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Intrasexual selection favours an immune-correlated colour
ornament in a dragonfly
M. P. MOORE & R. A. MARTIN
Department of Biology, Case Western Reserve University, Cleveland, OH, USA
Keywords:
encapsulation;
male–male competition;
melanin;
odonates;
sexual selection;
sexual signal;
viability selection.
Abstract
Sexual signalling is predicted to shape the evolution of sex-specific orna-
mentation, and establishing the costs and benefits of ornamentation and the
information that ornamentation provides to receivers is necessary to evalu-
ating this adaptive function. Here, we assessed the adaptive function of a
common colour ornament in insects, melanin wing ornamentation, using
the dragonfly Pachydiplax longipennis. We hypothesized that greater orna-
mentation would improve territory-holding success by decreasing aggression
that males receive from territorial rivals, but that more ornamented males
may have shorter lifespans. Using mark–recapture field observations, we
found that more ornamented males had greater territory-holding success
and that viability selection did not act on wing melanization. We then com-
pared the aggression of territorial rivals to decoy males before and after
experimentally augmenting wing melanization, finding that males signifi-
cantly reduced aggression following the manipulation. We next hypothe-
sized that wing melanization would signal fighting ability to territorial rivals
by reflecting condition via investment in the costly melanin synthesis path-
way. We observed a positive relationship between ornamentation and the
likelihood of winning territorial disputes, suggesting that wing melanization
provides information about fighting ability to rivals. We also found a posi-
tive relationship between melanin-based immune defence and ornamenta-
tion, supporting a link between the signal and condition. We conclude that
wing melanization is a condition-related signal of fighting ability and suggest
that this may be a common mechanism promoting the evolution of melanin
ornamentation.
Introduction
Sex-specific ornamentation is predicted to affect repro-
ductive success through sexual signalling (Darwin,
1871; Andersson, 1994; Maynard Smith & Harper,
2003). Specifically, sexual signals in animals function-
ally shape mating success by providing information
about the individual to rivals or potential mates,
thereby directly moderating the outcomes of beha-
vioural interactions over territories or copulation oppor-
tunities (Maynard Smith & Harper, 2003; Searcy &
Nowicki, 2005; Lailvaux & Irschick, 2006). For exam-
ple, sex-specific ornamentation may influence
competition over territories by signalling the bearer’s
fighting ability to rivals and facilitating the resolution of
disputes quickly and without injury (Maynard Smith &
Harper, 2003; Arnott & Elwood, 2009). As sexual selec-
tion should therefore strongly favour individuals with
large, conspicuous ornaments, the adaptive value and
evolutionary maintenance of signal function crucially
depends on its ability to convey reliable information
(Maynard Smith & Harper, 2003). Understanding the
adaptive function of sex-specific ornaments thus neces-
sitates directly evaluating the relationship between
ornamentation and components of reproductive suc-
cess, as well as characterizing the information being sig-
nalled to rivals and potential mates. However,
investigations of the adaptive function of sex-specific
ornamentation rarely integrate both approaches (Lail-
vaux & Irschick, 2006).
Correspondence: Michael P. Moore, Department of Biology, Case Western
Reserve University, 2080 Adelbert Road, Cleveland, OH 44106, USA.
Tel.: +1 216 368 2177; fax: +1 216 368 4672;
e-mail: mpm116@case.edu
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1
JOURNAL OF EVOLUTIONARY BIOLOGY ª2016 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
doi: 10.1111/jeb.12953
Melanin is a phylogenetically widespread pigment
underlying sex-specific ornaments in many animals, yet
its role in sexual signalling has been relatively contro-
versial (Stoehr, 2006; Roulin, 2016). While any positive
covariance between ornamentation and aspects of con-
dition may promote sexual signalling functions, insects
use the melanin synthesis enzymatic pathway not only
for colouration but also for directly mounting immune
responses and repairing wounds (Schmid-Hempel,
2005; Siva-Jothy et al., 2005), providing a strong proxi-
mate link by which ornamentation may intrinsically
reflect aspects of condition (Hill, 2011; Roulin, 2016).
