Supplemental InformationNestling polymorphism in a cuckoo-host system: a consequence of an escalating coevolutionary arms raceNozomu J. Sato, Keita D. Tanaka, Yuji Okahisa, Masato Yamamichi, Ralph Kuehn, Roman Gula, Keisuke Ueda, Jörn Theuerkauf

Supplemental Experimental ProceduresMaterials and Methods

Study Sites, Species and Measurements

We conducted field investigations at three sites on the main island (Grande Terre) of New Caledonia

during three breeding seasons (September-January) from 2011 to 2014: Parc provincial des Grandes

Fougères (main study site) and near surroundings (21°37’39.44” S, 165°45’41.75” E), approx. 40

km west (21°35’58.89” S, 165°23’55.61” E) of the main study site, and approx. 130 km northwest

(20°41’45.55” S, 164°59’38.41” E) of the main study site.

The fan-tailed gerygone Gerygone flavolateralis flavolateralis is a passerine bird of the

family Acanthizidae and an endemic subspecies to New Caledonia. The shining bronze-cuckoo

Chalcites lucidus layardi is a brood parasitic cuckoo of the family Cuculidae, and also an endemic

subspecies to New Caledonia. Both are abundant and widespread throughout Grande Terre year-

round, and the gerygone is exclusively parasitised by the bronze-cuckoo [S1].

To measure reflectance, we captured 32 gerygone chicks from 18 broods (dark: N = 5;

bright: N = 11; polymorphic: N = 2), out of which 22 were of the bright morph and 10 were of the

dark morph. We also captured 3 shining bronze-cuckoo chicks to measure reflectance, which all

were of the bright morph. We measured reflectance spectra of light from 300 nm to 700 nm

wavelengths from their skin (Figure S1a) using spectrophotometers (USB-2000 and Jaz-EL-200,

Ocean Optics, Dunedin, Florida, USA) with light irradiated by a deuterium-tungsten-halogen light

source (DT-MINI-2-GS, Ocean Optics, Dunedin, Florida, USA). Before measurements, we

calibrated the spectrometer with a diffuse reflectance standard (WS-1, Ocean Optics, Dunedin,

Florida, USA). Measurements were carried out in a film-changing bag (E-7041, Etsumi, Tokyo,

Japan) to block ambient light. While holding chicks by hand, we placed the probe vertically above

their skin, keeping an approximately 2-mm distance ensured by insulating tape winded around the

probe. To avoid injuring gerygone chicks, we conducted measurements when they were at least 3

days old, while cuckoo chicks were measured on the day of hatching before host parents ejected

them. We measured reflectance spectra of bare skin on the back of each chick twice (except for 4

chicks that we could only measure once). This usually took only few minutes, thereby avoiding

potential negative effects on chicks. From the 35 chicks, we obtained 66 reflectance spectra in total

(dark: n = 18; bright: n = 42; cuckoo: n = 6).

Because host parents were likely to eject cuckoo chicks, we artificially incubated 2 cuckoo

eggs to measure reflectance spectra of hatchlings (Mini Advance Incubator, Brinsea, Wiscombe,

North Somerset, UK). We conducted measurement for one cuckoo chick that hatched naturally in

the host nest before being ejected by the host. All artificially hatched cuckoo chicks were

reintroduced in the nearest active nest on the day of hatching in the case that the original nest was

depredated. We placed an artificial cuckoo egg in the nest at least 24 h before we replaced it with a

cuckoo chick.

Skin Colour Polymorphism in Chicks

There was neither a sign of colour polymorphism in adults nor a sign of assortative mating in

relation to the chick skin colour polymorphism in the gerygone since there were no detectable

differences in adult songs and plumages between parents of dark and bright chicks (authors’

unpublished data), which are known as indices of prezygotic isolation in birds [S2, S3]. Both dark

and bright gerygone chicks were found sympatric in all study sites.

The skin colour polymorphism of host chicks was most remarkable just after hatching, and

weakened with chick age (authors’ unpublished data). It became almost indistinguishable in chicks

of ca. 13-15 days of age. We occasionally measured reflectance from chicks older than 5 days (2

bright and 3 dark chicks in 3 nests), but not from chicks 10 days or older. The polymorphism was

not related to deformation of pigmentation such as albinism [9], since no bright chick had red irises

or a pinkish bill, or fibromelanosis [S4].

