Functional Ecology 2013, 27, 1176–1185 doi: 10.1111/1365-2435.

12123

Eggshell coloration reflects both yolk characteristics

and dietary carotenoid history of female mallards
Michael W. Butler *,† and Kevin J. McGraw
School of Life Sciences, Arizona State University, PO Box 874601, Tempe, Arizona 85287-4601, USA

Summary

1. Avian eggshell coloration has frequently been examined in a functional context (e.g. mim-
icry, camouflage), but in recent years, an interest has emerged in identifying the mechanisms
that drive eggshell colour variation.
2. Eggshell coloration is predominately caused by pigment deposition; one such pigment is the
antioxidant biliverdin, and deposition of biliverdin into eggshells may be costly to mothers due
to depletion of their antioxidant reserves. Previous work has shown that dietary supplementa-
tion during laying with another type of antioxidant – carotenoid pigments – induces females to
produce more biliverdin-rich eggshells. However, the impact of pre-laying nutrition – including
the developmental period early in life – on eggshell coloration has not been investigated.
3. Here, we raised female mallards (Anas platyrhynchos) from hatching, supplemented their
diets with carotenoids during early-, mid- or late-developmental periods, and at adulthood
measured female circulating carotenoid levels, yolk carotenoid levels, and eggshell coloration.
We found that carotenoid supplementation during the late stages of development (transitional
period from juvenile to adult plumage) promoted the laying of eggs with more biliverdin-rich
eggshells. Independent of developmental dietary treatment, females with higher circulating
carotenoid levels at the time of egg laying produced more biliverdin-rich eggshells and more
carotenoid-rich yolks. When controlling for female identity, we found that more biliverdin-rich
eggshells were associated with more carotenoid-rich, but smaller, yolks. We also detected a lay-
ing order effect; later-laid eggs had larger, less carotenoid-rich yolks and less biliverdin-rich
eggshells.
4. Taken together, these results demonstrate that eggshell coloration reveals carotenoid status
of both mothers and yolks and that diet quality more than 1 month prior to laying can affect
eggshell coloration in a waterfowl species. As mallards are considered to be capital breeders in
terms of lipid stores, our findings provide a new developmental perspective on the carryover of
lipid-soluble and antioxidant nutrient reserves for breeding.

Key-words: Anas platyrhynchos, auto-communication, biliverdin, capital breeding, carote-

noids, developmental plasticity, mallards, yolk size

patches (De Ayala et al. 2007) that are used in

Introduction
Behavioural ecologists have traditionally devoted most of
their research attention on exaggerated traits to adult ani-
mals (e.g. sexual selection; Andersson 1994), but increas-
ingly, studies are focusing on elaborate features of
neonates and juveniles (e.g. Boncoraglio, Caprioli & Saino
2012). Such features include vocalizations (Dreiss, Lahlah
& Roulin 2010), odours (C
el
erier et al. 2011) and colourful
*Correspondence author. E-mail: butlermw@lafayette.edu
† and hosts (Honza & Polacikov
a 2008). However, there is
Present address. Lafayette College, Easton, Pennsylvania, USA
offspring–offspring or parent–offspring communication
(Shizuka & Lyon 2010). For oviparous species, such com-
munication may occur even earlier in life, via coloration of
eggshells.
Eggshell coloration has long been viewed as a trait that
enhances camouflage (Lee, Kwon & Yoo 2010), plays a
thermoregulatory (Maurer, Portugal & Cassey 2011) or
structural-integrity role (Gosler, Higham & James
Reynolds 2005; Cherry & Gosler 2010), or is the product
of a co-evolutionary arms race between brood parasites