For instance, allocation trade-offs of melanin precursors
(e.g. tyrosine) may promote signal reliability such that
only males in the best condition can invest precursors
in both immunocompetence and ornamentation (i.e. Y-
shaped acquisition–allocation model; Stoehr, 2006; Hill,
2011). Additionally, the high energetic costs of melanin
synthesis may maintain signal reliability (e.g. Moret &
Schmid-Hempel, 2000; Fedorka et al., 2004; Schwarzen-
bach & Ward, 2006), whereby only males with the
greatest energetic reserves can activate and maintain
high overall levels of melanin synthesis (Gonz
alez-San-
toyo & C
ordoba-Aguilar, 2012). As a consequence of
this proximate link, melanin ornamentation may often
reliably signal information about an individual’s condi-
tion, directly moderating the outcomes of interactions
with rivals or potential mates, and causally shaping
reproductive outcomes (Hooper et al., 1999; Siva-Jothy,
2000; Wittkopp & Beldade, 2009). However, the rela-
tively few tests of this sexually selected function of mel-
anin colouration in insects have been equivocal
(Lawniczak et al., 2007; Punzalan et al., 2008b; Izzo &
Tibbetts, 2012), and its explicit function in intrasexual
selection has received little attention in particular.
Given this, and the growing recognition of similar
pleiotropic effects in invertebrates and vertebrates
(Ducrest et al., 2008; Roulin, 2016), studies that directly
estimate phenotypic selection on, and the information
content of, melanin ornaments remain critical to
understanding the adaptive function of this widespread
pigment.
Using the sexually dimorphic dragonfly, Pachydiplax
longipennis (Burmeister), we tested the adaptive
function of a common melanin ornament in arthro-
pods, wing melanization (Fig. 1). Similar to damselflies
that display red wing ornaments (e.g. Hetaerina ameri-
cana, Grether, 1996b), we hypothesized that wing
melanization improves territory-holding success, a vital
component of mating success in odonates (reviewed in
Koenig, 2008; Suhonen et al., 2008), by decreasing
energetically costly and potentially injurious aggressive
interactions with rivals. We also predicted that males
with greater wing melanization would be more suscep-
tible to predators and have higher mortality (e.g.
Grether, 1997; Kuchta & Svensson, 2014) and therefore
have shorter reproductive lifespans. We further hypoth-
esized that wing melanization signals fighting ability to
rivals by proximately reflecting energetic reserves via
the shared melanin synthesis pathway. We thus pre-
dicted that ornamentation would be positively associ-
ated with the likelihood of winning territorial contests
and with melanin-based immune defence.
Materials and methods
Study system
Pachydiplax longipennis (Burmeister) is a medium-sized
dragonfly distributed broadly across North America.
Females are brown with longitudinal yellow stripes,
whereas males have blue abdomens and, in the eastern
extent of their range where our study was conducted,
express melanin colouration on the distal portion of
their wings (Fig. 1; Paulson, 2012). Detailed descriptions
of the mating system can be found elsewhere (Johnson,
1962; Robey, 1975; Fried & May, 1983; Sherman,
1983). Briefly, on sunny days between mid-June and
early August, males arrive at the pond between 0800
and 0900 EST and defend territories on emergent vege-
tation around the perimeter of the pond until approxi-
mately 1530 EST. Males spend time in their territories
perching on emergent vegetation and patrolling. Males
frequently encroach upon rivals’ territories, engaging in
aggressive disputes where the winner retains the terri-
tory and the loser either searches for a new territory or
leaves the pond entirely. Once males have established at
a pond for breeding, they rarely disperse (McCauley,
(a) (b)
Fig. 1 Wings of two mature males with
relatively low (a) and high (b) wing
melanization. Note that the
pigmentation at the base of the wing is
less variable among males and is also
expressed in females. Pigmentation in
the distal portion of the wing is
sexually dimorphic and highly variable
among males.
ª2016 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY. J. EVOL. BIOL. doi: 10.1111/jeb.12953
JOURNAL OF EVOLUTIONARY BIOLOGY ª2016 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
2M. P. MOORE AND R. A. MARTIN
2010). Females arrive at the pond throughout the day,
mate with a male, and then oviposit in his territory
while he hovers above her (Sherman, 1983). There are
seldom more than one or two females on a pond at a
given time, and copulations are very short, rarely lasting
more than 2 min (Sherman, 1983; Paulson, 2012).
Study site, population monitoring and phenotypic
measurements
We conducted this study at a small (perime-
ter =140.2 m) research pond at Case Western Reserve
University’s Squire Valleevue Farm (Hunting Valley,
OH, USA). Males defend territories along two regions of
emergent vegetation on opposite sides of the pond (pri-
marily Typha sp.; 13.8 and 35.7 m, respectively). We
captured males and uniquely marked their abdomens
with four dots of acrylic paint (randomly chosen from
among five different colours; Anderson et al., 2011).