We have no evidence for sympatric polymorphism in the bronze-cuckoo but photo

evidence of a dark morph (Figure 1C) was published 35 years ago [S5] and taken in Parc

Provincial de la Rivière Bleue, approx. 100 km southeast of the main study site. Chick colour

polymorphism is known in the shining-bronze cuckoo, though in different subspecies, and

respective cuckoo morphs mimic chicks of respective hosts in different colours [7, 9, S6].

Avian Visual Model

Birds are thought to have two distinct pathways to perceive a colour, i.e., chromatic (hue) and

achromatic (luminance, or perceived lightness) [S7-S9]. We estimated both chromatic [S7] and

achromatic [S9] discrimination thresholds of the chick skin colours, respectively, based on the

Vorobyev-Osorio model [S7, S9]. Birds of the genus Gerygone have VS (violet-sensitive) vision

[S10]. However, because data of the visual performance of the study species were not available, we

applied hitherto available single-cone sensitivity of a VS-type bird, the wedge-tailed shearwater

Puffinus pacificus [S11], the double-cone sensitivity of the blue tit Cyanistes caeruleus [S12], and

the single-cone abundance in the posterior dorsal area of retina of the satin bowerbird

Ptilonorhynchus violaceus [S13], all phylogenetically closest to the gerygone among species with

available information.

We first calculated photon capture Qi with sensitivity of respective photoreceptors Ci(λ)

and measured reflectance spectra R(λ) according to the following equation:

Qi=∫300

700

R ( λ )⋅C i ( λ ).

We then calculated the colour discrimination threshold, i.e., just noticeable difference (jnd),

between a given pair of colours. Jnds were obtained from the following equations:

jnd hue=√ (ωUV ωS )2 ( Δf L−Δf M )2+(ωUV ωM )2 ( Δf L−Δf S)2+(ωUV ωL )2 ( Δf M−Δf S )2+ ¿

(ωS ωM )2 ( Δf L−Δf UV )2+(ωS ωL )2 ( Δf M −Δf UV )2+(ωM ωL )2 (Δf S− Δf UV )2 ¿(ωUV ωS ωM )2+ (ωUV ωS ωL )2+(ωUV ωM ωL )2+ (ωS ωM ωL )2

(S1a)for hue [S6], and

jnd luminance=ΔS=|Δf D

ωD|

(S1b)

for luminance [S7], where ∆f denotes the log ratio of photon captures of the focal pair of measured

colours by a given type of photoreceptors:

,and ω denotes the relative abundance of each single-cone type in the posterior dorsal area of the

retina, with incorporating the Weber fraction of 0.05, the conventionally adopted error rate (i.e.,

noise-to-signal ratio) in the Weber-Fechner law [S7]. Since fan-tailed gerygones build domed nests

in which the inside is dimly lit, we considered fluctuation of the number of photons captured by

cone cells (i.e., shot noise) as a relatively great quantal flux of 103 [S14]. We did not consider colour

constancy because the ambient light condition should be very similar for all chick types.

Statistics

We first calculated jnds between all possible combinations of measured colours for both hue and

luminance respectively based on equations S1a and S1b [S5, S7]. Next, we converted these jnds

into respective distance matrices, from which we calculated tridimensional coordinates through a

principal coordinate analysis (PCoA, or multi-dimensional scaling, MDS) [S15, S16].Each principal

coordinate consists of a set of data, each of which corresponds to each photospectral measurement,

and thus the total number of replicates analysed was n = 66. Each coordinate value indicates the

relative position of each datum among the whole dataset, i.e., distance from the centroid, on each

coordinate axis in its unit (jnd in this case) (see Figure S1d). Unlike jnd (i.e., psychophysical

distance), all coordinate values are geometrically independent of each other, and thus the dataset is

compatible with linear models [S15, S16]. Eigenvalues were used to assess the accuracy of primary

eigenvectors (i.e., first principal coordinates). The rationale underlying this procedure is illustrated

in [S15] and [S16].

