing model can be employed to calculate the relative percentage of C3 and C4 consumption. For example, Lee-Thorp and colleagues (1989b) used enamel 13C values to verify that Papio ursinus consumes some C4 resources in South Africa. Interpreting 13C values for fossils requires some assumptions about plant isotope values. Nevertheless, this approach has been useful for distinguishing C3 and C4 consumption among fossil African cercopithecoids (Fourie et al. 2008; Lee-Thorp et al. 1989b). It has also been informative for interpreting early hominin diets (Lee-Thorp et al. 2003). Isotopic evidence suggests that australopithecine diets more closely resembled those of baboons rather than chimpanzees. Like baboons, both robust (Paranthropus robustus, P. boisei) and gracile (Australopithecus africanus) australopithecines may have been opportunistic dietary generalists regularly exploiting C4 resources (Cerling et al. 2011; Lee-Thorp et al. 2003). Distinguishing C3 and C4 plant consumption could also prove useful when applied to other fossil ape or monkey species but only for the last ca. 8 million years. Earlier than this, C4 plants were not abundant (Kohn 2010). Because each tooth erupts at a slightly different time and takes several months to fully erupt and mineralize, it may be possible to use isotope values in enamel hydroxyapatite to track seasonal or annual dietary shifts within an individual (Fricke and ONeil 1996; Kohn et al. 1998). This could be achieved by 1) measuring and comparing isotope values from teeth that erupted at slightly different times, or 2) sequentially sampling the same tooth via laser ablation. Although this method has yet to be applied to modern nonhuman primates, variability in enamel 18O values has been used to infer seasonality for Paranthropus and Sivapithecus (Nelson 2007; Sponheimer et al. 2006b). As demonstrated by Cerling et al. (2004), combining enamel 13C and 18O values can differentiate detailed ecological differences among sympatric taxa. This dual isotope approach has been used to answer questions about both the diet and paleoenvironment of fossil species (Lee-Thorp et al. 2003; Nelson 2007; White et al. 2009). For example, the combination of low 13C values and high 18O values in Sivapithecus sp. relative to sympatric taxa suggests that this extinct ape lived in a forested environment and fed on upper canopy vegetation (Nelson 2007). Similarly, a combination of low 13C and intermediate 18O values relative to sympatric taxa suggests that Ardipithecus ramidus consumed a range of C3 plant foods, and possibly animals, in a dry woodland environment (White et al. 2009). Analysis of Bone Carbonate (13C and 18O) Similar to tooth enamel, the 13C values of bone carbonate reflect a mix of dietary lipids, carbohydrates, and proteins (Jim et al. 2004). Oxygen isotope values reflect both diet and drinking water (Koch 2007). However, two major differences distinguish enamel and bone carbonate. First, bone is more porous, less crystalline,

