PLANT-ANIMAL INTERACTIONS - ORIGINAL RESEARCH

A faecal index of diet quality that predicts reproductive successin a marsupial folivore

Hannah R. Windley • Ian R. Wallis •

Jane L. DeGabriel • Ben D. Moore •

Christopher N. Johnson • William J. Foley

Received: 31 July 2012 / Accepted: 5 February 2013

� Springer-Verlag Berlin Heidelberg 2013

Abstract Estimating the nutritional value of a herbivore’s

diet is difficult because it requires knowing what the animal

eats, the relative quality of each component and how these

components interact in relation to animal physiology.

Current methods are cumbersome and rely on many

assumptions that are hard to evaluate. We describe a new

method for estimating relative diet quality directly from

faeces that avoids the problems inherent in other methods.

We combine this method with near infrared reflectance

spectroscopy (NIRS) to analyse many samples and thus

provide a technique with immense value in ecological

studies. The method stems from the correlation between the

concentrations of dietary and faecal nitrogen in herbivores

eating a tannin-free diet, but a weaker relationship in

browsers that ingest substantial amounts of tannins, which

form complexes with proteins. These complexes reduce the

availability of nitrogen and may increase faecal nitrogen

concentrations. Using the tannin-binding compound, poly-

ethylene glycol, we showed that tannin-bound nitrogen is a

significant and variable part of faecal nitrogen in wild

common brushtail possums (Trichosurus vulpecula). We

developed a technique to measure faecal available nitrogen

and found that it predicted the reproductive success of

female brushtail possums in northern Australia. Faecal

available nitrogen combined with NIRS provides a power-

ful tool for estimating the relative nutritional value of the

diets of browsing herbivores in many ecological systems. It

is a better indicator of diet quality than other commonly

used single-nutrient measures such as faecal nitrogen and

foliage analysis paired with observed feeding behaviour.

Keywords Nutrition � Tannins � Available nitrogen �PEG � Browser

Introduction

It is widely assumed that the quality of foliage, incorpo-

rating both nutrients and plant secondary metabolites

(PSMs), should influence the life history strategies of wild

browsers. Presumably, animals feeding on higher quality

diets can invest more nutritional resources in reproduction

and growth and proportionately less in detoxification and

maintenance (Zera and Harshman 2001). Nevertheless, it

has proven difficult to demonstrate this link in populations

of wild mammalian herbivores. Nitrogen (N) is generally

Communicated by Joanna Lambert.

H. R. Windley (&) � I. R. Wallis � J. L. DeGabriel � W. J. Foley

Evolution, Ecology and Genetics, Research School of Biology,

Australian National University, Canberra, ACT 0200, Australia

e-mail: hannah.windley@anu.edu.au

J. L. DeGabriel � B. D. Moore � C. N. Johnson

School of Marine and Tropical Biology, James Cook University,

Townsville, QLD 4811, Australia

Present Address:J. L. DeGabriel

Landscapes and Ecosystems Conservation Branch,

Office of Environment and Heritage, PO Box A290,

Sydney South, NSW 1232, Australia

Present Address:B. D. Moore

Hawkesbury Institute for the Environment, University of

Western Sydney, Locked Bag 1797, Penrith, NSW 2751,

Australia

Present Address:C. N. Johnson

School of Zoology, University of Tasmania, Sandy Bay Campus,

Hobart, TAS 7001, Australia

123

Oecologia

DOI 10.1007/s00442-013-2616-9

regarded as a limiting nutrient (e.g. White 1993), and many

studies have attempted to relate total N concentrations in

foliage to the distribution and abundance of herbivore

populations (e.g. Braithwaite et al. 1988; Chapman et al.

2002, 2004; Cork and Catling 1996; Fashing et al. 2007;

Oates et al. 1990; Wasserman and Chapman 2003).

Recently, DeGabriel et al. (2009) and McArt et al. (2009)

used similar approaches to demonstrate that reductions in

protein availability, due to tannins, were correlated with

poorer reproductive success in two species of browsers.

Specifically, DeGabriel et al. (2009) showed that the con-

centration of available nitrogen in leaves (AvailN; the N

available to the animal measured in an in vitro digestion

that accounts for the negative effects of tannins and fibre),

rather than total N, was correlated with the reproductive

success of a population of common brushtail possums

(Trichosurus vulpecula) in northern Australia.

Predicting an animal’s potential reproductive success

from the nutritional quality of its food is not simple. One

problem is measuring and determining the nutritional

quality of what an animal eats while another is obtaining

and analysing hundreds of samples. Near infrared reflec-

tance spectroscopy (NIRS) is a common analytical method

that enables many samples to be analysed rapidly, cheaply

and repeatedly, solving the latter problem (Foley et al.

