Discrimination factors of carbon and nitrogen stable ...Peromyscus maniculatus 19.4 0.3 3.2 5 Herbivore Hwang, Millar & Longstaffe (2007) Myodes gapperi 19.4 0.3 4.2 5 Herbivore Hwang,

Submitted 22 August 2016Accepted 17 May 2017Published 13 June 2017

Corresponding authorShaena Montanarishaemontanarigmailcom

Academic editorRita Grandori

Additional Information andDeclarations can be found onpage 11

DOI 107717peerj3436

Copyright2017 Montanari

Distributed underCreative Commons CC-BY 40

OPEN ACCESS

Discrimination factors of carbon andnitrogen stable isotopes in meerkat fecesShaena MontanariSchool of GeoSciences University of Edinburgh Edinburgh United Kingdom

ABSTRACTStable isotope analysis of feces can provide a non-invasive method for tracking thedietary habits of nearly any mammalian species While fecal samples are often collectedfor macroscopic and genetic study stable isotope analysis can also be applied to expandthe knowledge of species-specific dietary ecology It is somewhat unclear how digestionchanges the isotope ratios of animalsrsquo diets so more controlled diet studies are neededTo date most diet-to-feces controlled stable isotope experiments have been performedon herbivores so in this study I analyzed the carbon and nitrogen stable isotope ratios inthe diet and feces of the meerkat (Suricata suricatta) a small omnivorousmammal Thecarbon trophic discrimination factor between diet and feces (113Cfeces) is calculated tobe 01plusmn 15h which is not significantly different from zero and in turn not differentthan the dietary input On the other hand the nitrogen trophic discrimination factor(115Nfeces) is 15 plusmn 11h which is significantly different from zero meaning it isdifferent than the average dietary input Based on data generated in this experimentand a review of the published literature carbon isotopes of feces characterize diet whilenitrogen isotope ratios of feces are consistently higher than dietary inputs meaning adiscrimination factor needs to be taken into account The carbon and nitrogen stableisotope values of feces are an excellent snapshot of diet that can be used in concertwith other analytical methods to better understand ecology diets and habitat use ofmammals

Subjects Animal Behavior Biogeography Ecology Ecosystem Science ZoologyKeywords Stable isotopes Ecology Dietary ecology Mammalogy Meerkats Trophic ecologyFeces

INTRODUCTIONSmall mammals are often overlooked in favor of larger more charismatic species but theyfill vital roles as ecosystem engineers prey base and seed dispersal agents (Huntly amp Inouye1988 Brown amp Heske 1990 Davidson Detling amp Brown 2012) They can live in coloniesor in large numbers therefore their plentiful modern and historical remains can providerecords of changing environments and shifting ecological conditions (Terry 2010) Non-invasive monitoring is an ideal way to track changes in modern mammalian communitiesas shed hair and feces can provide a substrate for examining population trends dietsand health of groups using genetic or chemical methods (Crawford McDonald amp Bearhop2008 Pompanon et al 2012 Rodgers amp Janečka 2013) Specifically examining feces isuseful as it can be collected from rare or cryptic species that are hard to monitor and oftenavoid humans Also feces is plentiful and relatively inexpensive to analyze Traditionally

How to cite this article Montanari (2017) Discrimination factors of carbon and nitrogen stable isotopes in meerkat feces PeerJ 5e3436DOI 107717peerj3436

vertebrate diets have been assessed macroscopically through physical examination of gutcontents and feces (eg Hermsen Kerle amp Old 2016) With the advent of affordable high-throughput sequencing fecal studies are becoming more common for barcoding dietaryDNA which allows for a more complete dietary picture not as easily biased by differingdigestibility of food (eg Shehzad et al 2012Kartzinel et al 2015) Stable isotopemethodscan provide a useful complement to these barcoding studies that are growing in popularityFecal stable isotopes have been useful for detecting dietary shifts in mammals such asgorillas (Blumenthal et al 2012) for understanding weaning time in primates (Reitsema2012) and as a climate record from bats (Royer et al 2015) so understanding isotopicdiscrimination and variability of this material is vital for future research

Stable isotope ratios of nitrogen (15N14N written δ15N) and carbon (13C12C writtenδ13C) are incorporated in animal tissues and excretions following digestion of foodproducts Changes in stable isotope ratios can elucidate information about food webstrophic structure and habitat use (Ben-David amp Flaherty 2012) It is assumed that carbonisotope ratios do not change drastically as they propagate through the food web (DeNiroamp Epstein 1978) and in terrestrial ecosystems these ratios are generally used to indicatethe primary production at the base of the food web Carbon isotope ratios of plants thatuse different metabolic pathways (C3 C4 or CAM) are systematically different and thisdifference is incorporated into the tissues or byproducts of consumers On the other handδ15N values are known to become enriched as trophic level increases (DeNiro amp Epstein1981) and differences in δ15N of tissue over time or between populations can be used todiscern changes in food webs habitat or prey composition (Post 2002)