Stable Isotope Techniques and Applications

and more organic than tooth enamel, resulting in bone being more prone to diagenetic alteration (Kohn and Cerling 2002; Lee-Thorp and van der Merwe 1991; Nelson et al. 1986; Tuross et al. 1988). Unless sample alteration can be assessed using a technique such as X-ray diffraction or Fourier transform infrared spectroscopy, analyzing carbonate in subfossil or fossil bones is not recommended (Koch et al. 1997). Second, unlike enamel, bone is continually remodeled during an individuals lifetime (Gannes et al. 1998). As a result, bone carbonate 13C and 18O values for an adult individual reflect the adult diet while enamel isotope values reflect the infant or juvenile diet, regardless of the individuals age. Sample Collection and Preparation Pretreatment methods for bone carbonate vary both in oxidizing reagent and the acid concentration. Because sodium hypochlorite can alter the 13C value of bone carbonate (B. E. Crowley, pers. obs.), I advocate following the protocol presented in Clementz et al. (2009), which uses hydrogen peroxide. Start with 20 mg of flesh-free, dry bones. Grind bone samples into a fine powder using either an agate mortar and pestle or a dremel equipped with a dental drill bit. Next transfer the powdered samples into a 1.5- or 2-ml microcentrifuge tube and record the combined mass of the sample and the empty tube. First, oxidize organic materials by adding 30% laboratory-grade hydrogen peroxide (H2O2) to the powdered bone samples. Follow the ratio of 0.5 ml solution per 10 mg of bone. Thoroughly mix the samples using a vortex device so that they are well mixed, and leave them, lids loose, for 48 h at room temperature. Agitate the samples two or three times to confirm that they stay well mixed. After 48 h, all bubbling should have ceased. Once the samples have completely oxidized, centrifuge and carefully remove H2O2 using a pipette. Add 11.5 ml of ultrapure water, agitate and centrifuge the samples, and then remove the first water rinse. Repeat this rinse process five times. Next, remove nonlattice-bound carbonates. Add 1 M acetic acid (buffered to pH 5.0 with calcium acetate) to each sample, following the ratio of 0.5 ml per 10 mg of sample. Thoroughly mix the samples and allow them to react for 24 h at 4C. Centrifuge, remove the buffered acid, and rinse samples five times with ultrapure water following the procedure outlined in the preceding text. After samples have been rinsed, they should be lyophilized for 12+ h. Record the mass of each dried sample + microcentrifuge tube. This documents how much material was lost during preparation and may help determine if the samples are contaminated or altered. Store the samples in a dry place, preferably a desiccator. Applications Bone carbonate is more obtainable than tooth enamel, and is, therefore, preferable for quantifying ecological differences among sympatric species. Yet surprisingly, only two studies have used isotope values in bone carbonate to examine the feeding ecology of nonhuman primates (Carter 2001; ORegan et al. 2008). Neither of these studies applied both 13C and 18O values. Carbon isotope values in bone carbonate have revealed dietary variability both within and between populations of Macaca mulatta. Whereas tropical populations consume similar C3based diets, a population living in a more temperate climate exhibits greater dietary diversity, possibly in response to a more seasonal or stressful environment (ORegan et al. 2008). Oxygen isotope values in bone carbonate have been used to distinguish sympatric forest-dwelling African primates consuming a variety of foliage and fruits (Carter 2001).

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Analysis of Bone Collagen (13C and 15N) Carbon isotope values in bone collagen reflect a mix of dietary macronutrients, but are biased toward dietary protein (Froehle et al. 2010; Hedges and van Klinken 2000; Jim et al. 2004). Nitrogen isotope values in collagen reflect dietary protein (Hedges and van Klinken 2000; Martnez del Rio et al. 2009). Similar to bone carbonate, collagen is continuously regenerated during an individuals lifetime (Gannes et al. 1998). As a result, isotope values in bone integrate several years of dietary and habitat information. Isotope values in bone collagen are not useful for examining short-term changes in diet or habitat. On the other hand, they may be helpful for determining long-term dietary differences among individuals or between populations. Sample Collection and Preparation Bone should be dried and flesh-free. To isolate collagen, modern bones must be demineralized and degreased. Removing lipids is critical because they have lower 13C values than proteins (Jim et al. 2004). Older bones, e.g., subfossils and fossils, need to be demineralized but generally do not retain enough lipids to require degreasing (Clementz et al. 2009; Tuross et al. 1988). Two chemicals are frequently used to decalcify bone: dilute hydrochloric acid (HCl) and ethylenediaminetetraacetic acid (EDTA), each with both pros and cons. Whereas HCl treatment is quicker, EDTA yields more collagen. EDTA may also remove potential contaminants such as humic acids that can accumulate in samples that were buried for long periods of time (Tuross et al. 1988). The two yield similar isotope values for modern and well-preserved fossil collagen (Tuross et al. 1988). To prepare modern bone, coarsely crush 3050 mg of defleshed bone using a mortar and pestle and place crushed samples into 5-ml glass scintillation vials. Add 5 ml of 0.5 N HCl to each sample and allow the samples to react for 2448 h at 4C (Clementz et al. 2009). Allow more decalcification time if samples are still hard when checked with a clean probe. Remove the HCl, add fresh acid, and let the samples react at 4C for an additional 2448 h. Once the samples are pliable, they are completely decalcified. After removing HCl from samples using a pipette and rinse them five times with ultrapure water, centrifuging between rinses if necessary. Next, thoroughly dry samples using a freeze drier. To remove lipids from the samples, add 5 ml of petroleum ether to each sample and cover the scintillation vials with foil. Because petroleum ether is extremely toxic and volatile, all steps involving this solvent must be completed under a fume hood. Avoid using a chloroformmethanol mixture to extract lipids. This solvent is not lipid specific and can remove substantial amounts of protein (Dobush et al. 1985). Sonicate samples for 15 min and carefully pipette off the petroleum ether and any liberated lipids. Repeat this process until lipids are no longer released from the samples (usually three to five cycles). Then rinse samples five times using ultrapure water and lyophilize them for 12+ h. To isolate collagen from older bone and subfossil specimens, it is best to use cortical bone, e.g., a long bone shaft, which is dense and relatively resistant to diagenetic alteration. Because mass loss can be substantial during sample preparation, it is ideal to start with at least 150 mg of bone. Do not mix bones from more than one individual to reach 150 mg. If mandibles or maxillae are sampled, make sure that all teeth are removed, including dentine roots. If the surface of the bone shows signs of remineralization or is highly stained, carefully clean the surface using sandpaper or a dremel. If a