We analysed the first principal coordinates for hue and luminance with linear mixed

models (LMMs), in which brood ID and nestling ID nested within brood were assigned as random

effects to avoid pseudoreplication [S15]. We assigned dummy variables [S16, S17] to each chick

type, i.e., dark chick, bright chick and cuckoo chick, and set bright chick as the intercept in the

LMMs. Representative values for respective chick types were calculated as the absolute value of the

difference of partial coefficient from the intercept, i.e., |cuckoo chick – bright chick| and |dark chick

– bright chick|. These values represent the perceivable difference of respective chick types from

bright chick on average, and thus can be interpreted as chromatic (hue) or achromatic (luminance)

discriminability [S15, S16]. We assumed jnd > 3 as discriminable by convention [S9]. Goodness of

fit was tested by the likelihood ratio (2) test.

For the analyses of the observed data, we conducted a contingency (2) test, a binomial

test, and a generalised linear model (GLM) with a likelihood-ratio (2) test. All statistical

procedures were conducted in R [S18]. PCoAs/MDSs were conducted with the cmdscale function,

setting the dimensional parameter k as 3 because jnds existed in a tridimensional colour space.

LMMs were conducted with the lmer function in the lme4 package [S19] and the GLM the glm.nb

function in the MASS package [S20]. Likelihood ratio tests were conducted with the Anova function

in the car package [S21]. Figure S1a was drawn with the aggplot function in the pavo package

[S22].

Population Genetics Model

We assumed 1-locus-2-allele complete dominance as the inheritance mechanism, which was most

probable in this case because the colour difference appeared to be discrete, as we have not observed

hatchlings of an ambiguous type. The observed frequency of within-nest polymorphism might be

biased or obscured in several ways: an inevitable bias caused by small brood sizes, i.e., no

polymorphism in single-chick broods irrespective of parental genotypes; an observational bias, i.e.,

before we found a nest, polymorphism in there might have already vanished due to randomly

caused partial brood mortality (including that caused by brood parasitism); and a small sample size.

To deal with such bias and uncertainty, we simulated stochastic distributions for expected

frequencies of phenotype, monomorphic brood of each morph, and polymorphic brood at Hardy-

Weinberg equilibrium. These values represent the frequency of phenotype and respective brood-

types, ideally obtainable in a limited number of observations and at a low average brood size of the

population when the population is at the equilibrium. We assumed genetic monogamy of parents

(i.e., chicks in a nest have the same genetic parents) and our observation to be random sampling for

simplicity.

On allele frequencies at even intervals, offspring phenotypes were randomly sampled

assuming a binomial distribution with coefficients for the emergence probabilities of respective

brood types based on equations described below. Parameter values were set at 33 for sample size

assuming a Poisson distribution, and set for brood size ranging from 1.525 to 3.350 in integer

assuming a uniform distribution (average 1.93), in reference to our observation (see Results). We

iterated the sampling 500 times in R [S18].

We extracted a pair of simulated samples of phenotype frequency, each assuming the dark

morph as either recessive or dominant, from samples in each iteration, among those which were

nearest to the observed phenotype frequency of chicks. The brood-type frequencies and allele

frequencies corresponding to the extracted phenotype frequencies were also extracted. We

compared the position (i.e., percentile) of the observed frequencies of phenotype and respective

brood-types within the corresponding simulated distributions of expected frequencies between

recessive and dominant (see Figure S2). These simulated percentiles were resampled with the

parametric bootstrapping method (iteration = 1000). The minimum requirement for Hardy-

Weinberg equilibrium here is that all the observed frequencies are simultaneously contained within

the corresponding simulated distributions respectively.

Equations for Population Genetics Model

When the genotype frequency in the parental generation is x for dominant homozygotes (AA), y for

heterozygotes (Aa), and z for recessive homozygotes (aa), the expected genotype frequency in the

offspring generation under Hardy-Weinberg equilibrium is X = , Y = , and

Z = , where X denotes dominant homozygotes, Y heterozygotes, and Z recessive

homozygotes; note that x + y + z = X + Y + Z = 1. The emergence of polymorphic broods is

potentially limited in those from parents of the Aa-Aa pair (both heterozygotes; y2) and the Aa-aa

pair (heterozygote and recessive homozygote; 2yz). However, the emergence of polymorphic broods

is also limited in situations that brood size, C, is two or greater, and thus, the emergence probability

of each brood type is:

bD: (S2a)

b

p: (S2b)

bm:

(S2c)

bR: z2 (S2d)

where bD denotes dominant monomorphic broods, bp polymorphic broods, bm monomorphic broods

of either from the potential mating combinations, and bR recessive monomorphic broods (Figure

S2a). bp and bm are complementary to each other and thus sum up to y2 + 2yz (“potential” in Figure

S2a); when C is 1, bp is always 0 and bm is always y2 + 2yz (Figure S2a).