1998). There are many problems associated with quanti-

fying the diets of wild herbivores (e.g. Shrestha and Wegge

2006), but analysis of faeces may circumvent these by

providing an interpretable sample reflecting an animal’s

diet at a specific point in space and time (Kohn and Wayne

1997; Leslie et al. 2008; Putman 1984).

While it may be difficult to measure how much N an

animal ingests using observational data and diet samples, it

is often easy to measure the N in the faeces. The reason for

doing so is that studies of both domestic and wild mammals

(Hodgeman et al. 1996; Kamler and Homolka 2005;

Kucera 1997; Leslie et al. 2008; Leslie and Starkey 1987;

Verheyden et al. 2011) suggest a positive relationship

between dietary N and faecal N (FaecN), indicating that

FaecN might be a useful proxy for dietary N.

Although Leslie and Starkey (1987) provided a clear

description of when FaecN could be used legitimately in

studies of nutritional ecology, many have ignored their

recommendations while others have warned of the meth-

od’s failings (Hobbs 1987; Robbins et al. 1987). After

measuring marked effects of tannins on N digestion and

excretion, Robbins et al. (1987) stated that FaecN ‘‘is not a

precise indicator of any dietary parameter and should not

be used in ecological studies’’. Clearly, there are competing

factors: diets with more N lead to faeces with more N,

while tannins may also elevate concentrations of FaecN.

Many animal nutritionists have assumed that the only

undigested dietary N in faeces is that associated with plant

cell walls (Schwarm et al. 2008), but this is not true when

tannins are present, and tannins are ubiquitous in woody

plants (Ayres et al. 1997). For example, tannins in the diet

of howler monkeys (Alouatta spp.) increase their faecal

excretion of N (Milton et al. 1980). This is because indi-

gestible complexes form between tannins and proteins

(Bilgener 1988; Hagerman et al. 1992; Makkar 2003).

Another example is that some animals eating tannin-rich

diets secrete more digestive enzymes, increasing FaecN

(Schwarm et al. 2008). Verheyden et al. (2011) concluded

that FaecN bore no relationship to dietary N when there

was more than 2 % condensed tannin in the diet, implying

that measuring FaecN is futile in the presence of tannins.

Clearly, the development of a faecal index that reflects the

nutritional quality of diets that contain tannins would rep-

resent an important advance in ecology.

DeGabriel et al. (2008) developed an in vitro assay to

quantify the amount of N in foliage that is available to

browsers (AvailN), using polyethylene glycol (PEG),

which preferentially binds to tannins and prevents them

binding to protein. Tannin–protein complexes can pass to

the faeces (Makkar 2003). Thus, if we subject a sample of

faeces from an animal that ingested tannins to the in vitro

protein digestibility assay of DeGabriel et al. (2008) to

measure faecal available N (FaecAvailN), we should find

that treatment with PEG releases the protein from any

tannin–protein complexes. This would indicate the capacity

of the ingested tannin to bind protein and quantify the loss

of protein due to tannins. Several factors complicate this

scenario. First, the method depends on faeces varying in

both N and FaecAvailN. Second, PEG must liberate tannin-

bound N in faeces, as it does from foliage, so that samples

incubated with and without PEG differ in their concentra-

tions of FaecAvailN. Finally, it is necessary to demonstrate

that increasing concentrations of dietary tannins reduce the

concentration of FaecAvailN when dietary N remains

constant.

As far as we are aware, there are no published accounts

that assess dietary quality by measuring the concentration

of tannin-bound protein in the faeces. This is surprising

because doing so would solve many of the problems cur-

rently associated with FaecN analysis. Faeces contain

dietary residues, microbes and endogenous material and

thus represent an animal’s diet and physiological state at a

particular time and place. Therefore, where N is limited, an

index based on quantifying the amount of tannin-bound N

relative to the total N concentration in faeces would be a

better indicator of the nutritional status of wild mammals

than observations of feeding and analyses of plants within

the animal’s home-range. Furthermore, this approach

combined with NIRS allows ecologists to link the nutri-

tional status of free-ranging herbivores to their demogra-

phy in many plant–mammal systems.

Oecologia

123

The broad aim of this study was to evaluate the use of

faeces for studying the nutritional and population ecology

of herbivores while accounting for the effects of tannins.

We did this by testing three hypotheses. First, we manip-

ulated tannin concentrations in the diets of captive brushtail

possums to test the hypothesis that the concentration of

FaecAvailN reflected the dietary composition of tannins (a

validation experiment). Second, we obtained a large sam-

ple of faeces from wild brushtail possums living in dif-

ferent nutritional environments to test the hypothesis that

they vary in their concentrations of both total N and

FaecAvailN, and that a substantial amount of this variation

is due to tannins. Finally, we examined whether the con-

centration of FaecAvailN measured in wild brushtail pos-

sums reflected the reproductive success of individual

females and the growth rates of their young.