We kept males on ice in plastic bags before processing
to slow their movement and facilitate safe handling
(McCauley, 2010). To assess variation in wing
melanization, body size and wing size (two traits associ-
ated with odonate reproductive success; Koenig, 2008),
we took digital photographs (Canon G15; Canon, USA,
Inc., Lake Success, NY, USA) of each male’s wings and
body. We standardized the lighting conditions by taking
pictures of males against a standard white background
(DGK Color Tools
; Fig. 1) in a dark box that excluded
ambient light. We attempted to include only males with
little wing wear and fully developed abdomen coloura-
tion to minimize potential variation due to age differ-
ences (Grether, 1996a; Contreras-Gardu~
no et al., 2008;
McCauley, 2010). In two cases, we recaptured and
reprocessed males that had not developed their full
abdomen colouration. Following processing, we
released males from a common location approximately
10 m from the pond. While several ponds were within
the average dispersal distance of breeding males
(430 m, McCauley, 2010), no marked males were ever
observed at any of these other ponds.
We quantified all traits from digitized photographs in
ImageJ (Rasband, 2012). Body size was calculated as
the distance (mm) from the front of the head to the
tips of the cerci. Wing area was scored as the total area
(mm
2
) of all four wings. Wing area and body size are
highly correlated (r=0.834, P<0.001), and thus, we
calculated relative wing size by taking the standardized
residuals from the linear regression of body size on total
wing area. To quantify the extent of wing melanization,
we identified the highest mean grey value (0–255,
0=most opaque; 255 =most transparent) in ImageJ of
the pigmented portion of each wing (i.e. least darkly
pigmented), converted the photograph to binary black
and white with this value as the threshold for black
and calculated the size (mm
2
) of the digitized
black area. Wing melanization was then estimated as
the proportion of the total wing area that was pig-
mented. To ensure the consistency of measurements,
we calculated repeatability by randomly resampling a
subset of 16 individuals and blindly rescoring each trait.
The repeatability was high for all traits (all R>0.99, all
F
15,16
>259.2, all P<0.001; Lessells & Boag, 1987).
We evaluated the phenotypic variation in the popula-
tion by considering how wing melanization varied with
body size and relative wing size using Pearson’s pro-
duct-moment correlation coefficients, with individuals
included as the unit of replication.
Do territory-holding success and longevity vary with
wing melanization?
We examined the fitness costs and benefits of our focal
traits using behavioural observations of marked individ-
uals. One observer (MPM) continuously circled the
pond during the peak activity hours (0900–1530 EST)
on all sunny days between 23 June and 24 July
(n=18), recording each marked male’s location, terri-
torial behaviour and the time. On most days, approxi-
mately 50% of the territorial males at the pond were
marked. Territorial males exhibit characteristic, unam-
biguous behaviours, such as perching in an obelisk
position or chasing other males (see Johnson, 1962;
Robey, 1975). We quantified a male’s within-day terri-
torial tenure as the amount of time (minutes) that it
was territorial during that day. Males that were sighted
only once were assigned a territorial tenure of zero
minutes. We could not directly evaluate mate choice as
we observed only four marked males copulating with
females. However, as females choose among territorial
males, a male’s territorial tenure is strongly correlated
with mating success (Sherman, 1983).
We assessed how daily territorial tenure varied with
wing melanization, body size and relative wing size
using a generalized linear mixed-effects model with a
negative binomial error distribution to account for
overdispersion. We did not test interactive effects
among traits because models including these terms did
not converge, and preliminary visual assessment of the
interactions suggested that there were no strong effects
between any combinations of the traits. To account for
multiple territorial tenures of the same male among
days, and the nonindependence among males within a
given day, we included random intercepts for individ-
ual identity and observation date, respectively. As terri-
torial tenures of zero minutes were potentially
misidentified males, we excluded these observations
from analyses. However, our results are qualitatively
robust to their inclusion. Using individuals’ daily terri-
torial tenures, as opposed to total time spent defending
a territory over the flight season, reduces bias resulting
from variation in longevity (Hamon, 2005).
We evaluated how wing melanization, body size and
relative wing size varied with minimum-estimated
longevity using a generalized linear model with a
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Sexual selection on dragonfly ornamentation 3
quasipoisson error distribution to account for overdis-
persion. Each male’s minimum-estimated longevity
(hereafter: ‘longevity’) was calculated as the number of
days between marking and the last day it was observed.
As we conducted field observations regularly through
the end of the flight period, this sample of days reflects
a realistic period over which viability selection may act
through differences in longevity. This metric is com-
monly used in odonate studies (e.g. Grether, 1996a;
C
ordoba-Aguilar, 2002) and is relatively robust to low
resighting probabilities (Waller & Svensson, 2016). We
only included males marked prior to 5th July to control
for differences in the available number of days between
marking date and the end of the flight season. We only
considered males that were observed at least twice after
marking to ensure that we exclusively included resident
males.