© 2013 The Authors. Functional Ecology © 2013 British Ecological Society


mounting interest in the intraspecific quality-signalling role
of conspicuous eggshell colours (Moreno & Osorno 2003).
For example, producing exaggerated shell coloration is
argued to be costly and honestly reveals maternal quality
and offspring quality (i.e. variation in egg contents) to
fathers, who may vary paternal investment accordingly
(English & Montgomerie 2010).
To date, all attention on costliness of quality-revealing
eggshell colours has been placed on the environmental
and physiological conditions of the laying mother
(e.g. Hargitai, Her
enyi & T€or€
ok 2008; Hargitai, Mateo &
T€or€
ok 2010; Honza et al. 2011). For example, investiga-
tors have examined body condition (Mart
ınez-De La
Puente et al. 2007) and manipulated dietary antioxidant
levels (Morales, Velando & Torres 2011; Dearborn et al.
2012) of females during egg production and measured
aspects of eggshell coloration (Morales, Velando &
Torres 2011), egg size (Walters & Getty 2010), and yolk
nutritional profile (Navarro et al. 2011). This emphasis
on assessing or manipulating conditions during adulthood
makes sense given that this is the time period when shells
are pigmented and eggs are laid (shells are generally col-
oured within 24 h of laying; Sparks 2011), but it is also
important to consider conditions during the long period
leading up to egg production due to potentially long-
lasting effects of developmental plasticity (Monaghan
2008).
There has been some work to date on developmental
plasticity of pigment acquisition and utilization in birds,
but it has largely focused on the diet-derived carotenoid
pigments that are present in egg yolk (Blount et al.
2003). In contrast, eggshells are coloured by porphyrin
pigments (Kennedy & Vevers 1976; Gorchein, Lim &
Cassey 2009), which can be synthesized de novo (Maurer,
Portugal & Cassey 2011) and thus may carry different
costs and benefits of synthesis and allocation to eggs.
Many studies on eggshell coloration focus on one class of
porphyrins – the bilins (e.g. biliverdin, which creates blue-
green shell colour; Riehl 2011) – because they can have
antioxidant properties (Kaur et al. 2003). Some have
hypothesized that it may be costly for mothers to allocate
this pigment to eggshells, and thus, colour intensity is a
function of maternal antioxidant supplies (Hanley, Heiber
& Dearborn 2008). In support of this relationship, biliver-
din-based eggshell coloration reflects yolk antioxidant lev-
els (e.g. in spotless starlings, Sturnus unicolor; Navarro
et al. 2011), and supplementation of dietary antioxidants
such as carotenoids to laying mothers can result in
increased biliverdin-pigmented eggshell coloration (blue-
footed boobies, Sula nebouxii; Morales, Velando & Torres
2011) and in yolks with increased carotenoid con-
centrations (zebra finches, Taeniopygia guttata; McGraw,
Adkins-Regan & Parker 2005). Moreover, exposure to
carotenoids early in life can affect adult carotenoid
assimilation ability (Blount et al. 2003), yielding a sys-
tem wherein nutritional conditions during development
may affect adult pigment-associated traits, including
© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology, 27, 1176–1185
Developmental plasticity and eggshell coloration 1177
allocation of carotenoids to egg yolk and of biliverdin to
eggshells.
Given the paucity of data on how pre-laying conditions
of females affect eggshell coloration, we experimentally
tested the effect of developmental nutrition on yolk and
eggshell characteristics in female mallards (Fig. 1; Anas
platyrhynchos). Mallard eggs range from grayish to green-
ish in colour (Drilling, Titman & McKinney 2002;
Kreisinger & Albrecht 2008), and clutch sizes typically
range from 6 to 13 (Drilling, Titman & McKinney 2002).
This egg laying comes at a great cost to females, as they
lose considerable lipid reserves and half of their body mass
during this time (Krapu 1981). Some female mallards can
lay up to three clutches in a year, with renesting occurring
after prior nest failures (Arnold et al. 2010). We raised
female mallards from hatch and supplemented their diets
with carotenoids at three different points throughout devel-
opment, allowing us to evaluate whether a critical develop-
mental window exists for affecting adult egg-laying
characteristics. After laying, we measured yolk and egg
size, eggshell coloration, and yolk carotenoid concentra-
tion as additional indices of reproductive investment (Batt
& Prince 1978; Eldrigde & Krapu 1988). First, we pre-
dicted that carotenoid supplementation would increase bili-
verdin-based eggshell coloration and yolk carotenoid
concentration (i) based on the previously documented rela-
tionship between carotenoids and biliverdin-pigmented
eggshells in birds (Morales, Velando & Torres 2011); and
(ii) because female mallards are considered to be capital
breeders in terms of lipid reserves (Boos et al. 2002), which
may also include lipophilic carotenoids. If early-life carot-
enoid access shapes adult carotenoid physiology (Blount
et al. 2003) more so than later-life carotenoid access, carot-
enoid supplementation early during development would
have the greatest effects on egg characteristics. Alterna-
tively, if recent access to carotenoids is more important,
then carotenoid supplementation later in development (i.e.
closer to adulthood) would have the greatest effects on egg
traits. Because previous work with lesser black-backed
Fig. 1. Female mallard in nuptial plumage (photo: M.W. Butler).
1178 M. W. Butler & K. J. McGraw
gulls (Larus fuscus) and red-legged partridges (Alectoris
rufa) showed that carotenoid supplementation to laying
mothers had no effect on egg or yolk size (Blount et al.
2002; Bortolotti et al. 2003), but work with Japanese quail
(Coturnix japonica) showed that yolks became smaller after
carotenoid supplementation (McGraw 2006), we did not
develop predictions related to the effects of supplementa-
tion on egg or yolk size. Lastly, to test for relationships
between carotenoid-related traits in mothers and their eggs,
we also quantified circulating carotenoid levels and carot-
enoid-pigmented bill coloration in females, which is a sig-
nal of sexual attractiveness in male mallards (Omland
1996a,b) but has no known signalling function in females.
Materials and methods
EXPERIMENTAL PROTOCOL AND BLOOD COLLECTION
We acquired 46 one-day-old female ducklings from Metzer Farms
(Gonzales, CA, USA) in December 2009 and housed them as in
Butler & McGraw (2009, 2012). Ducklings were reared indoors in