Stable Isotope Techniques and Applications

sample contains exogenous material such as dirt, briefly sonicate the sample in ultrapure water and then lyophilize the sample. Using a mortar and pestle, coarsely crush bone samples, record the mass of each sample, and place the fragments in a 15-ml plastic centrifuge tube with a screw-cap lid. This is an opportune time to gauge the quality of each sample. Does the bone break easily or crumble into dust? Does it sound like it might be made out of porcelain? If the answer to any of these questions is yes, then it is unlikely that this particular sample will yield any collagen (Tuross et al. 1988; B. E. Crowley, pers. obs.). If, however, the bone is difficult to break and does not appear to be completely permineralized, then it may produce a good collagen yield. Collagen extraction methods for older bone are modified from Hare et al. (1991). These methods, developed by Kena Fox-Dobbs, are briefly presented in Fox-Dobbs et al. (2006) and Crowley et al. (2011a). To demineralize older bone, add 14 ml of 0.5 M EDTA (pH 8) to each centrifuge tube. Sonicate the samples for 1520 min, and then allow them react at 4C for 7 d. Periodically invert the tubes so that the EDTA solution stays well mixed. After 7 d, decant the solution, add fresh EDTA, sonicate, and allow the samples stand at room temperature for 10 additional days. Using clean forceps or a probe, periodically check to see if the sample fragments are still hard or if they are pliable and gummy. If samples are still hard after 10 d at room temperature, add fresh EDTA and sonicate the samples a third time. Once samples are pliable, they have been demineralized. If a sample is still hard or crunchy after 20+ d at room temperature, it will probably not yield any collagen. It is very important to completely remove all EDTA from the collagen residue. This requires rinsing the samples with ultrapure water 10 times, allowing the samples to stand in ultrapure water overnight, and sonicating the samples after the 2nd and 9th rinses. Unlike modern samples, older demineralized bone fragments can sometimes lose their structure and turn into a thick goo (B. E. Crowley, pers. obs.). If this occurs, centrifuge samples at a high speed for 5 min to minimize loss of small particles and rinse samples very carefully. Some authors soak decalcified samples in dilute NaOH to remove organic acid contaminants (Ambrose 1990). This step can be risky. Soaking in NaOH may, indeed, remove contaminants, but it might also leave salt residues (Ambrose 1990) or completely dissolve the sample (B. E. Crowley, pers. obs.). Because EDTA can also remove humic and fulvic acids, exposure to NaOH is unnecessary (Tuross et al. 1988). After rinsing, samples should be lyophilized for 12+ h. To remove any remaining noncollagenous residues, samples need to be gelatinized and filtered. Label 10-ml glass culture tubes using a permanent marker and cover the writing with a piece of clear tape so the writing does not rub off. Transfer the dried samples into preweighed and labeled 10-ml glass culture tubes, fill the tubes with 0.01 N HCl, loosely cover the tubes (to prevent evaporative moisture loss), and heat the samples at 57C for 1215 h on a heating block. Depending on the amount of collagen, samples may not be completely dissolved. Using a vacuum, pull the sample solution through a 1.5-m glass fiber filter, e.g., Whatman 934-AH. Retain the filtered solution and discard removed solids. Some authors choose to ultrafilter samples (using a Centriprep centrifugal ultrafilter) to isolate the collagenous residue that is >30 kDa (Fox-Dobbs et al. 2006). The benefit of ultrafiltration is that many noncollagenous proteins are removed. This step is particularly useful if samples will be radiocarbon dated. The disadvantages of ultrafiltering are that it is time consuming, expensive, and there is a chance that the ultrafilter will contaminate the samples. Ensure that the ultrafilters