To estimate the strengths of intrasexual and viability
selection on our focal traits, we used standard regres-
sion techniques to calculate selection gradients (Lande
& Arnold, 1983), dividing each individual’s fitness com-
ponent (territorial tenure or longevity) by the popula-
tion mean (i.e. relative fitness) and converting
phenotypic values to mean of 0 and unit variance. To
compare overall fitness variation acting through intra-
sexual selection vs. viability selection, we also calcu-
lated the opportunities for sexual and viability selection
by dividing the variance in each fitness component by
its squared mean (Arnold & Wade, 1984).
Do males with greater wing melanization receive less
aggression from territorial rivals?
To assess whether wing melanization has direct effects
on the aggression received from rival males, we pre-
sented decoys (previously frozen males) to territorial
males, experimentally augmented the ornamentation of
the decoys, presented them to new males and com-
pared the aggression received before and after the
experimental manipulation (see also Anderson &
Grether, 2010; Guillermo-Ferreira et al., 2015). We
tethered decoys to a 2-m aluminium pole using clear
nylon line (diameter =0.18 mm), and presented them
to territorial males. Typical of natural encounters
between rivals (Suhonen et al., 2008; McCauley, 2010),
males engaged the decoys by aggressively making
direct, physical contact multiple times (mean number
of strikes SD =4.4 5.1) over short durations
(mean number of seconds SD =6.2 4.6). For each
presentation, we evaluated the following: whether the
territorial male engaged the decoy (‘engagement’, y/n);
the time between the territorial male engaging the
decoy and returning to a perch in its territory (‘engage-
ment duration’, seconds); how many times it struck the
decoy (‘strikes’, n); and the number of strikes per sec-
ond (‘aggression rate’, strikes per second). After pre-
senting the decoy to several different territorial males
(median: 4, range: 2–5), we augmented the
ornamentation by homogenously colouring the wings
distally from the nodus (Fig. S2) with a felt-tip marker
chosen to approximate the natural colour (Crayola
Cuppa’ Cappucino;sensu Anderson & Grether, 2010;
Guillermo-Ferreira et al., 2015). This degree of orna-
mentation is within the natural phenotypic range. We
then presented the decoys to a different set of territorial
males and again evaluated the interactions. While the
same territorial males were often presented multiple
decoys, many of these males were presented post-
manipulation decoys before premanipulation decoys,
and thus, any observed effects of the manipulation on
territorial male aggression are not confounded with ter-
ritorial males becoming acclimated to the decoys.
We compared interactions before and after the
manipulation using mixed-effects models with decoy
identity as a random effect to account for multiple pre-
sentations of the same decoy to the different males. For
error distributions, we specified the binomial for
engagement, negative binomial for number of strikes
and gaussian for engagement duration and aggression
rate. Engagement duration was natural log-transformed
to improve normality of the residuals. Significance was
assessed with likelihood ratio tests of models with and
without the fixed effect for generalized linear mixed-
effects models and F-tests with the Kenward–Roger
degrees of freedom approximation for linear mixed-
effects models (Kenward & Roger, 1997).
Is wing melanization associated with fighting ability?
To assess the potential for wing melanization to signal
fighting ability to territorial rivals, we observed natu-
rally occurring, aggressive interactions (e.g. chasing,
striking) between males during the field observations
described above. We defined the winner of a territorial
contest as the male that succeeded in forcing the other
male out of the territory. Many contests included
marked males, and we attempted to capture all
unmarked males from contests. However, as contest
losers frequently fly to another location on the pond or
off the pond altogether, we rarely knew the phenotypic
values of both males in a contest.
We analysed the likelihood of winning a territorial
contest using a generalized linear mixed-effects model
with a binomial error distribution and contest outcome
(win =1, lose =0) fitted as the response variable. We
included wing melanization, body size and relative wing
size as fixed effects. To account for repeated measures of
some individuals across disputes, and the nonindepen-
dence of two individuals in the same contest when both
were known, we included random intercepts for both
individual and contest identity, respectively.
Does wing melanization correlate with melanin-based
immune responses?