Materials and methods

Validation of relationship between diet AvailN, FaecN

and FaecAvailN

To test whether faeces reflect the changes in the AvailN of a

diet, we fed six adult male common brushtail possums

leaves from two Eucalyptus melliodora and from one

E. polyanthemos under controlled conditions, following

procedures described previously (Wallis et al. 2002). We

placed branches in buckets of water within 20 min of cutting

and stored them in a dark room maintained at 5 �C. We

weighed bunches of foliage and placed them in water-filled

feeding tubes provided for each possum at 1700 hours.

We manipulated the AvailN content of foliage by dip-

ping branches in a 20 % polyethylene glycol (PEG) (MW

*4,000) solution following the procedure of Marsh et al.

(2003). Possums were offered untreated foliage from one

tree for 7 consecutive days. PEG-treated foliage from this

tree was then offered to the possums for another 7 days.

Faeces from the last 2 days of each 7-day period were

collected for analysis. This was repeated for untreated and

PEG-treated foliage from the other two trees (six diets in

total).

Possums failed to maintain weight when eating only

E. polyanthemos foliage so we supplemented it with fruit.

Fruit was not expected to have a marked influence on the

AvailN of the diet due to its very low N concentration

(\0.1 % wet matter). Instead, we expected that the fruit

would increase the digestibility of the diet. We collected

faeces from each possum on the last 2 days of the exper-

iment and dried them at 40 �C to constant mass. We

determined concentrations of N and AvailN in faeces and

on freeze-dried samples of the foliage according to

DeGabriel et al. (2008).

Variation in N and AvailN concentrations of faeces

collected from wild possums

We measured the N and AvailN concentrations in the

presence and absence of PEG in 337 faecal samples col-

lected from 180 wild common brushtail possums (93 males

and 87 females) that we trapped between 2004 and 2006 at

six sites in north Queensland, Australia. We refer to these

measures in faeces as FaecN, FaecAvailN and Faec-

AvailNPEG, to distinguish them from similar measures on

plant material. We collected pellets from the floor of the

trap on the first night that an individual was caught to

minimize the presence of bait in the faeces. We stored the

faeces at -20 �C before freeze-drying and grinding them

to pass a 1-mm sieve in a Tecator Cyclotec Mill (Foss,

Hillerød, Denmark).

We carried out a single trapping session (4–5 nights) at

five of the sites as part of a study to determine possum

densities (Moore, unpublished data), but trapped the

remaining site (Tabletop Station; TT1) at approximately

6-weekly intervals from October 2004 to January 2006 as

part of a more detailed demographic study (DeGabriel et al.

2009). We sampled most of the sites in the dry season

(April–November) in 2005, a dry year. Site BB4 was the

exception, being sampled in November 2006, which,

although dry, followed heavy rain in the first 4 months of

the year. The sites were all open Eucalyptus woodlands

dominated by species from the red ironbarks (series Sid-

erophloiae). In a separate study, DeGabriel et al. (2008)

reported that the AvailN concentration of eucalypt leaves at

these sites varied markedly both within species and

between sites.

Analytical

We used the in vitro assay of DeGabriel et al. (2008) to

determine concentrations of FaecAvailN and FaecAvailN-

PEG. Briefly, we weighed 0.8050 ± 0.0050 g of dried,

ground faeces into four filter bags (ANKOM F57; ANKOM

Technology, Macedon, NY, USA) that we heat-sealed. We

incubated two of the bags for 24 h in 0.05 M Tris-BASE

buffer, pH 7.1 (Sigma), and the other two with the same

buffer containing 33.3 g l-1 PEG (both 25 mL per bag).

Incubating with PEG releases protein from tannin–protein

complexes. We then incubated both sets of samples, still

separated, for 24 h with pepsin [2.00 g l-1 1:10,000 pep-

sin;DifcoTM Bacto Laboratories, Mt Pritchard, Australia; in

0.1 N HCl; 25 mL per bag] and then for 48 h with cellulase

(6.25 g l-1 cellulase; Onazuka 3S, Yakult Japan; in 0.1 M

acetate buffer pH 4.75, 25 mL per bag) with thorough

washing with distilled water between incubations. Pepsin

digests protein and the cellulase preparation digests much

of the fibre, releasing fibre-bound N that is not associated

Oecologia

123

with indigestible lignin. After digestion, we washed the

bags thoroughly and dried them to constant mass before

reweighing them to determine the remaining dry matter.