To use stable isotopes in dietary ecology there has to be a comprehensive understandingof the difference in isotope ratios between diet and tissue (or feces) this difference is calledthe trophic discrimination factor (also fractionation factor or discrimination factor) and iscaused by isotopic fractionation during digestion and metabolism Trophic discriminationfactor is denoted as 1 and defined as 1= δtissuendashδdiet (Martiacutenez del Rio et al 2009)Trophic discrimination factors (abbreviated TDFs) are widely variable among animalsand differ depending on species tissue examined and diet type and quality (Caut Anguloamp Courchamp 2009) General TDFs are used in many mixing model studies for animaldiet reconstruction but it has been shown that small differences in TDFs in these types ofstudies can lead to vastly different conclusions about dietary makeup (Ben-David amp Schell2001) Experimental work by Caut Angulo amp Courchamp (2008) shows that mixing modelsare most accurate when species-specific TDFs are obtained

In this study I calculate TDFs for carbon and nitrogen isotopes in meerkat (Suricatasuricatta) feces in order to obtain fecal TDFs in a small mammalian omnivore Meerkatsare small diurnal mammals that are members of Carnivora and are herpestids closelyrelated to mongoose They have generalist diets and in the wild consume insects berriesreptiles and other small invertebrates (Doolan amp Macdonald 1996) The diets of generalistconsumers are difficult to study in the wild due to unknown variability in their diet so azoo-based study of meerkats in this instance will provide dietary control allowing for lessvariation in the determination of a TDF Studies like this one are necessary as TDFs arenot often determined for terrestrial mammalian omnivores and carnivores or even feces in

Montanari (2017) PeerJ DOI 107717peerj3436 215

Table 1 Fecal trophic discrimination factors of carbon (113C) from this study and from the literature Average δ13C of diet used to calculateeach TDF is included from the original listed publication All isotope values are presentedplusmn1 standard deviation if it was available in the originalpublication Asterisks () indicate when one species from the same experiment was divided into different diet treatments The column lsquolsquonrsquorsquo repre-sents the number of fecal samples used to calculate the TDF in each experiment The 113C values in bold are statistically significantly different fromzero in their original experiments (in some cases this was not specifically tested)

Species δ13C diet 113C n Diet class Reference

Peromyscus maniculatus minus194plusmn 03 minus32 5 Herbivore Hwang Millar amp Longstaffe (2007)Myodes gapperi minus194plusmn 03 minus42 5 Herbivore Hwang Millar amp Longstaffe (2007)Microtus longicaudus minus194plusmn 03 minus27 5 Herbivore Hwang Millar amp Longstaffe (2007)Microtus pennsylvanicus minus194plusmn 03 minus36 5 Herbivore Hwang Millar amp Longstaffe (2007)Tamias amoenus minus194plusmn 03 minus30 5 Herbivore Hwang Millar amp Longstaffe (2007)Zapus princeps minus194plusmn 03 minus59 5 Herbivore Hwang Millar amp Longstaffe (2007)Lama glama minus133plusmn 03 minus12plusmn 04 4 Herbivore Sponheimer et al (2003a)Lama glama minus270plusmn 02 minus04plusmn 05 4 Herbivore Sponheimer et al (2003a)Capra hircus minus270plusmn 02 minus08plusmn 01 4 Herbivore Sponheimer et al (2003a)Capra hircus minus133plusmn 03 minus10plusmn 04 4 Herbivore Sponheimer et al (2003a)Bos taurus minus133plusmn 03 minus09plusmn 02 4 Herbivore Sponheimer et al (2003a)Bos taurus minus270plusmn 02 minus100plusmn 02 4 Herbivore Sponheimer et al (2003a)Oryctolagus cuniculus minus270plusmn 02 minus03plusmn 01 4 Herbivore Sponheimer et al (2003a)Vicugna pacos minus270plusmn 02 minus04plusmn 04 4 Herbivore Sponheimer et al (2003a)Vicugna pacos minus133plusmn 03 minus13plusmn 02 4 Herbivore Sponheimer et al (2003a)Equus caballus minus270plusmn 02 minus05plusmn 04 4 Herbivore Sponheimer et al (2003a)Equus caballus minus133plusmn 03 minus07plusmn 02 4 Herbivore Sponheimer et al (2003a)Uncia uncia minus2361plusmn 315 230plusmn 166 10 Carnivore Montanari amp Amato (2015)Panthera tigris minus2361plusmn 315 125plusmn 062 7 Carnivore Montanari amp Amato (2015)Gorilla gorilla minus284 03 121 Herbivore Blumenthal et al (2012)Clethrionomys gapperi minus2923 minus051plusmn 119 11 Herbivore Sare Millar amp Longstaffe (2005)Clethrionomys gapperi minus2549 minus195plusmn 104 10 Herbivore Sare Millar amp Longstaffe (2005)Clethrionomys gapperi minus2822 024plusmn 120 10 Herbivore Sare Millar amp Longstaffe (2005)Myotis myotis minus2454plusmn 076 minus017plusmn 110 15 Insectivore Salvarina et al (2013)Myotis myotis minus2050plusmn 081 minus025plusmn 075 21 Insectivore Salvarina et al (2013)Rhinolophus ferrumequinum minus2454plusmn 076 0 15 Insectivore Salvarina et al (2013)Rhinolophus ferrumequinum minus2050plusmn 081 009plusmn 039 21 Insectivore Salvarina et al (2013)Suricata suricatta minus249plusmn 33 01plusmn 15 24 Omnivore This study

general (Tables 1 and 2) Even though feces represent a snapshot of diet it is often collectedin sizeable quantities for non-invasive studies This can provide important ecological andenvironmental records and elucidate short-term and seasonal dietary variability Changes inisotope ratios from diet to feces need to be calculated as there may be isotopic fractionationfrom the process of digestion and waste excretion