To evaluate whether wing melanization proximately
signals an individual’s condition via investment in the
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4M. P. MOORE AND R. A. MARTIN
shared melanin synthesis pathway (sensu Siva-Jothy,
2000), we considered the relationship between a male’s
wing melanization and its ability to mount a melanin-
based immune response. We captured and assayed the
melanin-based immune responses of 33 territorial males
over 2 days (28 July and 3 August). To assay immuno-
competence, we inserted a piece of sterilized nylon
monofilament (mean length SD =2.70 0.38 mm,
diameter =0.18 mm) into the body cavity dorsally
between the fifth and sixth abdominal segments and
allowed the males’ immune systems to react to it for
24 h. A pilot study indicated that 24 h provided the
strongest and most variable immune responses in this
species (M. P. Moore, unpublished). During this period,
males were kept in plastic bags in a dark climate cham-
ber set to 8.2 °C. While this treatment may have slo-
wed the rate of melanin immune responses, it was
necessary to prevent adults from injuring themselves
during or after the surgical implantation, and we do
not expect that any temperature effect will have differ-
entially affected males with different phenotypes. We
then dissected out the implants and stored them in a
freezer set to 22.8 °C. In four cases, the implant fell
out or was accidentally inserted into the midgut, and
these males were not considered further.
We quantified the melanin-based immune responses
from digitized photographs of the implants. Using a dis-
secting microscope with a bright field background, we
took one photograph of the implant, rotated the
implant 90°and then took another photograph. In
every photograph, we also had a nylon monofilament
that was not inserted into any males as a negative con-
trol. We used ImageJ to assess the mean grey values
(0 =opaque, 255 =transparent) of each implant. To
calculate an immune response score for each male, we
subtracted the mean grey values of the implant from
the control nylon for each picture and averaged the
two pictures. Higher scores indicate a darker implant
and therefore a stronger melanin-based immune
response, and previous work indicates this standard
technique reflects resistance to naturally occurring
pathogens (Rantala & Roff, 2007). Using the procedure
described above (see also Lessells & Boag, 1987), the
repeatability of this metric was high (R=0.995,
F
15,16
=404.00, P<0.001).
We used a linear model to consider how immune
response scores varied with wing melanization, body
size and their interaction. To account for potential vari-
ation between the two dates when males were cap-
tured, we also initially included date and its
interactions in the model. As these interactions were
not significant (all F<1.75, P>0.201), we removed
them from the model and retested effects. The immune
score from one very large male was an outlier and was
not included in the analysis to improve the normality
of model residuals. However, our results are qualita-
tively robust to its inclusion.
Statistical analyses
All statistical analyses were conducted using R v. 3.1.2
(R Development Core Team, 2014). Mixed-effects mod-
els were fit using the ‘lme4’ package (Bates et al.,
2014). To account for large scaling differences among
three focal phenotypes, wing melanization, body size
and relative wing size were z-transformed for all analy-
ses (Schielzeth, 2010). All model parameter estimates
and selection gradients are reported as estimate SE.
Results
Phenotypic variation
We first assessed the phenotypic variation of males in
our population. The mean body size SD of males was
38.06 2.18 mm, and the mean wing melaniza-
tion SD was 0.41 0.09 (proportion of melanized
win area; n=115). There was no relationship between
body size and wing melanization (r=0.090,
t
114
=0.96, P=0.339; Fig. S1a), or between relative
wing size and wing melanization (r=0.143,
t
114
=1.54, P=0.126; Fig. S1b).
Do territory-holding success and longevity vary with
wing melanization?
In total, we observed 126 territorial tenures across 45
males (mean SD per individual: 2.8 2.2 tenures),
with a mean tenure SD of 104.9 84.2 min. Males
with greater wing melanization had longer territorial
tenures (0.348 0.090; v
2
=13.99, d.f. =1, P=0.002;
Fig. 2). In contrast, territorial tenure did not vary with
body size (0.023 0.077; v
2
=0.09, d.f. =1,
P=0.767) or relative wing size (0.057 0.088;
v
2
=0.43, d.f. =1, P=0.515). Overall, the opportunity
for intrasexual selection was 0.707, and the estimated
strength of intrasexual selection on wing melanization
was relatively strong (b=0.326 0.104; c.f. King-
solver et al., 2012). We report nonsignificant intrasex-
ual selection gradients in Table S1.
Of the 24 males included in the longevity analyses,
the mean longevity SD was 17.4 5.4 days. Longev-
ity was not associated with wing melanization
(0.078 0.066, v
2
=1.43, d.f. =1, P=0.232), body
size (0.096 0.079, v
2
=1.49, d.f. =1, P=0.222) or
relative wing size (0.022 0.093, v
2
=0.05, d.f. =1,
P=0.817). Overall, the opportunity for viability selec-
tion was 0.097, and we report the nonsignificant viabil-
ity selection gradients in Table S1.
Do males with greater wing melanization receive
less aggression from territorial rivals?
Regardless of experimental treatment, decoys were
equally likely to be engaged by territorial males (before
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Sexual selection on dragonfly ornamentation 5
vs. after: 0.719 0.479, v
2
=2.31, d.f. =1, P=0.129).