We determined the N concentrations of the original

faecal material and the residues after digestion using a

Dumas procedure on a LECO TruSpec� CN analyser

(LECO, St Joseph, MI, USA) using 200 mg of faeces or

100 mg of residue. We then calculated the concentrations

of FaecAvailN and FaecAvailNPEG in the original

material.

FaecAvailN or FaecAvailNPEG ð% DMÞ¼ Ns � N digestibility ð1Þ

for samples incubated without and with PEG where,

N digestibility ð% DMÞ ¼ 100 � ½½ðDry sample mass

� NsÞ � ðDry residue mass

� NrÞ�=ðDry sample mass

� NsÞ�ð2Þ

where Ns and Nr are the N concentrations (% DM) in the

original faecal sample and in the digestion residue,

respectively.

Faecal available N: an integrative faecal index

The AvailN concentration of a herbivore’s diet depends on

the N concentration of the diet and its digestibility. Two

main factors reduce the digestibility of N in foliage that

animals eat—tannins and fibre. Thus, a faecal index of diet

quality in animals ingesting tannins must account for the

overall quality of the diet, as indicated by the total N

concentration in the faeces, the protein-binding effect of

the tannins ingested and the amount of N bound by fibre.

FaecAvailN does this: diets richer in N should increase

FaecAvailN while increasing concentrations of dietary

tannins and N-binding fibre will decrease it.

We quantified these effects by incubating samples with

and without PEG.

Effect of tannis¼ FaecAvailNPEG � FaecAvailN ð3ÞEffect of fibre ¼ Faecal N ð%Þ � FaecAvailNPEG :

ð4Þ

Near infrared spectroscopy (NIRS)

We developed calibrations using NIRS to predict the con-

centrations of N, FaecAvailN and FaecAvailNPEG. We

collected NIRS spectra (400–2,500 nm) of all faecal sam-

ples using a FOSS-NIR Systems 6500 scanning monochro-

mator with a spinning cup attachment (Foss, Silver Spring,

MD, USA), following the procedure described by Moore

et al. (2004). We selected the calibration set for chemical

analysis using the programme SELECT implemented in

WinISI III (Infrasoft International, Port Matilda, PA, USA).

We developed calibrations using WinISI by following

the procedures described for quantitative infrared analysis

(American Society for Testing and Materials 1995). We

selected equations for the parameters of interest based on

the standard error of calibration (SEC), the standard error

of cross validation (SECV), the coefficient of determina-

tion between the spectra and the analytical values (the r2 of

calibration), the proportion of variation explained by cross-

validation (1-VR) and the standardised SECV—the ratio of

the standard deviation of the sample set to the SECV.

Preferred equations gave both low values and agreement

between values for SEC and SECV; similarly, we looked

for agreement between r2 and 1-VR but with values close

to 1 (i.e. explaining most of the variance). There was

excellent agreement between the laboratory values and the

NIRS values predicted from the partial least squares

regression of the spectra with the calibration set for FaecN,

FaecAvailN and FaecAvailNPEG (Table 1). Thus, we used

the calibration equations to estimate these parameters for

all of the samples.

Table 1 The statistical results relating near infrared spectra of common brushtail possums (Trichosurus vulpecula) faeces to analytical values

(% dry matter)

Factor (% DM) n Mean Data processing SD SEC SECV r2 1-VR SD/SECV

FaecN 184 2.885 2, 8, 8, 1 0.821 0.103 0.142 0.98 0.97 5.7

FaecAvailN 190 1.190 2, 4, 4, 1 0.579 0.115 0.152 0.96 0.93 3.8

FaecAvailNPEG 186 1.945 2, 4, 4, 1 0.654 0.097 0.126 0.98 0.96 5.2

FaecDMD 187 27.8 1, 4, 4, 1 6.3 2.4 2.7 0.85 0.81 2.3

FaecDMDPEG 188 30.8 2, 8, 8, 1 6.6 1.8 2.5 0.92 0.86 2.7

Data processing provides details of the derivation and smoothing of the spectra (derivative, gap, first smoothing, second smoothing). Thus,

‘‘2,8,8,1’’ refers to using the second derivative, leaving a gap of eight wavebands between calculated values, doing a first smoothing over eight

wavebands and then a second smoothing over one waveband

n number of samples used in the equation, SEC standard error of calibration, SECV the standard error of cross validation, r2 the coefficient of

determination between the spectra and the analytical values, 1-VR coefficient of determination of cross validation and SD/1-VR

Oecologia

123

Predicting reproductive success and growth of pouch

young from FaecAvailN

We used life history data collected by DeGabriel et al.