Here I calculate trophic discrimination factors for captive meerkats by measuringthe isotopic composition of both the diet and the feces I also describe how fecal stableisotope values vary over short periods of time so that more information can be gleanedabout the measured variability in generalist diets during wild studies Focusing on smallmammals with generalist diets will be key to uncovering ecological factors that cause shifts


Table 2 Fecal trophic discrimination factors of nitrogen (115N) from this study and from the literature Average δ15N of diet used to calculateeach TDF is included from the original listed publication All isotope values are presented 1 standard deviation if it was available in the original pub-lication Asterisks () indicate when one species from the same experiment was divided into different diet treatments The column lsquolsquonrsquorsquo representsthe number of fecal samples used to calculate the TDF in each experiment The 115N values in bold are statistically significantly different from zeroin their original experiments (in some cases this was not specifically tested)

Species δ15N diet 115N n Diet class References

Peromyscus maniculatus 36plusmn 002 21 5 Herbivore Hwang Millar amp Longstaffe (2007)Myodes gapperi 36plusmn 002 22 5 Herbivore Hwang Millar amp Longstaffe (2007)Microtus longicaudus 36plusmn 002 22 5 Herbivore Hwang Millar amp Longstaffe (2007)Microtus pennsylvanicus 36plusmn 002 25 5 Herbivore Hwang Millar amp Longstaffe (2007)Tamias amoenus 36plusmn 002 14 5 Herbivore Hwang Millar amp Longstaffe (2007)Zapus princeps 36plusmn 002 22 5 Herbivore Hwang Millar amp Longstaffe (2007)Lama glama 04 29plusmn 03 4 Herbivore Sponheimer et al (2003b)Lama glama 58 30plusmn 04 4 Herbivore Sponheimer et al (2003b)Bos taurus 07 20 4 Herbivore Steele amp Daniel (1978)Bos taurus 06 17 4 Herbivore Steele amp Daniel (1978)Equus caballus 04 26 Unknown Herbivore Sponheimer et al (2003b)Equus caballus 58 33 Unknown Herbivore Sponheimer et al (2003b)Ovis aries 08 30 Unknown Herbivore Sutoh Obara amp Yoneyama (1993)Capra hircus 15 36 3 Herbivore Sutoh Koyama amp Yoneyama (1987)Uncia uncia 895plusmn 073 249plusmn130 10 Carnivore Montanari amp Amato (2015)Panthera tigris 895plusmn 073 157plusmn 204 7 Carnivore Montanari amp Amato (2015)Sus scrofa 46plusmn 03 12 3 Omnivore Sutoh Koyama amp Yoneyama (1987)Gorilla gorilla 32 06 121 Herbivore Blumenthal et al (2012)Clethrionomys gapperi minus042 176plusmn 126 11 Herbivore Sare Millar amp Longstaffe (2005)Clethrionomys gapperi 145 117plusmn 168 10 Herbivore Sare Millar amp Longstaffe (2005)Clethrionomys gapperi 400 127plusmn 206 10 Herbivore Sare Millar amp Longstaffe (2005)Myotis myotis 531plusmn 063 181plusmn 128 15 Insectivore Salvarina et al (2013)Myotis myotis 1288plusmn 116 234plusmn 217 21 Insectivore Salvarina et al (2013)Rhinolophus ferrumequinum 531plusmn 063 053plusmn 054 14 Insectivore Salvarina et al (2013)Rhinolophus ferrumequinum 1288plusmn 116 097plusmn 045 21 Insectivore Salvarina et al (2013)Suricata suricatta 46plusmn 18 15plusmn 11 24 Omnivore This study

in mammalian biodiversity as small mammals are accurate recorders of environmentalchange over a variety of time scales (Barnosky Hadly amp Bell 2003 Terry 2010)

MATERIALS AND METHODSDiet and feces samplesThe meerkats in this study are maintained at the Edinburgh Zoo (Royal Zoological SocietyScotland) Fecal samples were taken randomly from an enclosure containing seven adultfemale meerkats over the course of April 2016 Subsamples of diet were collected oncea week for four weeks to account for variability in diet items Per the information ofEdinburgh Zoo animal care workers the meerkats are fed different combinations of thefood items each day but over the course of a week the amount of each item they eat isroughly equal Each animal receives the same amount of each food item by weight A total


of 24 fecal samples were collected along with 2ndash4 subsamples of each diet item carrotsand apples horsemeat dog biscuits whole frozen small mice and whole frozen chicksThe above items were used to calculate the trophic discrimination factor from diet to fecesHomogenized bulk muscle with attached skin was sectioned from the whole chicks andmice for analysis