Ethical Notes

We conducted fieldwork under permissions from Province Sud and Province Nord of New

Caledonia. No chick died because of our treatments. The research protocol complies with the

current laws in New Caledonia, and was approved by the Ethical Committee of Life-Sciences at

Rikkyo University in Tokyo, Japan, and the First Warsaw Local Ethics Committee for Animal

Experimentation in Warsaw, Poland.

Results

Colour Discriminability

We described the results for achromatic discriminability in the main text. The distribution of

measured colours (in hue) overlaps in the tetrachromatic colour space of VS birds irrespective of

chick types (Figure S1b, c). Eigenvalues of first principal coordinates were 3.00 (100% of variance

explained) for luminance and 2.47 (82% of variance explained) for hue (Figure S1d, e), and thus

they well represented the distribution of measured colours.

Chromatic discriminability for both dark host and (bright) cuckoo chicks was neither

greater than 3 on average nor statistically different from that for bright chick (intercept: 0.033, SE =

0.60) (Figure S1d, e): dark chick, 0.18 jnd (partial coefficient = -0.15, SE = 0.92), 21 = 0.027, P =

0.87; cuckoo chick, 1.69 jnd (partial coefficient = -1.65 SE = 1.50), 21 = 1.21, P = 0.27. These

results suggest that the colours of respective chick types are indiscernible in terms of hue.

Observed Frequencies

We found 149 gerygone nests over three breeding seasons. We found eggs in 68 nests, out of which

18 were parasitised, and 42 nests with chicks. We were not able to determine the skin colour in 9

nests because chicks were too old, and thus the skin colour was known from 33 nests, in which we

included parasitised nests. Out of them, 23 broods consisted only of bright chicks and 8 broods

exclusively of dark chicks, while 2 nests contained both types of chicks, one nest with 1 chick of

each morph, and the other with 1 bright and 2 dark chicks. The observed brood type frequency of

bright monomorphic, polymorphic, and dark monomorphic broods was 0.70:0.06:0.24 (Figure S2d-

f). The number of bright chicks was 39 and that of dark chicks 16 in total. The overall phenotype

frequency of bright and dark chicks was 0.71:0.29 (Figure S2c).

Out of 42 nests containing chicks, we could not reliably assess brood size in 12 nests due

to brood parasitism (3), unhatched eggs (3), egg disappearance (2), partial predation (1), the

location of the nest preventing close inspection (1), and clutch or brood reduction for unknown

reasons (2). The clutch and brood sizes were thus known from 50 and 30 non-parasitised nests,

respectively. The estimated mean clutch and brood sizes were 1.82 (log-linear coefficient: 0.60 SE =

0.11; GLM, family = negative binomial, link = log) and 1.93 (0.66 SE = 0.17) respectively, which

were not statistically different (21 = 0.13, P = 0.72). Frequencies of each size class did not differ

between egg and nestling stages as well (contingency test, 22 = 0.75, P = 0.69; Figure 1F).

We successfully monitored the transition from egg to nestling stages in 18 out of 30 non-

parasitised nests, of which brood size was consistent with clutch size in 10 nests. Among 8 nests of

clutch-brood size discordance, the causes of the discordance were unknown in 2 nests but known in

6 nests as above mentioned. After excluding these 6 nests, the probability of concordance, 10 out of

12 nests, was significantly greater than chance (one-sided binomial test, P = 0.019).

Observed Frequencies among the Expected Frequencies Inferred by Simulation

Assuming the dark morph to be dominant, all observed frequencies (red arrows in Figure S2c-f)

were within the range of simulated distributions for their corresponding expected frequencies (all

within 40-60%ile). In contrast, assuming the dark morph to be recessive, observed frequencies for

bright monomorphic and polymorphic broods greatly diverged from those expected (namely,

3.7%ile and 90.6%ile) (Figure S2c-f). This trend was consistent in the outcome of the parametric

bootstrap: mean percentiles were 4.6 and 90.3 respectively for bright monomorphic and

polymorphic broods if recessive, while those for bright monomorphic and polymorphic broods if

dominant were 39.3 and 55.0, respectively. The mean percentiles for the rest of frequencies were all

between 35%ile and 65%ile. These results suggest that the dominant dark morph is more consistent

with the assumption for Hardy-Weinberg equilibrium than the recessive one. The putative dark-

morph allele frequency was inferred at 0.63 (SD = 0.070) if recessive, and at 0.11 (SD = 0.035) if

dominant.