randomly selected groups of five ducklings per cage
(60 9 60 9 60 cm) until they were 2 weeks old, then three per
cage until they were 4 weeks old, and then two per cage until they
were 7 weeks old, at which point all birds were moved outside and
individually housed to allow for normal sexual maturation (Butler
& McGraw 2009, 2012). Light : dark regime was 13L : 11D when
ducklings were housed indoors, and natural photoperiod thereaf-
ter (10
5L : 13
5D at 7 weeks old to 13
5L : 10
5D at 20 weeks
old).
Individuals were randomly assigned to one of four treatment
groups that varied in dietary carotenoid content. Individuals
received carotenoid-supplemented diets during either the period of
maximal growth (EARLY; 3–6 weeks old; N = 10; Butler &
McGraw 2012), minimal growth and minimal nuptial plumage
acquisition (MIDDLE; 8–11 weeks old; N = 12), or complete
acquisition of nuptial plumage (LATE; 13–16 weeks old; N = 12).
CONTROL (N = 12) birds did not receive carotenoid-supple-
mented diets at any point. We prepared diets by mixing a base diet
of dry food (Mazuri Waterfowl Starter: Richmond, IN, USA,
weeks 0–7; Mazuri Waterfowl Maintenance thereafter) with
ORO-GLO dry pigmenter (2% carotenoids by mass, predomi-
nately lutein; Kemin AgriFoods North America, Inc., Des
Moines, IA, USA) suspended in sunflower oil to achieve concen-
trations of 25 lg g 1 of carotenoids (upper quartile of carotenoids
in mallard duckling diets in the wild; Butler & McGraw 2010).
Whenever any treatment group was receiving carotenoid-supple-
mented diets, all other individuals received food mixed with
sunflower oil as a sham control.
We measured body mass to the nearest g four times during a
10-day adult immune assessment period that was part of a sepa-
rate study (Butler & McGraw In Press). During this immune
assessment period, we collected 600 lL of whole blood at four dif-
ferent time points (Days 0, 1, 6 and 10) that was used to quantify
circulating carotenoid concentration (see below). We calculated
mean adult body mass as the average of the four body mass values
that were obtained concurrent with blood collection. Blood was
stored on ice for several hrs, centrifuged for 3 min at 10 000 rpm
and then stored at 80 °C until analysis.
EGG COLLECTION AND ANALYSIS
We collected all eggs (N = 59) on a daily basis starting when
the first female laid an egg (on 27 April; 20 weeks old) until
© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology, 27, 1176–1185
all birds were euthanized at 22
5 weeks old for a separate
study (Butler & McGraw In Press), thus creating a time period
when only a subset of individuals were able to lay eggs
(EARLY: N = 3 birds; MIDDLE: N = 4; LATE: N = 5;
CONTROL: N = 2). Eggs were stored in a refrigerator for
1 week, at which point we used an Ocean Optics (Dunedin,
FL, USA) USB2000 spectrophotometer with a PX-2 pulsed
xenon light source to measure reflectance from k = 300–700 nm
on 5 randomly selected areas of the eggshell. We binned all
measurements into 1 nm increments using CLRfiles (CLR ver-
sion 1.05; Montgomerie 2008) and then used CLRvars (CLR
version 1.05; Montgomerie 2008) to calculate brightness (total
amount of reflected light; B1) and saturation (proportion of
reflectance within a specific portion of the spectrum, see below;
S1U and S1v) for each egg.
Biliverdin, which is the only pigment in eggshells that produces
blue-green coloration (Sparks 2011), has a peak absorbance at
365 nm (Mizobe et al. 1997), so a decrease in eggshell reflectance
around this wavelength relative to reflectance at other wavelengths
should be due to increased biliverdin concentration. Thus, a
reduced S1U and S1v (which measure the saturation in the
300–400 nm and 300–415 nm range, respectively) should corre-
spond to a greater biliverdin concentration; in fact, this relation-
ship has been demonstrated empirically in other bird species
(Hargitai, Mateo & T€ or€
ok 2010) and corresponded with our visual
assessment of eggshell coloration (M.W. Butler, pers. obs.). Addi-
tionally, this methodology is analogous to work with other pig-
ments, wherein the colour metrics that are most closely associated
with pigment concentrations are those that quantify saturation
around the pigments’ peak absorbance (Butler, Toomey &
McGraw 2011).
We then weighed each egg to the nearest 0
01 g, measured egg
width and length to the nearest 0
1 mm and used the formula
described in Hoyt (1979) to calculate egg volume. We then
cracked each egg into a translucent weighing boat and took digital
photographs (Nikon Coolpix P3; Nikon Inc., Melville, NY, USA)
of the egg contents with a ruler in the background. With Adobe
Photoshop v. 8 software (Adobe Systems Inc., San Jose, CA,
USA), we used the lasso marquee to encircle the yolk and measure
the number of pixels occupied by the yolk (sensu McGraw 2006).
We then measured the area standard in the photo so that we could
convert pixel number to mm2. Because we could not wholly sepa-
rate yolk from albumen in these eggs, we were unable to quantify
yolk mass.
We prepared yolks for extraction by placing 200 lL of yolk in
a screw-top 8-mL glass tube, drawing an additional 200 lL
ddH2O into the same pipette tip to remove any yolk residue, and
then vortexing the combined yolk and water mixture for 10 s. We
stored this suspension at 80 °C until further analysis. To extract
carotenoids, we added 2 mL of ethanol to the thawed suspension,
vortexed the solution for 10 s, added 2 mL of 1 : 1 hex-
ane : methyl tert-butyl ether, vortexed the solution for an addi-
tional 10 s and then centrifuged the samples for 5 min at
3000 rpm. We then drew off the supernatant, evaporated it to
dryness under nitrogen and resuspended samples in 42 : 42 : 16
methanol : acetonitrile : dichloromethane mobile phase for high-
performance liquid chromatography analysis (see methods in
McGraw, Tourville & Butler 2008). We primarily detected the
carotenoids lutein, zeaxanthin, a lutein isomer and b-cryptoxan-
thin in mallard egg yolk. Because concentrations of each of these
carotenoids were significantly positively correlated with total yolk
carotenoid concentration (all r > 0
92, all P < 0
0001), we used
total yolk carotenoid concentration in all further analyses. To esti-
mate relative carotenoid amount per egg, we multiplied total yolk
carotenoid concentration by yolk area (as determined from digital
photographs). Because one egg contained only albumen and no
yolk, sample sizes are reduced by one for yolk-dependent
statistics.
 Developmental plasticity and eggshell coloration 1179