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have been thoroughly cleaned. Force ultrapure water through the filter membranes using a centrifuge and sonicate several times. After filtering, lyophilize the samples. Record the final mass of each dried sample + culture tube and calculate the final sample yield. In combination with C:N ratios and 13C and 15N values, sample yield helps assess collagen integrity (Ambrose 1990). Store samples in a cool, dry place. Applications Researchers have used carbon and nitrogen isotope values in bone collagen to examine sex- and age-related patterns in Pan troglodytes (Smith et al. 2010), as well as to establish inter- and intrapopulation isotopic variability for baboons (Papio cynocephalus ursinus) and macaques (Macaca mulatta) (ORegan et al. 2008; Thackeray et al. 1996). Documenting this variability establishes baseline isotopic variability among habitats and enables a quantitative comparison of dietary differences among populations. These patterns can then be used to better understand the diets and habitats of extinct species. For example, collagen 13C values have shed light on the dietary preferences of extinct lemurs from southwestern Madagascar. The extinct taxa occupied a variety of niches that differed from both extant lemur and nonlemur taxa. Several species regularly consumed C3 plants, and one species, Hadropithecus stenognathus, was a CAM or C4 specialist. Nitrogen isotope data suggest that all of these extinct taxa lived in arid habitats (Crowley et al. 2011a; Godfrey et al. 2011). Analysis of Fur or Hair Keratin (13C, 15N and 18O) Similar to collagen, keratin 15N values reflect those in dietary protein and keratin 13C values predominantly reflect protein rather than whole diet (Gannes et al. 1998; Hedges and van Klinken 2000; Martnez del Rio et al. 2009). Oxygen isotope values reflect both diet and water source (OBrien and Wooller 2007). Unlike many other tissues, keratin is isotopically inert; once formed, each fur or hair strand maintains its isotopic signature (Martnez del Rio et al. 2009). Keratin isotope values do not immediately record dietary shifts. Instead, there is an isotopic inertia, where isotope values equilibrate with changes in diet over a period of several weeks (Sponheimer et al. 2003b). As a result, brief shifts in diet or water source may not be discernible using keratin isotope values. Nevertheless, depending on diet and keratin growth and loss patterns, e.g., continuous growth vs. growing and shedding seasonal coats, it should be possible to examine seasonal dietary or habitat shifts for a particular individual (Dammhahn and Kappeler 2010; OBrien and Wooller 2007). This approach is most promising in primates that have long, continuously growing hair, or primates that have very seasonal diets, such as baboons (Codron et al. 2008). Sample Collection and Preparation Collecting fur or hair keratin samples is minimally invasive. Samples can be collected even from sleeping sites, provided it is possible to identify where particular individuals sleep. Keratin is easy to store, and it remains isotopically stable for long periods of time. Pelts from museum collections provide an excellent resource for examining niche partitioning among species or historical dietary shifts in extant primates (ORegan et al. 2008). To prepare keratin samples for 13C, 15N or 18O analysis, samples should be wiped clean using ethanol, methanol, or petroleum ether, then air-dried. Particularly