Stable isotope analysisStable isotope analysis was conducted at the Wolfson Laboratory in the School ofGeosciences at the University of Edinburgh The analysis of carbon and nitrogen isotoperatios was performed on a CE Instruments NA2500 Elemental Analyzer and the effluentgas was analyzed for its carbon and nitrogen isotopic ratios using a Thermo ElectronDelta+ Advantage stable isotope ratio mass spectrometer Sediment standard PACS-2(δ15N 5215h (Air) and δ13C- 22228h(VPDB)) from the National Research CouncilCanada was used for isotopic analyses Acetanilide standard (C 7109 and N 1036) wasused for elemental compositions Isotopic data were determined relative to CO2 and N2

reference gases whose mean values are derived from the average value of PACS-2 sampleswithin each daily run

The standard deviation for five analyses of the PACS-2 standard run over the same timeperiod as the study samples was plusmn007h for δ13C (VPDB) and plusmn014h for δ15N (Air)Elemental analysis measuring the percentage of carbon and nitrogen in the samples hadan error of 1 for carbon and 4 for nitrogen The stable isotope ratios are reported instandard notation and referenced to air for δ15N values and Vienna Pee Dee Belemnite(VPDB) for δ13C values Ratios are defined as δ= (RsampleRstandardminus1) where R= 13C12Cor 15N14N

Samples were prepared first by lyophilization followed by manual crushing to forma homogenized powder for isotope analysis Feces and diet samples were subsampledand analyzed both with and without lipid extraction treatment The samples were lipidextracted by immersion in a 21 ratio of chloroformmethanol for 12 h using a Soxhletapparatus Following lipid extraction samples were dried in a 50 C oven for at least 24 hto evaporate any remaining solvent

Statistics and data analysisStatistical tests and analyses were performed in R (R Core Team 2016) and plots werecreated in ggplot2 (Wickham 2009) Trophic discrimination factors were calculated usingthe averages of all subsampled dietary material TDFs were calculated for both δ13Cand δ15N using the aforementioned equation 1Xfeces = δXfecesndashδXdiet where 1Xfeces iscalculated in per mil (h) δXdiet is the value of the average of all non-lipid-extracteddietary samples and δXfeces is each individual fecal stable isotope value

Parametric statistical tests were performed as the data are normally distributed as shownthrough a ShapirondashWilk test (δ13C W = 0946 p= 0057 δ15N W = 0976 p= 0544)Before hypothesis testing an F-test was performed to see if a t -test for equal or unequalvariance (Welch two sample t -test) should be done based on the results F-tests show thevariance was equal for comparisons between lipid-extracted and non-lipid-extracted feces


for both δ13C and δ15N (δ13C F823= 1409 p= 0491 δ15N F823= 0408 p= 0191)Variance for comparisons between diet and feces for both δ13C and δ15N are unequal(δ13C F1523= 5001 p= 00006 δ15N F1523= 2633 p= 0036) All means are reportedplusmn standard deviation (SD) and all significance is reported for α= 005

To obtain carbon and nitrogen fecal trophic discrimination factors from other studiesfor comparison to results from this study a literature search was conducted using GoogleScholar and through mining references in other feces trophic discrimination factor papersThese were all of the papers with diet-feces trophic discrimination factors found as ofAugust 2016 searching keywords such as lsquolsquofeces stable isotopesrsquorsquo and lsquolsquoscat stable isotopesrsquorsquoThese values appear in Tables 1 and 2

RESULTSA summary of the stable isotope results is presented in Table 3 and a summary of t -tests isreported in Table 4 All raw isotope data can be found in the supplementary information(Data S1)

A subset of feces and diet samples were lipid extracted to see if this changed the resultsof the analysis however using a t -test no significant difference was found between theδ13C and δ15N of either material between lipid extracted and non-lipid-extracted samples(Table 4) As in my previous study of fecal TDFs (Montanari amp Amato 2015) I have optedto use only non-lipid-extracted materials for sampling as a meta-analysis of carbon andnitrogen discrimination factors from Caut Angulo amp Courchamp (2009) has shown thatlipid extraction has no significant effect on calculated TDFs

The mean δ13C value for each food item is as follows chick (n= 2 minus261plusmn12h)mouse (n= 2 minus234plusmn17h) horsemeat (n= 4 minus271plusmn13h) carrots and apple mix(n= 4 minus277plusmn15h) and dog biscuits (n= 4 minus202plusmn05h) The average for all dietitems (n= 16) is minus249plusmn 33h Mean δ15N values are chick (45plusmn 003h) mouse(52plusmn 06h) horsemeat (70plusmn 13h) carrots and apple mix (35plusmn 10h) and dogbiscuits (29plusmn05h) The average δ15N for all diet items is 46plusmn18h The mean diet CNratio is 271plusmn381 The mean δ13C value of the feces is minus248plusmn15h and the mean δ15Nvalue is 61plusmn11h The CN ratio is 69plusmn 16 The ranges and variability of δ13C and δ15Nfor all materials are presented in Table 3 and the variation of fecal isotope values over themonth they were collected is presented in Fig 1 When the fecal samples were placed intobins by weeks they were collected and subjected to an ANOVA there was no differencebetween the average weekly scat values over the course of the month (δ13C F419= 0539p= 0709 δ15N F419= 0886 p= 0491)