PLASMA CAROTENOID AND BILL COLOUR ANALYSES

Results

We quantified plasma carotenoid concentration by extracting car-
otenoids from 50 lL of plasma using a 1 : 1 hexane : methyl
tert-butyl ether method and analysing the extracts using high-per-
formance liquid chromatography (McGraw, Tourville & Butler
2008). Detectable amounts of lutein, zeaxanthin and a lutein isomer
were present in plasma from birds at all four time points of the
immune assessment period, while smaller amounts of b-cryptoxan-
thin were frequently detected. Within each adult sampling time
point, amounts of lutein, zeaxanthin, the lutein isomer and
b-cryptoxanthin were positively correlated with total carotenoid
titre (all r > 0
624, all P < 0
0001). We thus calculated the average
of total circulating carotenoid levels from these four samples as a
metric of circulating carotenoid levels at adulthood.
Carotenoid-based bill coloration in mallards begins to develop
by 10 weeks of age (Drilling, Titman & McKinney 2002) and is
completed in all birds by 16 weeks (M.W. Butler, pers. obs.; J.
Metzer, pers. comm.). When ducks were 19 weeks old, we mea-
sured carotenoid-based bill coloration of adults using the spectro-
photometer apparatus described previously. We measured a 1 cm
band of the right dorso-lateral surface of the bill between the
nares and the bill tip, and again used CLRfiles and CLRvars to
calculate the brightness (B1), saturation (S1B: proportion of
reflectance between 400 and 510 nm, the peak absorption area of
carotenoids), and hue (H4b) scores that are most closely corre-
lated with carotenoid content in the male mallard bill (Butler,
Toomey & McGraw 2011). While male (yellow) and female
(orange) bill coloration differs, both predominately use lutein and
zeaxanthin as pigments (M.W. Butler and K.J. McGraw, unpub-
lished data), and thus, we examined these same coloration metrics
for female mallards. Pilot work from females in this study demon-
strated that only S1B retained a significant relationship with
carotenoid content of the integument in females (r = 0
495,
P = 0
05); thus, we report all associated statistics for this colour
metric. We also performed analyses using both B1 and H4b, but
neither variable explained significant variation in any analysis (all
P > 0
15).
REPEATABILITY OF EGG CHARACTERISTICS WITHIN
FEMALES
All egg characteristic metrics were statistically significantly
repeatable within females (Table 1). Repeatabilities varied
substantially among characteristics, ranging from 0
23 to
0
75.
EFFECTS OF EARLY-LIFE DIETARY CAROTENOID
ACCESS ON EGG CHARACTERISTICS
Dietary carotenoid access during development did not sig-
nificantly affect likelihood of laying (v23 = 1
84, P = 0
61),
but did significantly affect eggshell brightness, saturation
in the ultraviolet region (S1U; Fig. 2) and saturation in
the violet region (S1v; all results, Table 2). LATE birds
had darker eggshells than all other treatment groups (all
P < 0
022; effect size relative to CONTROL = 1
03;
EARLY = 1
15; MIDDLE = 1
29) and less ultraviolet-
(effect size = 1
46) and violet-saturated (effect size = 1
29)
eggshells than CONTROL (both P < 0
0044) and
MIDDLE (both P < 0
027; effect size S1U = 1
05, effect
size S1v = 1
05) birds (Fig. 2). Dietary carotenoid access
during development also significantly affected yolk carot-
enoid concentration (Table 2), with LATE females having
more carotenoid-rich yolks relative to CONTROL birds
(P = 0
0058; effect size = 1
25). Dietary carotenoid treat-
ment during development did not significantly affect any
other aspects of average egg characteristics (Table 2; all
other effect sizes <1
0).