(2009) over 2.5 years for the population at Site TT1. They

radio-tracked all adult female possums in the population to

quantify patterns of tree use and determined the average

nutritional quality of trees within the home ranges. They

also trapped the population at 6-weekly intervals to collect

data on the reproductive success and growth rates of pouch

young (head length measurements during the 4–5 months

of pouch life) for up to five breeding seasons. We obtained

reproductive success data, defined as the percentage of

observed breeding seasons that a female successfully

reared a pouch young to pouch emergence, for 13 possums.

We collected between one and eight faecal samples for

each of these possums (n = 1, 2, 2, 2, 4, 4, 4, 5, 7, 7, 8, 8,

and 8 faecal samples for each possum, respectively) and

used a mean value of faecal composition in statistical

analyses.

Statistical

Validation experiment

We analysed the eucalypt validation experiment using one-

way ANOVA with diet as the treatment effect and possum

as the block.

Field studies

We used descriptive statistics and least squares regression

analysis to quantify the variation in FaecN, FaecAvailN

and FaecAvailNPEG and the relationships between the

parameters. We used an unbalanced ANOVA to examine

seasonal differences in faecal parameters at Site TT1 only

and to compare the influence of gender and site on faecal

composition for the dry season only.

For the samples collected at TT1, we used univariate

and multivariate statistics to test whether total FaecN or

FaecAvailN could explain variation in reproductive suc-

cess. The data were binomial (possums may or may not

reproduce in a particular breeding season) so a logistic

regression model was used, incorporating a logit-link

function, with reproductive success as the response vari-

able (DeGabriel et al. 2009). In some cases, there were

several faecal samples for a female so we used the mean

faecal measure (FaecN or FaecAvailN) for each individual.

In order to test whether components of the faeces can

predict the growth rates of pouch young, we used linear

mixed models with the mean growth rate of the young’s

head whilst in the pouch as the dependent variable and the

mother’s identity as a random term.

Results

Validation of relationship between diet AvailN, FaecN

and FaecAvailN

We successfully manipulated the composition of the faeces

of captive possums by changing the composition of a

foliage diet, specifically by altering the action of tannins.

We were able to see changes in FaecAvailN when the diet

varied naturally and when we artificially changed the

AvailN of the diet using PEG. We found our method,

measuring FaecAvailN, was more closely related to dietary

AvailN than was FaecN (Fig. 1). FaecN was always much

higher than both AvailN and FaecAvailN. FaecN concen-

tration was reduced when PEG was added to the diet while

FaecAvailN did not change.

Possums ate more foliage when it contained a higher

concentration of N or of AvailN both naturally and due to

manipulation with PEG. However, the amount eaten was

more closely related to the leaf AvailN (r2 = 0.64,

F1,34 = 63; P \ 0.001) than to the leaf total N (r2 = 0.30,

F1,34 = 16.0; P \ 0.001). Possums ate more of the foliage

treated with PEG but reduced their excretion of FaecN.

Even when supplementing the diet with fruit, the effects for

E. polyanthemos foliage were weaker than for E. mellio-

dora: treating E. polyanthemos foliage with PEG did not

stimulate feeding as it did for E. melliodora nor did it

reduce the N bound to tannin (tanninN) in the faeces by the

same degree.

Diet1 2 3 4 5 6

Nitr

ogen

(%

dry

mat

ter)

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Fig. 1 The influence of diet on the nitrogen chemistry of faeces

collected from common brushtail possums (Trichosurus vulpecula)

fed foliage from two E. melliodora and one E. polyanthemos fed

without PEG and with PEG. Diets 1 and 2 were E. melliodora one

without and with PEG respectively, Diets 3 and 4 were E. melliodoratwo without and with PEG, respectively, and Diets 5 and 6 were

E. polyanthemos without and with PEG, respectively. The triangles,

closed circles and open circles are dietary AvailN, FaecAvailN and

FaecN, respectively. Error bars SD (n = 6)

Oecologia

123

Variation in nitrogen and AvailN concentrations

of faeces collected from wild possums

Faeces from free-living brushtail possums varied widely in

their concentrations of FaecN between sites [1.27–5.33 %

DM, mean = 2.94, coefficient of variation (CV) = 29;