Cuckoo Chick Ejection by Host Parents

From more than 2000 h of video footage, we confirmed that all 8 cuckoo hatchlings that we found

in gerygone nests were ejected by host parents both from naturally parasitised nests and nests from

to which we introduced artificially incubated chicks (foster nests). Five out of 8 cuckoo chicks were

confirmed to be of the bright morph, 2 seemingly bright, and 1 was unidentified. Four out of 8 host

broods from these nests were composed only of bright chicks, 1 brood with only dark chicks, and 3

were unidentified. Two cuckoo chicks were fostered in 2 broods of the bright morph. The causes of

failure to identify the colour of chicks were ejection of cuckoo chicks by host parents, and failure of

eggs to hatch or predation of the host nests.

All parasitic eggs hatched earlier than host eggs, but host parents did not always eject

parasitic chicks before their own eggs hatched. Thus, cuckoo chicks coexisted with host hatchlings

in 2 nests (1 naturally parasitised and 1 fostered; both bright monomorphic broods). The shining

bronze-cuckoo is probably an evictor (a brood parasite whose chicks monopolise host nests by

evicting host eggs and chicks from the nests soon after hatching; Figure 1C) like Chalcites cuckoos

in other areas [4, 5, S1, S5]. We could however not confirm this because host parents ejected

cuckoo chicks soon after hatching (44 min. after hatching at the shortest). In foster nests, ejection

by host parents occurred later than in naturally parasitised nests (1 day after introduction at the

longest). We confirmed that host parents reared remaining host chicks until fledging (2 nests) or

until predation (3 nests), hence saving them from eviction by cuckoo chicks (c.f., [S6]).

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Supplemental Figures

Figure S1. a) Mean reflectance spectra from the skin of cuckoo chicks (green) and of both morphs

of gerygone chicks: bright (blue) and dark (red). Vertical widths in translucent colours represent

95% confidence intervals. b) Distribution of measured colours in the tetrachromatic colour space of

VS birds (b, c). Viewing angles are altered to illustrate the aspect of avian colour space that is

visible to humans (b) and that invisible to humans (c). Each apex indicates the coordinate for a

colour that stimulates solely the corresponding photoreceptor. First principal coordinates calculated

from distance matrices of pairwise hue jnds between all possible combinations of measured colours,

in relation to second principal coordinates (d), or chick types (e). Numbers represent the eigenvalue

of the first principal component, with the proportion of variances explained in parenthesis.

Figure S2. a) Expected brood-type frequency at Hardy-Weinberg equilibrium in relation to

recessive allele frequency, with a constant brood size C within population, according to the model

described in the supplementary text. y and z represent the frequencies of parental genotypes in

population, heterozygotes and recessive homozygotes, respectively. Red lines indicate the

frequencies of polymorphic broods (bp), and the blue line that of monomorphic broods (bm) among

the potential mating combinations (note that bp = potential – bm). b) Simulated expected frequencies

by the model in relation to recessive allele frequency. Line colours denote each brood type.

Horizontal lines indicate the observed frequencies of phenotype, assuming the dark morph as

dominant (solid) and as recessive (dashed); observed frequencies of brood types (red): polymorphic

(solid), dark monomorphic (dotted), and bright monomorphic broods (dashed). c-f Positions of the

observed frequencies (indicated by red arrows and percentile values) of respective categories among

the distributions of simulated expected frequencies, assuming the dark morph as dominant (shaded

bars) and as recessive (open bars): phenotype (c), dark monomorphic (d), bright monomorphic (e),

and polymorphic broods (f). Percentile values are underlined for dominant. Vertical lines indicate

the mean of simulated frequencies when assuming the dark-morph as dominant (dashed) and as

recessive (solid). Bin widths of all histograms were optimised by setting the breaks argument as

“Scott” for the hist function default in R [S18].