The δ13C and δ15N values of feces and diet were compared to establish if there is asignificant difference between them In the case of δ13C there is no significant difference(Welch two-sample t -test t =minus014 df = 1905 p= 089) while for δ15N the meansare significantly different (Welch two sample t -test t =minus297 df = 2261 p= 001)Discrimination factors were calculated by using the average value of all diet items subtractedfrom each individual feces sample in order to assess variance Average 113C for feces(113Cfeces) is 01 plusmn15h and 115N for feces (115Nfeces) is 15plusmn11h with only the115Nfeces being significantly different than zero


Table 3 Stable isotope results frommeerkat feces and diet samples (δ13C and δ15N) Means are shownplusmn1 standard deviation Stable isotopes are presented in delta no-tation (δ) and discrimination factors are noted by 1 All isotope values are presented in per mil (h) Trophic discrimination factor in bold is statistically significant fromzero

n δ13C (h) δ13C range δ15N (h) δ15N range CN C N 113C 115N

Meerkat scat 24 minus248plusmn 15 minus281minus207 61plusmn 11 4489 69plusmn 16 209plusmn 111 32plusmn 18 01plusmn 15 15plusmn 11Chick 2 minus261plusmn 12 minus270minus253 45plusmn 003 45 37plusmn 03 478plusmn 03 129plusmn 10Mouse 2 minus234plusmn 17 minus246minus222 52plusmn 06 4756 61plusmn 15 514plusmn 47 87plusmn 13Horse meat 4 minus271plusmn 13 minus282minus253 70plusmn 13 6190 35plusmn 004 460plusmn 19 133plusmn 04Fruit mix 4 minus277plusmn 15 minus298minus268 35plusmn 10 2849 886plusmn 218 385plusmn 10 05plusmn 01Dog biscuits 4 minus202plusmn 05 minus207minus195 29plusmn 05 2334 114plusmn 18 429plusmn 07 38plusmn 06Total diet 16 minus249plusmn 33 minus298minus195 46plusmn 18 2390 271plusmn 381 442plusmn 46 71plusmn 55

Montanari(2017)PeerJD

OI107717peerj3436

715

Figure 1 Variability in δ13C (A) and δ15N (B) values of meerkat feces over the month of samplingPoints on the line represented a measured fecal sample All isotope values are presented in per mil (h)

Table 4 Results from t -tests (p-value t df ) comparing δ13C and δ15Nmeans of stable isotope valuesfrom lipid and non-lipid-extracted meerkat feces and diet samples The t -test used (Welch or equal)was decided by a preliminary F-test to test for equal variances

Variable 1 Variable 2 Test p-value t df

δ13C Scat LE δ13C Scat t -test equal 012 159 31δ15N Scat LE δ15N Scat t -test equal 009 -174 31δ13C Diet LE δ13C Diet t -test equal 046 075 14δ15N Diet LE δ15N Diet t -test equal 079 027 14δ13C Diet δ13C Scat Welch t -test 089 minus014 1905δ15N Diet δ15N Scat Welch t -test 001 minus297 2261


DISCUSSIONTrophic discrimination factors of meerkat fecesFeces can be an extremely useful substrate for stable isotope analysis for estimating foodinput and these data show in meerkats that in relation to carbon isotopes feces are afair representation of diet but nitrogen isotopes of feces undergo isotopic enrichmentInvestigations into stable isotope discrimination factors show that herbivore feces arerepresentative of diet in large bodied animals (Sponheimer et al 2003a) but that in small-bodied herbivores (Hwang Millar amp Longstaffe 2007) and non-herbivores (Montanari ampAmato 2015) feces undergo enrichment of nitrogen isotopes within the digestive tract Inthis study the trophic discrimination factor of carbon isotopes from diet-to-feces is smallandnot statistically significant Similar results for113Cfeces are seen in studies of insectivores(bats Salvarina et al 2013) and carnivores (tigers and snow leopardsMontanari amp Amato2015) To this point experimental research on diet-to-feces discrimination factors showsmost calculated carbon TDFs are non-significant as referenced by the review of publishedstudies in Table 1 More data are needed on mammalian omnivores and carnivores tofurther assess this pattern

The means of δ15Ndiet and δ15Nfeces are different (Table 4) therefore the 115Nfeces

value (15plusmn11h) is significantly different than zero In this study and the other twonon-herbivore fecal TDF studies of Salvarina et al (2013) andMontanari amp Amato (2015)115Nfeces is of similar magnitude and also the only TDF that is different than zero although115Nfeces is only significant in one out of two species examined in each of these studiesNevertheless significance of 115Nfeces could indicate 15N enrichment occurs duringdigestion likely during transit through the gut as is seen in Hwang Millar amp Longstaffe(2007) This study investigated δ15N values at different parts in the digestive tract of volesand other small rodents and found digested material is enriched in 15N in the stomachintestine cecum and colon relative to the diet It has also been shown the δ15N of mucosalepithelium was higher than the diet input in some parts of the digestive tract of a sheepwhich suggests the enrichment in 15N in feces may be due to the presence of endogenousproteins (Sutoh Obara amp Yoneyama 1993) In general significant 115N values pointto some relationship between digestive processes and 115N but the cause is unknownand more experiments comparing 15N enrichment during digestion in herbivores andnon-herbivores are needed