Stable Isotope Techniques and Applications

dirty samples can be sonicated in ultrapure water, methanol, or a lipid-removing solvent such as petroleum ether (OConnell and Hedges 1999; ORegan et al. 2008). Cleaned samples can be stored indefinitely in bags, envelopes, or wax paper. If subsections of keratin samples will be used to examine seasonal or interannual isotopic patterns, be aware of fur or hair growth patterns for the species, and collect enough to analyze subsamples (plan for 500 g per subsample; Codron et al. 2008). Use a razor blade and a ruler to section keratin strands precisely. The length of each section will depend on the research question, growth rates, and the total length of the keratin strands. First, test if there are isotopic differences among a subset of the samples. This may save time and money. Applications Keratin 13C and 15N values have been used to distinguish the feeding ecology of New World monkeys (Schoeninger et al. 1997). Carbon isotope values separate species living in dry forest with an open canopy howlers (Alouatta palliata) and woolly spider monkeys (Brachyteles arachnoides) from those living in denser, more closed canopy forest capuchins (Cebus capucinus) and spider monkeys (Ateles geoffroyi). Nitrogen isotope values separate species with different dietary preferences; high 15N values in capuchins appear to support their faunivory, and low 15N values in howlers agree with their consumption of plant species with N-fixing symbionts (Schoeninger et al. 1997). Keratin 13C and 15N values have also been used to distinguish the feeding ecologies of sympatric groups of ring-tailed lemurs (Lemur catta: Loudon et al. 2007), and geographically diverse populations of macacques (Macaca mulatta), bush babies (Galago spp.), and mouse lemurs (Microcebus spp.) (Crowley et al. 2011b; Dammhahn and Kappeler 2010; ORegan et al. 2008; Schoeninger et al. 1998). Using simple mixing models, keratin isotope values can estimate the relative consumption of C3, C4, and CAM resources (Codron et al. 2008; Crowley et al. 2011b; Schoeninger et al. 1998; Sponheimer et al. 2006a). Incremental sectioning of baboon (Papio ursinus) hair strands documents substantial intra- and interindividual variation in the relative consumption of C4 vegetation (Codron et al. 2008). Although 18O values in keratin have not yet been employed in nonhuman primate research, research on humans (Homo sapiens) suggests that oxygen has great potential for tracking seasonal dietary or habitat changes and migrations (OBrien and Wooller 2007). Analysis of Blood (13C and 15N) Blood is the best tissue for examining short-term dietary changes. Isotope values in red blood cells (RBC) integrate dietary protein intake over several weeks, while blood plasma/serum reflects dietary protein intake over the previous several days (Gannes et al. 1998). Sample Collection and Preparation Blood collection can be performed on a routine basis. Collect 80 l of fresh blood (Herrera et al. 2006). Consult with a veterinarian to determine the best length and gauge needle to use for sampling blood from the species of interest. Although it is generally a good idea to avoid using additives for sample preservation, field collection of samples often occurs in remote areas, far from electricity, freezers, and centrifuges. Unless only whole blood will be analyzed or

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samples can be centrifuged immediately after collection, anticoagulation additives need to be used. Fortunately, sodium heparin does not affect 13C and 15N values in red blood cells or plasma (Kurle 2002). Samples should be centrifuged at high velocity for 5 min. If specific components, e.g., plasma, will be analyzed, do not freeze the samples until they have been centrifuged. Heparinized samples are stable if kept cool for 24+ h before spinning (C. Kurle, pers. comm.). After spinning, separate the plasma/serum from the red blood cells using a pipette. Samples can be stored in glass vials or on clean glass fiber filter paper and frozen, lyophilized or dried at 50C in a drying oven (Barrow et al. 2008). Once dry, samples should be stored in airtight containers. Whole blood can also be briefly preserved in 70% ethanol (Barrow et al. 2008). It is unknown, however, if ethanol affects the isotope values of plasma/serum. Applications Stable isotope values in blood have yet to be utilized in nonhuman primate foraging ecology research. Nevertheless, blood shows good potential. For example, blood 13C and 15N values have successfully quantified seasonal dietary shifts in tropical birds and distinguished dietary niches among sympatric bird species (Herrera et al. 2006). They have also been used to distinguish protein sources for sympatric frugivorous and nectivorous bats (Herrera et al. 2001). Analysis of Feces (13C and 15N) Feces can yield detailed information about short-term dietary changes (Sponheimer et al. 2003a). Isotope values in feces correlate with those in body tissues and diet. They may, however, be enriched in 13C and 15N due to excreted microbes and sloughed tissues (Barboza and Parker 2006; Sponheimer et al. 2003a). Sample Collection and Preparation Fresh or dried feces can be collected noninvasively without direct animal contact. Fresh feces should be dried in an oven <60C (Barboza and Parker 2006; Codron et al. 2006), air dried, or lyophilized until a constant mass is reached. Dried samples should be ground into a fine powder using a mill or a mortar and pestle. Some authors recommend passing the ground feces through a coarse sieve, e.g., 1 mm, to ensure that only small particulate matter is analyzed (Codron et al. 2008). Applications Stable isotope ratios in feces can be used to distinguish dietary preferences among sympatric species (Codron et al. 2006), but they can also be used to link biome type, food availability, nutritional quality, and dietary preference for a single species across heterogeneous landscapes (Codron et al. 2006; Sponheimer et al. 2009). For example, fecal isotopic analysis has shown substantial isotopic variability among South African chacma baboon populations (Papio ursinus: Codron et al. 2006) as well as dramatic short-term dietary shifts for single individuals (Codron et al. 2008). Analysis of Urine (15N and 18O) Because nitrogen in urine reflects amino nitrogen that is expelled as waste, urinary 15N values are closely tied to 15N values in body tissues (Koch 2007). Urinary 15N values can, therefore, be used to investigate energy balance and nutritional