Table 2], FaecAvailN (0.12–3.01 % DM, mean = 1.19,

CV = 49; Table 2) and FaecAvailNPEG (0.59–3.80 % DM,

mean = 1.96, CV = 35; Table 2). We observed a positive

relationship between the concentrations of FaecAvailN and

FaecN (r2 = 0.70, F1,184 = 440, P \ 0.0001; Fig. 2a), but a

stronger relationship between FaecAvailNPEG and FaecN

(r2 = 0.96, F1,184 = 4,368, P \ 0.0001; Fig. 2b). As the

FaecN increased so did the N bound to tannin (tanninN;

r2 = 0.30, F1,184 = 79, P \ 0.0001) and the N bound to

fibre (fibreN; r2 = 0.47, F1,184 = 163, P \ 0.0001). The

tannin and fibreN were also positively related (r2 = 0.31,

F1,184 = 82; P \ 0.0001). There was a positive relationship

between faecal dry matter digestibility (FaecDMD) and

FaecAvailN (r2 = 0.63, F1,184 = 314; P \ 0.0001). We

explored this relationship further through regression of the

residuals from the ‘‘tanninN versus FaecN’’ and ‘‘fibreN

versus FaecN’’ relationships against FaecDMD. There was a

negative relationship between the tannin residuals and

FaecDMD (r2 = 0.35, F1,184 = 101; P \ 0.0001) indicating

that faecal samples with lower concentrations of tanninN at

any concentration of FaecN have higher FaecDMD. In

contrast, the fibreN residuals explained less of the variation

in FaecDMD (r2 = 0.11, F1,184 = 22; P \ 0.0001).

Effects of season, site and sex on the composition

of faeces

The N concentration of faeces differed between sites, while

males produced faeces with more N than did females (2.91

vs. 2.78 %, F1,144 = 6.5, P = 0.012; Table 2). Due to the

positive relationships between most other faecal parame-

ters (FaecAvailN, FaecDMD, etc.) and FaecN, we included

FaecN as a covariate in the unbalanced ANOVAs that

examined the effects of site and sex. At TT1, the only site

with faeces collected in both seasons, faeces tended to

contain more AvailN in the wet season (1.31 vs. 1.09 %;

P = 0.065). There were no differences between seasons for

the other faecal parameters.

The variation in leaf AvailN between sites did not

explain the variation in FaecN or in FaecAvailN. Likewise,

in a model that also included the body mass of the mother,

leaf AvailN within a home-range at TT1 did not explain

differences in faecal chemistry of the female inhabiting the

home range.

Predicting reproductive success and growth of pouch

young using FaecAvailN

We found a positive relationship between FaecAvailN and

reproductive success (t = -2.76, P = 0.006, n = 13;

Fig. 3). In contrast, there was no relationship between

FaecN and reproductive success (t11 = 1.36; P = 0.172).

In models that also included body mass of the female

(Wald = 6.25; P = 0.031, n = 13), there was no rela-

tionship between various measures of a mother’s faeces

(such as total N or FaecAvailN) and the mean growth rate

of her young.

Discussion

Our results show that faeces produced by individual her-

bivores vary in composition and can provide a signature of

the nutritional status of the animal that deposited them.

This signature predicts the reproductive success of wild

brushtail possums.

Table 2 The composition of faeces collected during the dry season from male and female possums inhabiting six sites in north Queensland,

Australia

Parameter Site (n) Gender

BB4

(40)

MF1

(30)

TT1

(48)

TT2

(14)

TV1

(28)

TV2

(12)

P lsd

5 %

Male

(94)

Female

(78)

P lsd

5 %

FaecN 3.41 2.56 2.87 3.69 2.54 2.44 \0.001 0.17 2.98 2.86 ns

FaecAvailN 1.20 1.14 1.21 1.10 0.85 1.07 \0.001 0.076 1.14 1.09 ns

FaecAvailNPEG 2.00 1.91 1.97 1.91 1.85 1.84 \0.001 0.032 1.93 1.94 ns

FaecDMD 25.6 26.5 29.8 26.1 24.7 25.1 \0.001 1.16 27.3 26.1 0.042 0.58

FaecDMDPEG 29.7 27.8 32.7 29.8 28.4 26.7 \0.001 1.32 29.8 28.1 0.023 0.66

FaecFibreN 0.92 1.01 0.96 1.01 1.07 1.09 \0.001 0.032 0.99 0.99 ns

FaecTanninN 0.80 0.77 0.76 0.81 1.00 0.77 \0.001 0.063 0.80 0.84 ns

The values for FaecN are means. All other values are means (% DM or % for DMD), corrected for the highly significant effect of FaecN

(P \ 0.001 for all parameters)

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In an intensive study of one northern Australian popu-

lation, Degabriel et al. (2009) showed that both reproduc-

tive success and the growth of pouch young depended on

the average AvailN concentration of eucalypt foliage in a

possum’s home range. The total N concentration of the

foliage had no effect on reproduction, while subtle differ-

ences in AvailN proved the difference between success and

failure. Two factors largely dictate how much of the dietary

N is unavailable: fibre and tannins. The difference between

these fractions and the total N content of the diet is the

AvailN. We assumed that these factors result in a faecal

signature of diet quality. In grazing animals, which largely

avoid foods containing tannins, there is a strong positive

relationship between dietary N and FaecN (Leslie and

Starkey 1987; Hodgeman et al. 1996; Kucera 1997; Kamler

and Homolka 2005; Leslie et al. 2008). This relationship

deteriorates, however, in browsing animals that consume

large amounts of tannins (Verheyden et al. 2011),

prompting some authors to reject faecal analysis for

gleaning information about the nutrition and life-history

traits of animals that ingest tannins (e.g. Robbins et al.