A number of other factors may be also affecting nitrogen flux during digestion andexcretion Different biochemical pathways related to the changes in proteins occurringduring digestion (deaminationamination) could be the cause of 15N enrichment (HwangMillar amp Longstaffe 2007) Additionally the presence of microorganisms in the digestivetract could also enrich fecal matter compared to diet (Hwang Millar amp Longstaffe 2007Macko et al 1987) The 15N enrichment could be due to preferential absorption of 14Nby the animal during digestion or removal during urine excretion but experimental datafrom Sponheimer et al (2003b) seem to indicate that at least in herbivores 14N is notpreferentially excreted InMontanari amp Amato (2015) we cautioned that more data shouldbe collected to better understand TDFs of carnivores because the physiological mechanisms


are still unknown this study continues in the same vein and reinforces that there is apreferential loss of 14N andor enrichment in 15N occurring during movement throughthe gastrointestinal tract in relation to solid waste Further mammalian physiologicalexperiments may explain the mechanism

Variables that affect trophic discrimination factorsIt has been shown that TDFs vary due to a number of factors one of them being the initialisotopic ratio of the diet Significant relationships between both δ13C and δ15N of diets andTDFs have been shown in bears (Hilderbrand et al 1996 Felicetti et al 2003) rats (CautAngulo amp Courchamp 2008) and birds (Pearson et al 2003) A meta-analysis of TDFs anddietary isotope ratios values byCaut Angulo amp Courchamp (2009) finds significant negativerelationships between these variables in both carbon and nitrogen A similar study of thisrelationship with fecal TDFs cannot be completed due to the fact most of the calculatedfecal TDFs are not significantly different from 0 (data from Tables 1 and 2) This couldbe due to the fact that feces represents immediately ingested diet (within hours or days)as opposed to assimilated diet and is not subjected to as many physiological processes offractionation This is a promising result for the use of feces in isotope studies because thevariability of TDFs due to dietary input is a major issue for stable isotope food web studiesVariability that needs to be accounted for with other tissues (Caut Angulo amp Courchamp2009) appears to be a non-issue in feces

Mammalian body size might influence calculated TDF (Hwang Millar amp Longstaffe2007) A mechanism that could explain this is a general trend of higher mass-specificmetabolism in animals with smaller body mass (Kleiberrsquos law) which could in turn impactthe fractionation of isotopes that occurs after food ingestion (Pecquerie et al 2010)HwangMillar amp Longstaffe (2007) did ameta-analysis of fecal TDFs in the literature combined withtheir rodent TDFs and found significant differences in 113C between different body sizesbut only in herbivores A lack of published fecal TDFs for non-herbivorous mammals ofdifferent sizes means statistical test cannot be performed for herbivores vs non-herbivores

Isotopic variability over timeFeces can be used for point estimates of diet but long term collection is especially useful forfinding seasonal patterns in dietary variability (eg Blumenthal et al 2012) Mammaliancarnivores and omnivores tend to change their diets seasonally so it is important torealize the magnitude of fecal isotope variation day-to-day for wild studies (eg Kincaidamp Cameron 1982 Melero et al 2008) Other than this study Blumenthal et al (2012) andSalvarina et al (2013) track stable isotope ratios of animal feces atmonthly or daily intervalsrespectively

I have tracked meerkat fecal stable isotopes over the course of one month and it is clearthere is variation on any given day a sample is taken (Fig 1) as the combination of dietitems they eat changes daily There is a range of 73h in the δ13C and 45h δ15Nfeces

of meerkat feces over a month This suggests feces can be directly reflective of a highlyvariable diet and also shows δ13Cfeces may not be buffered in small mammals as muchagainst day-to-day variability as in larger mammals (Blumenthal et al 2012) It is not


known exactly how long it takes isotopes of feces to reach dietary equilibrium in meerkatsbut in Salvarina et al (2013) it was shown that bat feces acquired a new dietary signal in2ndash3 h so it stands to reason it is also within hours for a small mammal Examining the dailyfluctuations in δ13C and δ15N in feces indicates timing of major dietary changes can bepinpointed quite precisely using this method at least within days Due to the fact the TDFsin this study were calculated using an average diet value the TDF is also averaged and ismeant to act as a general guide for a TDF in the wild This variability is important to realizefor wild studies as it emphasizes the need for larger sample sizes of feces such as multiplesamples per day or week to lessen the impact of day-to-day variability if researchers areseeking a long-term ecological or environmental trend (eg Blumenthal et al 2012)