Stable Isotope Techniques and Applications

stress, particularly when combined with other measures such as urinary C-peptides or ketones (Vogel et al. 2011). Urinary 18O values closely track those in drinking water and provide the most direct means for measuring an animals body water isotope values (OBrien and Wooller 2007; OGrady et al. 2010a). Sample Collection and Preparation Isotopic studies using urine are rare, and methods for sample collection and preparation vary. Samples destined for 15N analysis: Because urine can be very dilute, collect several milliliters of fresh urine. Some authors argue that samples should be acidified with a small amount of dilute boric acid to prevent ammonia loss and bacterial ureolosis (Barboza and Parker 2006). Freezing after collection (< 20 C) is ideal (Barboza and Parker 2006; Parker et al. 2005), but not always practical. Cheryl Knott has demonstrated that urine can be dried and stored on filter paper in the field (Knott 1997). However, whether or not isotope values differ among storage methods remains untested. Regardless of the original collection methods, samples should be stored cool (ideally frozen) and ultimately dried in a forced air oven at ca. <65C (Parker et al. 2005). N loss is reportedly higher when samples are lyophilized (possibly due to ammonia volitalization; Parker et al. 2005). Depending on sample yield, dried urine can be directly weighed into tin capsules (Barboza and Parker 2006) or re-eluted in methanol, added to tin capsules in 1-ml aliquots, and evaporated at room temperature (Vogel et al. 2011). Samples destined for 18O analysis: Fresh urine samples can be collected in vials or centrifuge tubes and stored frozen. Some authors have simply analyzed 18O from liquid urine samples (OBrien and Wooller 2007). However, salts and organic compounds in the urine could affect the 18O value of the samples (OGrady et al. 2010a). Cryogenic distillation of the urinary water will remove some of these contaminants. Subsequent refrigeration and treatment with activated charcoal for 24 h appears to remove any remaining organic compounds (OGrady et al. 2010a). Before analysis, charcoal should be removed using a syringe or vacuum filter (OGrady et al. 2010a). Filtered samples should be transferred into crimp-top gas chromatography vials (without headspace) and sealed for isotope analyses. Check with the laboratory that will perform the analysis about vial size specifications. Applications During periods of nutritional stress, e.g., food scarcity, an animal may go into negative protein balance, in which nitrogen is recycled and both body and urinary 15N values increase (Gannes et al. 1998; Koch 2007). Preliminary research with captive Pan paniscus supports these predictions. As expected, during periods of food shortage, urinary 15N values increase, and during periods of food abundance, they decrease (Deschner et al. 2010). On the other hand, urinary 15N values from wild orangutans (Pongo pygmaeus wurmbii) are similar during periods of regular and low fruit abundance (Vogel et al. 2011), suggesting that orangutans do not experience negative protein balance during times of fruit scarcity. These apes may supplement their diet with protein rich bark or animal matter during periods of fruit scarcity. Orangutans may also be exceptionally protein efficient, not surprising considering their unpredictable environment (Vogel et al. 2011). Naturally occurring patterns in urinary 18O values have yet to be investigated to nonhuman primates. Research on humans (Homo sapiens) suggests that urinary 18O

B.E. Crowley

values can be used to track an individuals movement across a landscape, provided that the individual consumes isotopically distinct foods or drinks isotopically distinct water sources in different places (OBrien and Wooller 2007). At high levels of water flux, i.e., when animals drink more water, 18O of body water mirrors that of drinking water. However, when urinary output is lowered, 18O values of body water (and urine) increase (OGrady et al. 2010a). Urinary data could thus be useful for tracking water stress, the metabolic effects of water flux and energy expenditure, and distinguishing individuals with diseases that affect water homeostasis, such as diabetes (OGrady et al. 2010b; Simmen et al. 2010).