1987). We dissected the faecal signature using the method

of DeGabriel et al. (2008) and came to three conclusions.

First, the concentrations of FaecN and FaecAvailN varied

widely in samples collected from wild possums in different

seasons and from different places. Second, incubating

faecal samples with PEG released substantial protein from

tannin–protein complexes. Thirdly, studies with captive

possums showed that dietary tannins reduced the concen-

tration of FaecAvailN. Additionally, we could predict all of

these faecal traits using NIRS. The speed of NIRS enables

the researcher to obtain compositional data quickly and

then to link changes in composition to changes in the field,

such as an animal changing its diet or a small change in the

nutritional composition of a food.

The index that best reflects the diet and its interaction

with the animal, FaecAvailN, does not require one to

measure the amount of N bound to tannin. In this study,

however, we wanted to distinguish the components of

FaecN to understand why FaecAvailN varied. After cor-

recting for total N in the faeces, FaecAvailN was the main

component of FaecN at three sites, and what we assume to

be N bound to fibre, the largest at the other three sites.

However, the N bound to tannins was always substantial

accounting for 27–35 % of FaecN.

FaecN comprises three main components—undigested

dietary N, endogenous N and microbial N. The concen-

tration of microbial N in the faeces depends on several

factors including digestive strategy, plane of nutrition,

health, rate of passage of food through the gut and body

mass (Mason 1969). For instance, we would expect ca-

ecotrophic hindgut fermenters, such as ringtail possums, to

excrete less microbial and endogenous N than animals

Faecal nitrogen (% dry matter)

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5

Faec

al a

vaila

ble

nitr

ogen

PE

G (

% d

ry m

atte

r)

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

(r2 = 0.96; F1,184 = 4368; P < 0.0001)

b

Faec

al a

vaila

ble

nitr

ogen

(%

dry

mat

ter)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5 a

(r2 = 0.70; F1,184 = 440; P < 0.0001)

Fig. 2 The relationship between a FaecAvailN and FaecN and

between b FaecAvailNPEG and FaecN in faeces collected from 186

common brushtail possums in north Queensland, Australia

Faecal Available nitrogen (%)

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Rep

rodu

ctiv

e su

cces

s (%

)

0

20

40

60

80

100

Fig. 3 The relationship between reproductive success and Faec-

AvailN of 13 female common brushtail possums at a single site in

north Queensland, Australia. Reproductive success was the percent-

age of observed breeding seasons that a female successfully reared a

pouch young. FaecAvailN is the average of several faecal samples for

each female. The solid line shows the logistic regression model

Oecologia

123

occupying similar feeding niches that do not practice

caecotrophy (Schwarm et al. 2008). Also, increasing con-

centrations of dietary N result in faeces with more N.

While we are yet to test the method with a range of foregut-

and hindgut-fermenting animals, we argue that the in vitro

digestion process will eliminate the highly digestible

sources of N, such as endogenous N and microbial N, so

that N bound by fibre or by tannins explains all the vari-

ation in the N content of a sample following in vitro

digestion. Animals eating less nutritious food, in terms of

N, will produce faeces with more indigestible N—either

bound to fibre or to tannins. Incubating samples with PEG

enables us to identify the cause of the indigestible N.

We could predict the reproductive success of female

possums with FaecAvailN. DeGabriel et al. (2009) did the

same but based on extensive sampling of foliage. In that

study, females occupying home-ranges containing trees

with leaves higher in AvailN, bred more often and pro-

duced faeces with more FaecAvailN. This demonstrates a

link between the nutrition of the individual and demogra-

phy. We could not find a relationship between FaecAvailN

and the growth rate of pouch young. There are two related

explanations for this. The most likely is that the consid-

erable variation in FaecAvailN was much easier to asso-

ciate with the high degree of variability in fecundity but

much harder to link to the small variation in growth rates.

The second explanation is that the collection of faeces was

opportunistic and samples did not cover critical times in the

reproductive cycle. Nonetheless, the success of this study

warrants another with carefully planned collections of

faeces.

While the opportunistic nature of the field study may

explain the inability of foliar chemistry to directly explain

faecal chemistry, it is also likely that the foliage sampled

by Degabriel et al. (2008) did not completely reflect the

actual diet of the animals. Even at TT1, the site where

DeGabriel et al. (2009) mapped the home-ranges of radio-

collared possums and collected foliage from most of the

trees, foliar chemistry did not explain faecal chemistry.