CONCLUSIONSI found the δ13Cfeces of captive meerkats was not changed compared to the dietary inputwhile δ15Nfeces is higher than diet Compared with other published fecal TDFs the meerkatdata fit with observed trophic discrimination factors and also show that an enrichmentof 15N andor a depletion of 14N is occurring during digestion or in the gut duringgastrointestinal transit When these TDFs are compared to other fecal TDFs in publishedliterature they are similar in that they are mostly non-significant which removes onelayer of uncertainty when using feces in wild animal studies Looking at the stable isotoperatios for both carbon and nitrogen over the course of the month it is clear short-termnear-daily variability in diet can be captured using stable isotope analysis of meerkat fecesCaptive studies like this one with more controlled feces and diet collection parameters willhopefully lead to better understanding in other understudied groups like terrestrial smalland medium sized mammals with omnivorous diets so that stable isotope analysis of fecescan become a more common tool in mammalian stable isotope ecology

ACKNOWLEDGEMENTSThe author thanks the animal care staff at Edinburgh Zoo for collecting the diet andfeces samples and facilitating this research project Thanks are extended to Colin Chilcottand Steve Mowbray at the University of Edinburgh for help preparing and analyzing allsamples stable isotope analysis The author gives many thanks for reviews and commentsfrom Roberto Ambrosini and two other anonymous reviewers that greatly improved thismanuscript

ADDITIONAL INFORMATION AND DECLARATIONS

FundingFunding for this work was provided by the Royal Society Newton International FellowshipThe funders had no role in study design data collection and analysis decision to publishor preparation of the manuscript


Grant DisclosuresThe following grant information was disclosed by the authorRoyal Society Newton International Fellowship

Competing InterestsThe author declares that she has no competing interests

Author Contributionsbull ShaenaMontanari conceived and designed the experiments performed the experimentsanalyzed the data contributed reagentsmaterialsanalysis tools wrote the paperprepared figures andor tables reviewed drafts of the paper

Data AvailabilityThe following information was supplied regarding data availability

The raw data has been supplied as Data S1

Supplemental InformationSupplemental information for this article can be found online at httpdxdoiorg107717peerj3436supplemental-information

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varied temporal scales Journal of Mammalogy 84354ndash368DOI 1016441545-1542(2003)084lt0354MRTGWOgt20CO2

Ben-DavidM Flaherty EA 2012 Stable isotopes in mammalian research a beginnerrsquosguide Journal of Mammalogy 93312ndash328 DOI 10164411-MAMM-S-1661

Ben-DavidM Schell DM 2001Mixing models in analyses of diet using multiple stableisotopes a response Oecologia 127180ndash184 DOI 101007s004420000570

Blumenthal SA Chritz KL Rothman JM Cerling TE 2012 Detecting intraannualdietary variability in wild mountain gorillas by stable isotope analysis of fecesProceedings of the National Academy of Sciences of the United States of America10921277ndash21282 DOI 101073pnas1215782109

Brown JH Heske EJ 1990 Control of a desert-grassland transition by a keystone rodentguild Science 2501705ndash1707 DOI 101126science25049881705

Caut S Angulo E Courchamp F 2008 Discrimination factors (115N and 113C) in anomnivorous consumer effect of diet isotopic ratio Functional Ecology 22255ndash263DOI 101111j1365-2435200701360x

Caut S Angulo E Courchamp F 2009 Variation in discrimination factors (115N and113C) the effect of diet isotopic values and applications for diet reconstructionJournal of Applied Ecology 46443ndash453 DOI 101111j1365-2664200901620x

Crawford K McDonald RA Bearhop S 2008 Applications of stable isotope techniquesto the ecology of mammalsMammal Review 3887ndash107DOI 101111j1365-2907200800120x


Davidson AD Detling JK Brown JH 2012 Ecological roles and conservation challengesof social burrowing herbivorous mammals in the worldrsquos grasslands Frontiers inEcology and the Environment 10477ndash486 DOI 101890110054

DeNiroMJ Epstein S 1978 Influence of diet on the distribution of carbon isotopes inanimals Geochimica et Cosmochimica Acta 42495ndash506DOI 1010160016-7037(78)90199-0

DeNiroMJ Epstein S 1981 Influence of diet on the distribution of nitrogen isotopes inanimals Geochimica Et Cosmochimica Acta 45341ndash351DOI 1010160016-7037(81)90244-1

Doolan SP Macdonald DW 1996 Diet and foraging behaviour of group-livingmeerkats Suricata suricatta in the southern Kalahari Journal of Zoology239697ndash716 DOI 101111j1469-79981996tb05472x

Felicetti LA Schwartz CC Rye RO HaroldsonMA Gunther KA Phillips DL RobbinsCT 2003 Use of sulfur and nitrogen stable isotopes to determine the importanceof whitebark pine nuts to Yellowstone grizzly bears Canadian Journal of Zoology81763ndash770 DOI 101139z03-054

Hermsen E Kerle A Old JM 2016 Diet of an inland population of the commonringtail possum (Pseudocheirus peregrinus) Australian Mammalogy 38130ndash134DOI 101071AM15008