However, engagement durations were shorter following
the manipulation (0.429 0.201, F
1,46.9
=4.46,
P=0.040). Similarly, decoys received fewer strikes
(0.951 0.367, v
2
=6.12, d.f. =1, P=0.0134;
Fig. 3a) and lower aggression rates (0.441 0.121,
F
1,43.7
=12.98, P<0.001; Fig. 3b) after augmenting the
decoy’s ornamentation.
Is wing melanization associated with fighting
ability?
We observed 155 territorial contests (94 winners, 73
losers; mean contests SD per individual: 2.8 3.1).
Males with greater wing melanization (0.484 0.181;
v
2
=6.50, d.f. =1, P=0.011) and larger body sizes
(0.433 0.176; v
2
=5.39, d.f. =1, P=0.020) were
more likely to win contests (Fig. 4). Relative wing size
was not associated with the probability of winning con-
tests (0.213 0.166; v
2
=1.72, d.f. =1, P=0.189).
Does wing melanization correlate with melanin-
based immune responses?
Immune response scores increased with wing melaniza-
tion and marginally decreased with body size (Table 1).
Immune response scores were also marginally different
between the two capture dates (Table 1).
Discussion
We investigated the adaptive function of melanin orna-
mentation in the sexually dimorphic dragonfly,
P. longipennis. Following patterns in damselflies with
red wing ornaments (Grether, 1996b), we hypothesized
that greater wing melanization would improve terri-
tory-holding success by decreasing aggression that
males receive from territorial rivals, but that benefit
may come at the cost of a shorter reproductive lifespan
(Grether, 1997). We also hypothesized that wing
melanization would signal fighting ability to territorial
rivals by proximately reflecting condition via invest-
ment in the costly melanin synthesis pathway (Hooper
et al., 1999; Rantala et al., 2000; Siva-Jothy, 2000).
Overall, our results provide broad support for the
hypothesized adaptive function of male wing melaniza-
tion as a sexual signal of fighting ability to territorial
rivals.
Male fitness is determined by the combined effects of
intrasexual, intersexual and viability selection (Arnold
& Wade, 1984; Hamon, 2005). Intrasexual selection via
territorial occupancy is crucial to male reproductive
success in territorial odonates (Moore, 1990; Grether,
1996b) and especially in P. longipennis (Sherman,
1983). Among the traits considered, only wing
melanization improved territory-holding success
0
100
200
300
−2 −1 0 1
Wing melanization [z−transformed]
Territorial tenure (min)
Fig. 2 Territorial tenure increased with wing melanization. Each
point represents the duration of time within a day that an
individual held a territory, and points are jittered horizontally by
0.05 to improve visual clarity. The regression line is fitted from the
mixed-effects model reported in the Results, and wing
melanization was z-transformed to improve scaling among
explanatory variables.
0
5
10
15
Strikes (n)
(a)
(b)
0.0
0.5
1.0
1.5
2.0
Before After
Manipulation
Aggression rate (strikes * s–1)
Fig. 3 Territorial males exhibited reduced aggression (a: total
number of strikes; b: number of strikes per second) to decoy males
following experimental increase of wing melanization. Each circle
represents a presentation of a decoy to a territorial male, and
points in panel (a) are jittered vertically by 0.1 to improve visual
clarity. Squares represent the model-estimated means SE.
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JOURNAL OF EVOLUTIONARY BIOLOGY ª2016 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
6M. P. MOORE AND R. A. MARTIN
(Fig. 2), and our selection analysis indicated strong
intrasexual selection on this ornament (c.f. Kingsolver
et al., 2012). Although we are unable to assess the
strength of intersexual selection in this study, other
selection analyses in odonates have found positive (e.g.
C
ordoba-Aguilar, 2002) or no (e.g. Grether, 1996b)
intersexual selection on wing ornamentation. Contrary
to many studies of other odonates (reviewed in Koenig,
2008), and despite a positive relationship between body
size and the likelihood of winning territorial disputes
(Fig. 4b), we did not observe selection on body size. As
small males are likely to lose fights and never acquire a
territory, it is possible that, by including primarily
males that had already acquired territories, our esti-
mates of selection on body size (and all other traits)
may be conservative. While we also predicted that
increased melanization may come at the cost of repro-
ductive lifespan (e.g. Grether, 1997; Kuchta & Svens-
son, 2014), we found no evidence for viability selection
against wing melanization. However, the opportunity
for viability selection (0.097) was considerably lower
than the opportunity for intrasexual selection (0.707),
and therefore, intrasexual selection may be relatively
more important for generating variance in male fitness
in this population, further suggesting strong overall fit-
ness benefits of ornamentation.