Analyzing and Comparing Data After preparation, a subset of sample should be weighed for analysis using a precision microbalance. It is imperative to weigh prepared samples using a microbalance with a readibility of 0.001 mg, or 0.000001 g. These balances can be quite expensive. If a microbalance is not readily available, contact the laboratory that will be conducting the analyses to find out what they recommend. Sending prepared samples to be weighed at the laboratory may be worth the extra cost. Sample size depends on the laboratory that conducts the analysis. It is advisable to select and analyze a subset of pilot samples to ascertain the degree of isotopic variability among samples. If your pilot data exhibit very little isotopic variability this step will save you time and money. Dried plant, arthropod, collagen, keratin, blood, feces, and urine samples destined for carbon or nitrogen isotope analysis should be weighed and secured in tin capsules, e.g., 59 mm Costech no. 041060. Dried plant and keratin samples destined for oxygen isotope analysis should be weighed and secured in silver capsules, e.g., 3.5 5 mm Costech no. 041066. After placing the correct amount of sample into a capsule, compress the capsule into a small cube or ball using forceps and a clean working surface such as an agate or steel plate. Ensure that the sample is well secured and that there are no holes in the capsule. Never touch the boats with anything except clean forceps. If a capsule drops on the floor or tears, discard it and start over. Store secured samples in a 96-well untreated polystyrene culture plate, e.g., Costar no. 3790. Samples destined for oxygen isotope analysis should be kept under vacuum for 6 d before analysis, and need to be analyzed immediately after being removing from vacuum (Bowen et al. 2005). Enamel and bone carbonate samples destined for carbon or oxygen isotope analysis should be weighed into steel boats. Usually this step is completed at the laboratory where the samples will be analyzed. Immediately before analysis, carbonate samples should be dried at 65C for 1 h under vacuum to remove any water. There are several common issues that can affect isotope data. Before sending samples to an isotope laboratory, it is prudent to ask the laboratory about the analytical standards it uses and to request an estimate of the laboratorys analytical precision. Elemental composition of the standards should roughly match that of the samples. Confirm that the laboratory will analyze isotopic standards along with the samples. During an isotope run, every 810th sample should be a standard with a well-constrained isotope value. This is important because standards can be used to 1)

Stable Isotope Techniques and Applications

correct for both internal drift that can occur in the mass spectrometer during a run and 2) correct data so that they are comparable to data run in other laboratories. Carefully examine the data provided by the isotope laboratory after analysis, checking for discrepancies. Are the isotopic and elemental data for the analytical standards consistent throughout the run? Are the 13C and 15N values within the range expected for the system? Look at the amount of carbon, nitrogen, and oxygen (%C, %N, %O) yielded by the samples. Percent carbonate in treated enamel samples should be 3.5 4% (Koch et al. 1997). Collagen should be >0.5%N and >4.5%C, and atomic C:N ratios for collagen should be between 2.9 and 3.6 (Ambrose 1990). Similarly, atomic C:N ratios for keratin should be between 2.9 and 3.8 (OConnell and Hedges 1999). Atomic C:N ratios for red blood cells and blood plasma should be 3.6 to 4.2 and 4.2 to 4.9, respectively (C. Kurle, pers. comm.). Plant data are variable but most plants have atomic C:N ratios between 15 and 45 (e.g., Crowley et al. 2011b). Data can be considered reliable if 1) the %C and %N yields are reasonable, the 2) C:N ratios are within this range (or close), and 3) the 13C and 15N values are similar among individuals from the same species/ locality. If these conditions are not met, suspect samples should be reanalyzed. Bad data can occur if a sample was contaminated, poorly preserved, or simply did not run properly. Leaf or fecal material may mold if not dried completely, a sample may still contain chemicals used during sample preparation if not rinsed completely, and historic bone samples can have poor carbonate or collagen preservation. If sample size is too small for repeat analysis, consider omitting the data from statistical analyses. Mixing Models Mixing models can determine the relative contribution of isotopically distinct food resources, e.g., C3 and C4 plants. These models use the mean isotope values for the primate population and each dietary category (shifted to account for trophic discrimination between the animal and its diet) to determine the relative contribution of isotopically distinct food resources. For example, in a recent study (Crowley et al. 2011b), I used a single isotope mixing model for carbon to determine the relative proportion of CAM and C3 foods in mouse lemur diets using the following equations: d 13 Cmouse lemurs FractionC3 d 13 CC3 FractionCAM d 13 CCAM 1