STATISTICS Table 1. Intra-female repeatability of eggshell characteristics

within a clutch for female mallard ducks. All egg characteristics
These data have been deposited in the Dryad repository: http:// were significantly repeatable (R; Lessells & Boag 1987) within
dx.doi.org/10.5061/dryad.pt22p. We performed all statistical anal- female, although yolk carotenoid metrics were repeatable to a les-
yses with SAS 9.2 (Cary, NC, USA), and residuals from all models ser degree than the other variables. See text for descriptions of
were normally distributed. To test for inter-individual differences variables
in egg characteristics, we calculated repeatability (Lessells & Boag
1987) for eggshell hue, brightness, and saturation in the ultraviolet Variable d.f. F-statistic P-value R
(S1U) and violet (S1v) portions of the light spectrum, egg mass
and volume, yolk area, and total yolk carotenoid concentration Eggshell 13, 42 7
95 <0
0001 0
65
and amount. We performed a contingency table analysis to test brightness (B1)
whether likelihood of laying among all 46 females was affected by Eggshell 13, 42 3
23 0
0020 0
37
dietary carotenoid treatment. Within the subset of females that did saturation (S1U)
lay eggs, we then used mixed models with individual as a random Eggshell 13, 42 4
43 0
0001 0
48
effect to test for (i) effects of dietary carotenoid treatment, adult saturation (S1v)
body mass, adult bill coloration and average adult circulating Eggshell 13, 42 12
04 <0
0001 0
75
carotenoid levels on eggshell brightness, ultraviolet- and violet-sat- hue (H4b)
uration, egg mass and volume, yolk area and yolk carotenoid con- Egg mass 13, 41 5
38 <0
0001 0
54
centration; (ii) relationships between eggshell coloration and egg Egg volume 13, 41 4
54 <0
0001 0
49
characteristics (e.g. yolk carotenoid concentration, egg volume); Yolk area 13, 41 8
98 <0
0001 0
68
(iii) relationships between yolk area and yolk carotenoid concen- Yolk carotenoid 13, 42 2
20 0
027 0
24
tration; and (iv) laying order effects on eggshell coloration and egg concentration
characteristics. All comparisons between treatment groups utilized Total yolk 13, 41 2
12 0
034 0
23
LSMeans estimates and standard errors. Effect sizes were calcu- carotenoid amount
lated as Cohen’s d. Due to relatively small sample sizes, our power
to detect a significant effect may have been low in some analyses, S1U, saturation in the ultraviolet; S1v, saturation in the violet.
and thus, any relationships that are reported as not significant For this and all subsequent tables, we use boldfaced text to indi-
should be interpreted within this context. cate statistically significant P-values.

© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology, 27, 1176–1185
1180 M. W. Butler & K. J. McGraw

0·18 EGGSHELL COLORATION AS A PREDICTOR OF YOLK

CHARACTERISTICS

0·17 * Eggshell coloration significantly predicted yolk carotenoid

concentration, with brighter (F1,41 = 6
68, P = 0
013),
S1U (unitless)

0·16 more violet-saturated (F1,41 = 9
56, P = 0
0036) and more

0·15
0·14
0·13
Control Early Middle Late
Treatment
Fig. 2. Differences in eggshell coloration as a function of access to
carotenoid-supplemented diets during different periods of develop-
ment in female mallard ducks (Least Squares Means estimates
with 95% confidence interval). Individuals that received caroten-
oid-supplemented diets when 13–16 weeks old laid eggs with less
ultraviolet-saturated eggshells (P < 0
05: *) than those without
carotenoid-supplemented diets (CONTROL) and those that
received carotenoid-supplemented diets during 8–11 weeks (MID-
DLE) of age. There were no significant differences (all P > 0
05)
among CONTROL, EARLY or MIDDLE birds in the ultraviolet
saturation of their eggshells.
ultraviolet-saturated (F1,41 = 10
14, P = 0
0028) shells
associated with yolks that had lower carotenoid concentra-
tions (Fig. 4a). Eggshell coloration also significantly pre-
dicted total yolk carotenoid amount. Eggs with more
violet-saturated (F1,41 = 6
34, P = 0
016) and more ultravi-
olet-saturated (F1,41 = 6
97, P = 0
012) shells had yolks
that were less carotenoid-rich (Fig. 4b). There was no
significant relationship between eggshell brightness and
total yolk carotenoid amount (F1,41 = 2
87, P = 0
098).
Eggshell coloration also significantly predicted yolk area;
brighter (F1,41 = 20
38, P < 0
0001), more violet-saturated
(F1,41 = 9
92, P = 0
0030) and more ultraviolet-saturated
(F1,41 = 10
09, P = 0
00028) shells were associated with
yolks that had larger areas (Fig. 5).Yolks with larger areas
did not have significantly lower carotenoid concentrations
(F1,41 = 3
03, P = 0
089).
EFFECTS OF LAYING ORDER ON EGG
CHARACTERISTICS

Egg characteristics changed throughout the laying

Table 2. Differences in egg traits at sexual maturity in response to
sequence. Eggshells became significantly brighter
developmental dietary carotenoid manipulations in female mallard
ducks. Dietary carotenoid supplementation during different peri- (F1,42 = 5
09, P = 0
029) and more saturated in both the
ods of development significantly affected eggshell coloration and violet (F1,42 = 11
11, P = 0
0018; Fig. 6) and ultraviolet
yolk carotenoid concentration, but not other egg characteristics, (F1,42 = 15
10, P = 0
0004) portions of spectrum through-
among females out the laying sequence, and yolks became significantly lar-
ger (F1,41 = 19
29, P < 0
0001; Fig. 7), but with lower
Variable d.f. F-statistic P-value
carotenoid concentrations (F1,41 = 31
41, P < 0
0001) and
Eggshell brightness (B1) 3, 43 3
54 0
022 lower overall carotenoid amounts (F1,41 = 25
14,
Eggshell saturation (S1U) 3, 43 3
93 0
015 P < 0
0001). Egg size also significantly increased, as
Eggshell saturation (S1v) 3, 43 3
32 0
028 assessed by both mass (F1,42 = 8
31, P = 0
0062) and vol-
Egg mass 3, 43 2
49 0
073
ume (F1,42 = 8
36, P = 0
0060), throughout the laying
Egg volume 3, 43 1
68 0
19
Yolk area 3, 42 2
38 0
084 sequence.
Yolk carotenoid concentration 3, 42 2
92 0
045
Total yolk carotenoid amount 3, 42 1
70 0
18
Discussion
S1U, saturation in the ultraviolet; S1v, saturation in the violet.
We uncovered inter-individual differences in eggshell color-
ation and yolk carotenoid levels in female mallards that
CORRELATIONS BETWEEN FEMALE METRICS AND EGG
were due to dietary carotenoid access during development
CHARACTERISTICS
and that were correlated with circulating carotenoid levels
Neither average adult body mass nor female bill coloration just prior to laying. Yolk and eggshell characteristics also
was significantly related to egg mass or volume, yolk area covaried with laying order, with eggshells becoming less
or any metric of eggshell coloration (Table 3). Average biliverdin-rich and yolks becoming larger but less caroten-
adult circulating carotenoid level was significantly related oid-rich over the laying sequence, but still showing high
to multiple egg characteristics; it was negatively related to overall repeatability within females. Finally, intensity of
eggshell saturation in both the ultraviolet (Fig. 3a) and eggshell pigmentation was a reliable indicator of more
violet portions of the spectrum, and positively related to carotenoid-concentrated but smaller egg yolks. Together,
total yolk carotenoid concentration (Fig. 3b) and yolk these results show that eggshell coloration is linked not
carotenoid amount (Fig. 3c; Table 3). Average adult circu- only to egg contents, but also to female conditions during
lating carotenoid level was not significantly related to egg development, a period that occurred more than a month
mass or volume, yolk area or eggshell brightness (Table 3). prior to egg laying.

© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology, 27, 1176–1185
 Developmental plasticity and eggshell coloration 1181

Table 3. Maternal morphological and physiological traits as predictors of egg traits in laying female mallard ducks. Average adult circu-
lating carotenoid levels, assessed several weeks prior to laying, predicted average eggshell coloration and yolk carotenoid concentration
and amount. However, adult body mass and carotenoid-pigmented bill coloration during the same period did not predict any egg charac-
teristics

Independent variable Dependent variable d.f. F-statistic P-value Estimate

Average adult body mass Eggshell brightness (B1) 1, 42 0
08 0
78 0
019
Eggshell saturation (S1U) 1, 42 0
12 0
73 1E-6
Eggshell saturation (S1v) 1, 42 0
14 0
71 1E-6
Egg mass 1, 42 0
00 0
97 4
6E-4
Egg volume 1, 42 0
07 0
79 4
0E-3
Yolk area 1, 42 0
05 0
83 0
15
Yolk carotenoid concentration 1, 42 0
07 0
80 5
9E-3
Total yolk carotenoid amount 1, 42 0
15 0
70 10
4
Bill coloration (S1B) Eggshell brightness (B1) 1, 43 2
53 0
12 707
Eggshell saturation (S1U) 1, 43 2
09 0
16 0
28
Eggshell saturation (S1v) 1, 43 2
08 0
16 0
34
Egg mass 1, 43 0
17 0
68 42
3
Egg volume 1, 43 0
68 0
41 87
0
Yolk area 1, 42 1
78 0
19 5
9E3
Yolk carotenoid concentration 1, 42 0
09 0
76 50
5
Total yolk carotenoid amount 1, 42 0
54 0
47 1
5E5
Average adult circulating carotenoid level Eggshell brightness (B1) 1, 42 1
09 0
30 6
0
Eggshell saturation (S1U) 1, 42 6
83 0
012 5
2E-3
Eggshell saturation (S1v) 1, 42 4
87 0
033 5
9E-3
Egg mass 1, 42 0
79 0
38 1
05
Egg volume 1, 42 0
22 0
64 0
59
Yolk area 1, 41 0
51 0
48 39
9
Yolk carotenoid concentration 1, 41 13
52 0
0007 3
98
Total yolk carotenoid amount 1, 41 12
40 0
0011 5
4E3

S1U, saturation in the ultraviolet; S1v, saturation in the violet.

Dietary supplementation of carotenoids during develop-
ment affected eggshell coloration at adulthood, such that
birds that received carotenoid-rich diets from 13 to
16 weeks of age laid eggs when they were 20–22 weeks old
that had more biliverdin-rich (less bright, less ultraviolet-
saturated and less violet-saturated; Hargitai, Mateo &
T€or€
ok 2010) shells than those laid by both CONTROL
and MIDDLE birds. It is important to note that our sam-
ple size in this analysis was modest, with only four
MIDDLE birds and two CONTROL birds laying eggs
during this time period; however, those two CONTROL
birds laid a total of 20 eggs, suggesting that our calculation
of within-individual egg characteristic metrics for these
birds was robust. Together, these two results suggest that
it was carotenoid supplementation during the LATE per-
iod in particular that drove differences in eggshell colora-
tion, although we readily acknowledge that confidence in
this finding would be greater with a more robust sample
size. Unfortunately, our experimental design did not allow
us to detect whether treatment-based differences in eggshell
coloration were due to carotenoid supplementation during
a critical period of development (i.e. 13–16 weeks old) or
to recency of supplementation (i.e. approximately 6 weeks
prior to egg laying). However, these findings augment the
work of Morales, Velando & Torres (2011), corroborating
a link between dietary carotenoid levels and eggshell color-
ation, but extend this relationship to include the ability of
dietary carotenoid access over a month prior to laying to
affect shell pigmentation. To our knowledge, this is the
first study to show that nutrition during development and/
or significantly prior to laying can affect egg coloration.
Egg characteristics, including shell coloration and yolk
area, were highly repeatable within female mallards.
Dearborn et al. (2012) also found that maternal identity
was a strong predictor of eggshell coloration in chickens
(Gallus gallus domesticus), even under standardized hous-
ing conditions and despite differences in feed. This high
within-female repeatability is an important prerequisite for
both condition-dependent signalling hypotheses (e.g. sexu-
ally selected eggshell coloration hypothesis; Moreno &
Osorno 2003) and some condition-independent hypotheses
(e.g. detection of brood parasitism). Such consistent inter-
individual differences may be driven by female genetics
(Morales et al. 2010) or physiological conditions (Moreno
2006). Although we lack the data to test whether variation
in genotype or gene expression was associated with differ-
ences in egg characteristics, we did uncover links between
maternal physiological state and eggshell characteristics.
Specifically, we found that average adult circulating carot-
enoid levels collected when individuals were 18–20 weeks
old positively predicted both average yolk carotenoid
concentration and biliverdin-rich eggshell coloration.
Inter-individual differences in carotenoid assimilation or
transport ability could lead to increased circulating carot-
enoid levels, and may thus be associated with an increased
ability to deposit carotenoids into yolk (Karadas et al.

© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology, 27, 1176–1185
1182 M. W. Butler & K. J. McGraw
(a)
(b)
(c)
Fig. 3. Average eggshell coloration for each female as a function
of average adult circulating carotenoid levels within each female.
Females with greater plasma carotenoid levels laid eggs that
(a) were less saturated in the violet portion of the spectrum
(300–415 nm; i.e. more biliverdin-rich), (b) had yolks with a
greater carotenoid concentration and (c) had yolks with a greater
total carotenoid amount.
2006). These higher levels of circulating carotenoids may
also be linked to antioxidant status, either by directly
acting as antioxidants themselves (Woodall, Britton &
Jackson 1997; H~ orak et al. 2007) or indicating repletion of
other antioxidants (Hartley & Kennedy 2004). Because bil-
iverdin can act as an antioxidant (Kaur et al. 2003),
increased levels of circulating carotenoids may allow for a
greater amount of biliverdin to be directed towards egg-
shell pigmentation without compromising maternal anti-
oxidant status.
The relationship between biliverdin-rich eggshell colora-
tion and greater yolk carotenoid concentration held within
individual eggs, even when controlling for female identity,
further supporting the putative link between biliverdin
production and carotenoid physiology. However, we also
uncovered a relationship between more biliverdin-rich
eggshells and smaller, but more carotenoid-rich,
yolks. Although we did not detect an effect of carotenoid
© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology, 27, 1176–1185
(a)
(b)
Fig. 4. (a) Yolk carotenoid concentration and (b) total yolk carot-
enoid amount as a function of eggshell coloration. Eggshells with
less violet-saturated coloration (i.e. were more biliverdin-rich) con-
tained yolks that had a greater carotenoid concentration and total
carotenoid amount. In this and all subsequent figures, note that
these are the raw data, uncorrected for female identity.
supplementation during development on yolk carotenoid
levels, previous work has shown that feeding carotenoid-
rich diets to laying Japanese quail (Coturnix japonica)
induced females to produce more orange (and presumably
more carotenoid-rich), but smaller, yolks (McGraw 2006).
Because both larger yolks (Dzialowski & Sotherland 2004)
and more carotenoid-rich yolks (Saino et al. 2003) typi-
cally promote increases in offspring quality (e.g. larger
individuals with more robust immune systems), it is
unclear how the smaller, carotenoid-rich yolks laid by mal-
lards that had more colourful eggs would affect duckling
quality. This finding also raises the possibility that females
who obtain a carotenoid-deficient diet may compensate for
reduced yolk carotenoid concentration by increasing yolk
size (but see Safran et al. 2007).
Eggshell coloration, yolk size and yolk carotenoid con-
centration all varied according to laying order; later-laid
eggs were larger, contained larger and less carotenoid-
concentrated yolks and had shells that were less biliverdin-
rich. Such decreases in egg pigment or nutrient levels as a
function of laying order are frequently ascribed to resource
depletion (Rubolini et al. 2011). Under this framework,
intensity of eggshell coloration may indicate either biliver-
din (L
opez de Hierro & Neve 2010; but see Hanley &
Doucet 2009) or carotenoid (Blount et al. 2002; Saino
et al. 2002; Cassey et al. 2005; but see Newbrey et al.