Our results indicate that the functional benefit of wing
melanization in P. longipennis is through decreased
aggression received from rival males. Territorial disputes
in odonates are extremely energetically costly, and con-
sume most of an individual’s daily energy budget (Fried
& May, 1983; Koskim€
aki et al., 2004). As males do not
feed while they are defending territories (Fried & May,
1983), any decrease in the energy expended on battling
intruders may increase the time over which a male holds
its territory, and therefore also increase the likelihood of
mating (Suhonen et al., 2008). Moreover, in contrast
with many other odonates, P. longipennis males make
physical contact during territorial disputes, greatly
increasing the chances of severe injury or death (Sher-
man, 1983; McCauley, 2010; Paulson, 2012). Our results
suggest that rivals challenge males with greater ornamen-
tation less frequently and with lower intensity (Fig. 3),
potentially improving a male’s ability to remain in a terri-
tory and/or avoid injury. Similar to damselflies express-
ing red wing ornaments (Grether, 1996b; Guillermo-
Ferreira et al., 2015), it seems likely that a primary func-
tional advantage of greater melanin ornamentation is
through decreased aggression from territorial rivals.
The information provided to rivals and potential
mates is crucial to the evolution of sexual signals, and
those involved in male–male competition are predicted
to evolve to ensure that disputes are resolved as
cheaply as possible for both parties (Maynard Smith &
Harper, 2003; Searcy & Nowicki, 2005). In particular,
territorial males should assess the fighting ability of
their rival and avoid engaging them if they are unlikely
to win the dispute or if the cost of winning the dispute
is too high (Maynard Smith & Harper, 2003; Arnott &
Elwood, 2009). As wing melanization is associated with
a male’s ability to win contests (Fig. 4), rivals would
0.00
0.25
0.50
0.75
1.00
–3 –2 1 0 1 2
Body size [ztransformed (mm)]
Probability of winning contest
0.00
0.25
0.50
0.75
1.00
–2 –1 0 1 2
Wing melanization [ztransformed]
Probability of winning contest
(a)
(b)
Fig. 4 The likelihood of winning a territorial contest increased
with wing melanization (a) and body size (b). Each point
represents the outcome of a contest for an individual (0 =contest
lost, 1 =contest won), and points were jittered vertically by 0.1 to
improve visual clarity. Regression lines are fitted from the mixed-
effects model reported in the Results, and both explanatory
variables were z-transformed.
Table 1 Variation in immune response scores of 28 territorial
males as a function of wing melanization, body size and date
(multiple R
2
=0.306, F
3,24
=3.53, P=0.030). Model estimates
(SE) were obtained after removing the nonsignificant wing
melanization 9size interaction. Wing melanization and body size
were z-transformed prior to analysis, and date represents the
difference between the 2 days over which males were captured.
All partial F-tests were on 1 and 24 degrees of freedom.
Effect Estimate FP
Wing melanization 7.381 3.110 5.69 0.026
Body size 5.418 2.808 3.76 0.065
Date 9.466 5.414 2.94 0.100
Wing melanization 9body size 1.26 0.274
ª2016 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY. J. EVOL. BIOL. doi: 10.1111/jeb.12953
JOURNAL OF EVOLUTIONARY BIOLOGY ª2016 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
Sexual selection on dragonfly ornamentation 7
benefit from avoiding costly and/or potentially injuri-
ous disputes with highly ornamented males (Junior &
Peixoto, 2013; Guillermo-Ferreira et al., 2015). Indeed,
males with experimentally increased ornamentation
received significantly lower aggression from rivals
(Fig. 3). As the fitness benefits of displaying a large
ornament are great, the signal must be reliable, on
average, to be evolutionarily maintained, otherwise
males would cease to respond (Maynard Smith & Har-
per, 2003). In many cases, melanin ornaments in
arthropods may signal condition reliably because of the
biochemical link between immune defence and
colouration (Table 1). While a male’s immunocompe-
tence may not be specifically informative to rivals, the
high costs of the melanin synthesis pathway (Gonz
alez-
Santoyo & C
ordoba-Aguilar, 2012) will ensure that
only males in the best condition have the capacity to
allocate sufficient resources (e.g. energy, precursors,
enzymes) to support robust immune function and large
wing ornaments (e.g. Hooper et al., 1999; Rantala et al.,
2000; Siva-Jothy, 2000). The maintenance of melanin
wing ornaments in insects also requires some degree of
constant pigment deposition (Hooper et al., 1999; True
et al., 1999), further enabling the ornament to reflect
the individual’s present physiological state. As there is a
strong relationship between condition and fighting abil-
ity in insects (reviewed in Vieira & Peixoto, 2013), and
investment in many other condition-related traits ends
at metamorphosis or maturity (e.g. body size), the mel-
anin synthesis pathway may be a common mechanism
by which arthropods reliably signal proximate informa-
tion about their condition and fighting ability to rivals.