1 FractionC3 FractionCAM ; and after substitution and solving for FractionCAM 2

FractionCAM d 13 Cmouse lemurs d 13 CC3 =d 13 CCAM d 13 CC3

If a species consumes multiple food resources, e.g., arthropods, fruits, plant exudates, grasses, vertebrates, multiple isotopes are required to calculate the relative contribution of each food resource (Herrera et al. 2001). Although multiple isotope mixing models have been used to reconstruct the diets of ancient Homo sapiens (Phillips et al. 2005), they have been surprisingly underutilized in nonhuman primate foraging ecology. The general rule of thumb is that n different isotopes, e.g., 13C, 15N, 18O, are needed to determine the proportional contributions of n + 1

B.E. Crowley

isotopically distinct food categories (Phillips and Gregg 2003). Sometimes, the number of isotopically distinct food resources is larger than n+1 isotope systems. If this is the case, it will not be possible to calculate a unique solution (such as a mean contribution) for each dietary source. It is, however, possible to calculate a range of feasible contributions for each resource using a free online model such as isosource (http://www.epa.gov/naaujydh/pages/models/stableIsotopes/isosource/isosource.htm; Phillips and Gregg 2003). Recent more sophisticated mixing models such as MixSir (http://conserver.iugo-cafe.org; Moore and Semmens 2008) and SIAR (http://cran.rproject.org/web/packages/siar/index.html; Parnell et al. 2010) use a Bayesian approach to estimate the uncertainty surrounding the proportional contribution of each dietary source. Comparing Modern and Historical Data The burning of fossil fuels over the past 150 yr has steadily decreased the 13C value of atmospheric CO2 ca. 1.5 (Chamberlain et al. 2005; Francey et al. 1999). For recent trends see http://scrippsco2.ucsd.edu/graphics_gallery/isotopic_data.html). Because 13C values of atmospheric CO2 are passed up the food chain, modern plants and animals have increasingly lowered 13C values. The effects of this shift are trivial over short time scales of 510 yr (ca. 0.1). However, a decrease of 0.51.5 over longer periods of time could affect ecological interpretations. It is, therefore, essential to account for this shift when dealing with specimens that are different ages, e.g., keratin from modern animals vs. museum pelts. Failure to do so can result in inaccurate interpretations of diet or habitat. Even older fluctuations have been documented in atmospheric 13C and 18O values (Zachos et al. 2001). When investigating the isotope ecology of ancient specimens, it is important to consider the degree to which the 13C and 18O values during the period of interest may have differed from modern conditions. Although it is not always possible to account for these changes, it is critical to acknowledge the possibility that data may reflect regional or global temporal shifts in isotope values.

Conclusions The declining numbers of many primate species makes their conservation a priority, yet surprisingly little is documented about ecological requirements for their survival. Stable carbon, nitrogen, and oxygen isotope biogeochemistry can offer much needed data for quantifying the feeding ecology of many extant and extinct primate species. These isotopes help tease apart the relative contribution of a particular food resource, such as fruits or insects, and they can determine the habitat in which an animal feeds, or fed in the past. The applicability of isotopic patterns to studies on nonhuman primates has only recently been recognized. To date, isotope research on nonhuman primates has focused mainly on a handful of living species and extinct hominins. The potential benefits for distinguishing ecological differences among sympatric primate species, including habitat use, short-term dietary shifts, and trophic discrimination, have yet to be fully appreciated.

Stable Isotope Techniques and Applications Acknowledgments I thank Gabe Bowen, Kena Fox-Dobbs, Sora Lee Kim, Carolyn Kurle, and Patrick Wheatley, for consultation on sample preparation techniques, and Lawrence Crowley, Sally Goddard, Matthew Weirauch and three anonymous reviewers for critical review of the manuscript. I also thank Erin Vogel and Janine Chalk for inviting me to contribute to this special issue.

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