Fig. 5. Yolk area as a function of eggshell coloration. Eggs with
more violet-saturated (i.e. less biliverdin-rich) coloration contained
larger yolks.
Fig. 6. Eggshell coloration as a function of laying order. Shells
from later-laid eggs were more violet-saturated (i.e. less biliverdin-
rich) in coloration. If the data point in the upper-right corner is
removed, the slope remains significant (F1,41 = 7
96, P = 0
0074).
Fig. 7. Yolk area as a function of laying order. Later-laid eggs
had larger yolks, although these yolks also had lower carotenoid
concentrations, which resulted in an overall lower amount of car-
otenoids compared with earlier-laid eggs.
2008) limitations of mothers. However, our results also
show that females may have been compensating for a
reduction in yolk carotenoid concentration by producing
© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology, 27, 1176–1185
Developmental plasticity and eggshell coloration 1183
larger eggs with larger yolks, and in birds, egg size is a
significant predictor of not only size at hatching, but also
juvenile survival (Krist 2011). Without more precise data
regarding the relative benefits to offspring regarding both
yolk carotenoid concentration and/or amount and egg or
yolk size, it is difficult to assess how these competing met-
rics nested within laying order might affect offspring qual-
ity. It is possible that a continuum of egg size and
carotenoid investment results in offspring that are more
phenotypically diverse (i.e. differing levels of immunocom-
petence, variation in structural size), allowing females to
produce a suite of offspring phenotypes that are differen-
tially attuned to a diversity of future habitats (Monaghan
2008; i.e. bet-hedging: Childs, Metcalf & Rees 2010).
Female mallards are considered to be capital breeders in
terms of lipid reserves (Boos et al. 2002), relying on lipid
stores acquired prior to arrival on the nesting grounds to
produce clutches (Krapu 1981). However, few studies on
nutrition and reproduction consider the developmental
period in this capital-breeding context, and to our knowl-
edge, lipid-soluble nutrients like carotenoids have never
been subsumed into theories on capital-vs. income-breeding
investments. Previous research in other species has demon-
strated that carotenoid supplementation during laying
yields an increase in yolk carotenoid levels (Blount et al.
2002; Karadas et al. 2006), suggesting an income-breeding
investment with regard to these molecules. However,
because we found that yolk carotenoid concentration and
amount decreased throughout laying, and more impor-
tantly that pre-laying carotenoid status predicted subse-
quent eggshell coloration and yolk carotenoid levels, we
suggest that female mallards also use carotenoids as capital
resources during breeding. We encourage more work in this
area, with an emphasis on the possibilities that other lipid-
soluble yolk substances (e.g. vitamin E, steroids) may also
be subject to a capital-resource-investment paradigm.
Although the primary goal of this work was to evaluate
developmental and nutritional mechanisms underlying egg
characteristics, our findings have implications for how egg-
shell coloration may function in mallards. Unlike many of
the species for which quality-signalling hypotheses for egg
colour have been tested previously (i.e. where paternal care
is predicted to respond to maternal egg colour variation;
Moreno & Osorno 2003), male mallards provide no pater-
nal care to offspring and in fact often abandon mating
sites after insemination in anticipation of the upcoming
molt (Drilling, Titman & McKinney 2002). Thus, because
of their minimal to nonexistent opportunity to visually
assess a female’s clutch, it is highly unlikely that eggshell
coloration functions as a signal to mallard fathers. How-
ever, mallard females engage in intraspecific brood parasit-
ism, with almost one quarter of all nests experiencing egg
dumping (Kreisinger et al. 2010). Thus, the high repeat-
ability of eggshell coloration within females suggests that
females could use inter-individual differences in eggshell
coloration to increase likelihood of detection of brood
parasites. Still, eggshell coloration also reflects internal
1184 M. W. Butler & K. J. McGraw
yolk carotenoid concentration, demonstrating that eggshell
coloration has the potential to signal aspects of egg qual-
ity. Hence, our data led to the creation of a new, auto-
communication (or self-signalling) hypothesis of avian egg-
shell coloration (M. Meadows, pers. comm.); that is, a
female may use information from her own eggshell colora-
tion to make decisions about her subsequent reproductive
investment.
While our experiment was not designed to test this
hypothesis, it can inform several critical predictions that
may be applicable to many avian species. Under this
hypothesis, we predict that females would invest more
heavily in incubation of more colourful eggs or in
post-hatch maternal care of offspring hatched from more
colourful eggs. For example, under standardized thermal
conditions, a female with more colourful eggs would spend
a greater amount of time incubating and less time foraging
than a female in similar body condition but with less col-
ourful eggs, due to the greater expected benefit of maxi-
mizing optimal nesting conditions for high-quality eggs,
even at a cost of self-maintenance. We also predict that
self-signalling would be most beneficial in longer-lived spe-
cies. In such multiply-breeding species, reduced investment
in lower-quality young (i.e. reduced somatic cost to moth-
ers) during one breeding season could permit increased
investment during future breeding seasons. Lastly, we pre-
dict that self-signalling would have the greatest benefit in
species that have low paternal care, forcing incubating
females to directly allocate time and/or energetic reserves
between egg-tending behaviours and self-maintenance
behaviours, and thus maximizing the utility of information
of egg quality to females. Indeed, correlations between
eggshell coloration and female care of offspring (e.g. provi-
sioning rate) have actually been reported before (e.g. in
house wrens, Troglodytes aedon, Walters & Getty 2010),
but we now must employ experimental approaches to
determine whether females adjust maternal behaviour
according to their egg colour signals per se.
Acknowledgements
We would like to thank C. Ross for animal and laboratory assistance, and
R. Ligon, M. Meadows, D. Hanley, and two anonymous reviewers for
helpful comments on an earlier draft of this manuscript. Funding was pro-
vided to MWB by the Animal Behavior Society and the Facilities Initiative
Grant for Graduates in the School of Life Sciences and to KJM by the
National Science Foundation (IOS-0746364). MWB was partially
supported by the Arizona State University Graduate College Dissertation
Fellowship during this study. All work with animals was approved under
Arizona State University’s Institutional Animal Care and Use Committee
(protocol number 10-1094R).
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Received 12 February 2013; accepted 24 April 2013
Handling Editor: Tony Williams