Hilderbr GV Farley SD Robbins CT Hanley TA Titus K Servheen C 1996 Use ofstable isotopes to determine diets of living and extinct bears Canadian Journal ofZoology 742080ndash2088 DOI 101139z96-236

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Hwang T Millar S Longstaffe J 2007 Do δ15N and δ13C values of feces reflect theisotopic composition of diets in small mammals Canadian Journal of Zoology85388ndash396 DOI 101139Z07-019

Kartzinel TR Chen PA Coverdale TC Erickson DL KressWJ KuzminaML Ruben-stein DI WangW Pringle RM 2015 DNA metabarcoding illuminates dietary nichepartitioning by African large herbivores Proceedings of the National Academy of Sci-ence of the United States of America 1128019ndash8024 DOI 101073pnas1503283112

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Montanari S Amato G 2015 Discrimination factors of carbon and nitrogen stableisotopes from diet to hair and scat in captive tigers (Panthera tigris) and snowleopards (Uncia uncia) Rapid Communications in Mass Spectrometry 291062ndash1068DOI 101002rcm7194

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R Core Team 2016 R a language and environment for statistical computing Vienna RFoundation for Statistical Computing Available at httpswwwr-projectorg

Reitsema LJ 2012 Introducing fecal stable isotope analysis in primate weaning studiesAmerican Journal of Primatology 74926ndash939 DOI 101002ajp22045

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Salvarina I Yohannes E Siemers BM Koselj K 2013 Advantages of using fecal samplesfor stable isotope analysis in bats evidence from a triple isotopic experiment RapidCommunications in Mass Spectrometry 271945ndash1953 DOI 101002rcm6649

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vertebrate diets have been assessed macroscopically through physical examination of gutcontents and feces (eg Hermsen Kerle amp Old 2016) With the advent of affordable high-throughput sequencing fecal studies are becoming more common for barcoding dietaryDNA which allows for a more complete dietary picture not as easily biased by differingdigestibility of food (eg Shehzad et al 2012Kartzinel et al 2015) Stable isotopemethodscan provide a useful complement to these barcoding studies that are growing in popularityFecal stable isotopes have been useful for detecting dietary shifts in mammals such asgorillas (Blumenthal et al 2012) for understanding weaning time in primates (Reitsema2012) and as a climate record from bats (Royer et al 2015) so understanding isotopicdiscrimination and variability of this material is vital for future research

Stable isotope ratios of nitrogen (15N14N written δ15N) and carbon (13C12C writtenδ13C) are incorporated in animal tissues and excretions following digestion of foodproducts Changes in stable isotope ratios can elucidate information about food webstrophic structure and habitat use (Ben-David amp Flaherty 2012) It is assumed that carbonisotope ratios do not change drastically as they propagate through the food web (DeNiroamp Epstein 1978) and in terrestrial ecosystems these ratios are generally used to indicatethe primary production at the base of the food web Carbon isotope ratios of plants thatuse different metabolic pathways (C3 C4 or CAM) are systematically different and thisdifference is incorporated into the tissues or byproducts of consumers On the other handδ15N values are known to become enriched as trophic level increases (DeNiro amp Epstein1981) and differences in δ15N of tissue over time or between populations can be used todiscern changes in food webs habitat or prey composition (Post 2002)

To use stable isotopes in dietary ecology there has to be a comprehensive understandingof the difference in isotope ratios between diet and tissue (or feces) this difference is calledthe trophic discrimination factor (also fractionation factor or discrimination factor) and iscaused by isotopic fractionation during digestion and metabolism Trophic discriminationfactor is denoted as 1 and defined as 1= δtissuendashδdiet (Martiacutenez del Rio et al 2009)Trophic discrimination factors (abbreviated TDFs) are widely variable among animalsand differ depending on species tissue examined and diet type and quality (Caut Anguloamp Courchamp 2009) General TDFs are used in many mixing model studies for animaldiet reconstruction but it has been shown that small differences in TDFs in these types ofstudies can lead to vastly different conclusions about dietary makeup (Ben-David amp Schell2001) Experimental work by Caut Angulo amp Courchamp (2008) shows that mixing modelsare most accurate when species-specific TDFs are obtained

In this study I calculate TDFs for carbon and nitrogen isotopes in meerkat (Suricatasuricatta) feces in order to obtain fecal TDFs in a small mammalian omnivore Meerkatsare small diurnal mammals that are members of Carnivora and are herpestids closelyrelated to mongoose They have generalist diets and in the wild consume insects berriesreptiles and other small invertebrates (Doolan amp Macdonald 1996) The diets of generalistconsumers are difficult to study in the wild due to unknown variability in their diet so azoo-based study of meerkats in this instance will provide dietary control allowing for lessvariation in the determination of a TDF Studies like this one are necessary as TDFs arenot often determined for terrestrial mammalian omnivores and carnivores or even feces in

































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Documents

Discrimination factors of carbon and nitrogen stable ...Peromyscus maniculatus 19.4 0.3 3.2 5 Herbivore Hwang, Millar & Longstaffe (2007) Myodes gapperi 19.4 0.3 4.2 5 Herbivore Hwang,