While the shared biochemical pathway linking
immune defence and colouration promotes signal relia-
bility, intrasexual selection will also favour males that
maximize signal efficiency (Badyaev, 2004; Stoehr,
2006), which will have consequences across the mela-
nin synthesis pathway. Indeed, artificial selection
experiments (Armitage & Siva-Jothy, 2005) and studies
of natural populations experiencing divergent selection
pressures (Fedorka et al., 2013) have found that selec-
tion on melanin colouration often promotes the corre-
lated evolution of melanin immune defence. As with
most sexual signals (reviewed in Hill, 2011; Morehouse,
2014; but see Craig & Foote, 2001), the specific targets
of selection for improving the efficiency of melanogene-
sis are unknown. However, two evolutionary outcomes
for the melanin synthesis pathway seem most likely:
(1) energetic resources may be more efficiently allo-
cated to the production of melanin synthesis enzymes
(e.g. phenoloxidase), and (2) amino acid precursors
may be more readily available for conversion to mela-
nin. It remains to be seen which of these two outcomes
is more likely or whether either is general across spe-
cies, as some studies of the melanin synthesis pathway
indicate energetic limitations (e.g. Cotter et al., 2010),
while others report precursor limitations (e.g. Srygley
et al., 2009). Importantly, as both immune defence and
colouration depend on many of the same precursors
and enzymes, selection for increased signalling effi-
ciency may be unable to completely erode the associa-
tion between colouration and condition, and the signal
will remain reliable, on average. Nevertheless, if intra-
sexual selection favours greater efficiency of signal pro-
duction, then populations exhibiting stronger
intrasexual selection on wing melanization should
express more efficient melanogenesis.
The evolution of sex-specific ornamentation depends
in part on the functional mechanisms by which the
ornament affects reproductive success (Andersson,
1994; Lailvaux & Irschick, 2006). We found strong
support for a sexual signalling function shaping the
evolution of sex-specific wing melanization in a terri-
torial dragonfly. Melanin colouration exhibits a diverse
suite of adaptive functions in arthropods (e.g. Punza-
lan et al., 2008c; Fedorka et al., 2013; Debecker et al.,
2015), but given its frequently observed condition
dependence (e.g. Talloen et al., 2004; Punzalan et al.,
2008a), the ability to act as a sexual signal of condi-
tion may commonly shape its evolution. Although any
direct or indirect positive covariance between orna-
mentation and aspects of condition may ultimately
facilitate condition-related sexual signalling of an orna-
ment, linkages underlain by shared condition-depen-
dent developmental pathways are predicted to readily
evolve via sexual signalling functions (Hill, 2011; Rou-
lin, 2016). As the melanin synthesis pathway links
ornamentation and immunocompetence in arthropods,
sexual signalling, like that observed here, may indeed
prove to be a common adaptive function shaping the
evolution of sex-specific melanin colouration.
Acknowledgments
We thank M. Dugas for his invaluable assistance and
insight during all stages of the study. J. Larsen, H. Roll-
ins and A. Gilmore greatly assisted with the collection
of the data. A. Locci and the Squire Valleevue Farm
staff generously provided access to the field site. We
further appreciate comments from S. Diamond, P.
Lorch, M. Benard, K. Krynak, L. Chick, I. Ghergel, A.
Perez, T. Schwander, D. Punzalan, members of the Case
Western Reserve University Ecology and Evolution
group, and two anonymous referees that improved the
quality and presentation of the study.
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Supporting information
Additional Supporting Information may be found
online in the supporting information tab for this article:
Figure S1 There was no relationship between a male’s
wing melanization and its body size (a) or its relative
wing size (b). Each point represents an individual.
Figure S2 Rear wings of territorial Pachydiplax longipen-
nis showing examples of weak natural melanization
(top), experimentally manipulated melanization (mid-
dle) and strong natural melanization (bottom).
Table S1 All variance-standardized selection gradients
for intrasexual and viability selection (Lande & Arnold,
1983).
Data deposited at Dryad: doi: 10.5061/dryad.rc1k8
Received 6 May 2016; revised 3 July 2016; accepted 19 July 2016
ª2016 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY. J. EVOL. BIOL. doi: 10.1111/jeb.12953
JOURNAL OF EVOLUTIONARY BIOLOGY ª2016 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
10 M. P. MOORE AND R. A. MARTIN