Likewise, there was no relationship between these mea-

sures over all sites. Sometimes, we even found the opposite

of expectations. For example, FaecAvailN indicated that

site TV1 was extremely poor, whereas DeGabriel et al.

(2008) ranked it among the best based on the AvailN in the

foliage. This indicates the difficulty of relating demo-

graphic indices of animals to chemical attributes of a site

without knowing much about the animal population. Ani-

mals can adapt to nutritionally poor areas through physi-

ological and behavioural mechanisms, for instance by

increasing the size of their home range, slowing their

breeding (e.g. Krockenberger 2003) or by feeding on other

items such as fungi (How and Hillcox 2000). In this study,

there was also no control for differences in season or

rainfall or changes in the physiology of animals, such as

lactation.

It is likely that the foliage samples analysed from Site

TV1 did not reflect what the animals were eating because

we would expect the opposite response: high FaecAvailN

concentrations and low AvailN in foliage, indicating that

possums were finding relatively good food in an apparently

poor nutritional environment. Foliage samples provide only

an indication of diet and may even represent what the

animals chose not to eat! In contrast, there is no doubt that

faeces, although still only a snapshot in time, represent

what the animal ate and thus eliminate decisions about

what to sample.

The fact that FaecAvailN predicted reproductive suc-

cess, as did an extensive foliage sampling study, suggests

that this approach may benefit any nutritional study of

vertebrates that ingest tannins. We must acknowledge,

however, that there are explanations other than that Faec-

AvailN predicts the nutritional quality of the home range.

Apart from information about the diet, FaecAvailN also

contains the signature of the animal that deposited the

faeces. The foraging decisions of wild herbivores are

complex and there are several reasons why one possum

may have better nutrition than another and why one may

have higher reproductive success. For example, individual

possums may differ in their abilities to choose optimal

diets, as do sheep (Provenza et al. 1992, 1998, 2000).

Mammals are not born with ‘‘nutritional wisdom’’ and

learn much about foraging from their mothers, conspecifics

or through trial and error. This is hardly surprising when

we consider that folivores must gain protein, carbohydrates

and other nutrients from foliage while minimizing their

consumption of toxins and avoiding predators. There has

been little attention paid to individual variation in diet

selection, which is necessary for an evolutionary consid-

eration of the importance of nutrition on reproductive

success. Instead, logistical constraints in sampling and

analysis force most researchers to determine an average

diet for a population. The use of FaecAvailN removes these

constraints and easily provides information on individual

animals. Extensive sampling in conjunction with NIRS

makes a wide variety of studies possible, such as dietary

changes in response to season or reproductive status that

might otherwise go unnoticed. As we show in this paper,

measuring individual variation provides the crucial link

between nutrition and demography. It also confirms the

importance of bottom-up processes, in terms of nutrient

supply from plants, in the demography of a population of

brushtail possums in northern Australia (DeGabriel et al.

2009). This reinforces the theses of others (Braithwaite

et al. 1984; Cork and Catling 1996) who attributed the

patchy distribution of arboreal folivores to the concentra-

tion of nutrients in eucalypt leaves—‘‘the nutrient

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123

threshold hypothesis’’. It confirms their emphasis on N and

phenolic compounds but goes one step further by com-

bining these into a measure of available N measured in the

faeces.

It is important to note the difficulty of linking nutrition

and demography in wild herbivorous animals, with few

studies ever showing an association. Although we suc-

ceeded in predicting reproductive success in one popula-

tion, other factors, such as genetic variation between

mothers, must be important. Nevertheless, as female

brushtail possums inherit home-ranges from their mothers

(Johnson et al. 2001), there may be interactions between

genotype and nutrition. While it appears that a faecal

measure of nutrition can succeed only when nutrition

influences life-history traits, we cannot discount genotype

by nutrition interactions contributing significant variation.

This provides scope for FaecAvailN to explore these pro-

cesses in animals that ingest tannins.

Acknowledgments We thank R. and E. Fryer, Australian Wildlife

Conservancy, and other land owners for permission to use their land.

We thank Elesha Curran for assistance with field and laboratory work.

Funding was provided by grants from the Australian Research

Council to C.N. Johnson and W.J. Foley and the Ecological Society of

Australia and Royal Zoological Society of N.S.W. to J.L. DeGabriel.

Conflict of interest The authors declare that they have no conflict

of interest.

Ethical standards The procedures were approved by the Animal

Experimentation Ethics Committee of the Australian National Uni-

versity and conform to the Guiding Principles in the Care and Use of

Animals. Experiments comply with the current laws of Australia.

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