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Color as a reliable signal of fighting
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Calopteryx
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Donna Marie Fitzstephens

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COLOR AS A RELIABLE SIGNAL OF FIGHTING ABILITY IN MALE
DAMSELFLI ES, CALQPTERYX MACULATA

BY

Donna Marie Fitzstephens

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Zoology
1 994

ABSTRACT

COLOR AS A RELIABLE SIGNAL OF FIGHTING ABILITY IN MALE
DAMSELFLIES CALOPTERYX MACULATA

BY

Donna Marie Fitzstephens

Color may act as an important signal in agonistic encounters by providing
reliable information on the resource holding potential of opponents. Males of the
damselfly 9332ij magulata engage in costly agonistic contests which result in
the turnover of territory ownership. Male g; maculata exhibit structural
interference colors that vary with age and territorial status making color a prime
candidate to convey information as a signal. This study examines the
mechanisms and significance of color production and color change in a natural
population of _(_3_. m.

Physical and chemical cuticle tests are suggestive of constructive
interference colors produced by lamellae in the epicuticle. Transmission
electron micrographs provide direct evidence of a multilayer interference
reflector. The lamellae of blue males are thinner (6.2nm i 1.0SD) than the
lamellae of green males (6.4nm i 1.0SD). Wavelength of peak reflectance
calculated from Iamellar spacing yields values which correspond to the colors
observed in the field.

Male g magulata maintained in the laboratory on a high food diet have
significantly higher percent body fat (7.3% :t 2.28D) and exhibit a significantly
smaller color change than similar age/size color males maintained on a low food
diet (4.1% i 2.7SD). High food males also retain their initial color almost twice as

long as low food males.

In the field, territorial male Q. maculata were younger, reflect shorter
wavelength coloration, and were more successful at mating than nonterritorial
males. As male Q. maculata age, their color changes from short to long
wavelength. Fat analysis indicates that short wavelength males have
significantly higher fat levels (6.7% i 2.0SD) than do long wavelength males
(4.4% i 1.68D). The field and laboratory evidence indicates that fat content is
more important than age for determining male color.

Wavelength is reliably related to the measures of body condition (energy
reserves) which affect the outcome of male Q. maculata interactions. Path
analysis indicates that the wavelength of male color is the best predictor of male
territorial status. Therefore, color conveys reliable information about male

fighting ability as measured by fat content.

ACKNOWLEDGMENTS

I thank my committee members for their guidance and support. I received
numerous small grants from Don Straney and the Department of Zoology. Fred
Dyer kept me thinking about insect sensory systems and provided many helpful
suggestions on earlier drafts of this manuscript. I thank Jim Miller for leading me
to a most important literature set on insect structural colors and for always
offering encouraging words. I especially want to thank Tom Getty, who has been
an endless source of support, advice, guidance and patience particularly during
critical times. Thanks for keeping me on track, Tom!

Carolyn Hammerskjold provided superb library support. Thanks also to
Karen Klomparens, Director of the Center for Electron Optics at Michigan State
University, for the exquisite electron micrographs.

Most important, I wish to thank Scott Fitzstephens and my family for
providing emotional support and encouragement throughout my graduate career.
I especially want t thank my mom, Loretta Cicirello for always encouraging me to
choose my own path.

This work was financially supported by an NSF Graduate Fellowship, a
Grant-in-Aid of Research from Sigma Xi (The Scientific Research Society), and a
Theodore Roosevelt Memorial Fund Grant from the American Museum of Natural

History.

TABLE OF CONTENTS

LIST OF TABLES
LIST OF FIGURES

INTRODUCTION
93pm maculata natural history-

CHAPTER 1: A multilayer interference reflector in the epicuticle
of male Caloptem maculata

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

CHAPTER 2: Decreasing fat levels with age result in color
change in male Mew

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

CHAPTER 3: Color reliably signals fighting ability in male

Damage m_ac_ulafl.
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION

SUMMARY AND CONCLUSIONS
LIST OF REFERENCES

PAGE
vi

vii

3...;

AACD‘IO)
QM

TABLE

LIST OF TABLES

PAGE

Mean number and spacing of electron lucent and 16
electron dense lamellae in the epicuticle of blue versus

green male Calcium maculata.

Mean wing length and initial wavelength of color for 25
high food and low food male Qalgpteryx mam.

Mean latency to color change, final and change in 26
wavelength for high food and low food male

Caloptem maculata.

Mean fat and water content for high food and low food 27
male Qalgptegx maculata

Mean (1: SD) size (forewing length), age, and 40
wavelength of territorial versus nonterritorial male

Qilqgteryx maculata.

Mean It SD) size (forewing length), age, and 41
wavelength for successfully mated versus unsuccessful
male Calcium maculata.

Correlation and path coefficients for calculation of the 46
contributions of predictor variables to predicted
variables in Figures 5 and 6.

vi

FIGURE
1

LIST OF FIGURES

PAGE

The wavelengths of color exhibited by male Caloptem 14
maculata categorized as ‘blue’ or ‘green’. (N = sample
size)

Transmission electron micrographs of the outer 17
exoskeleton of blue (top) and green (bottom) male

Qaloptem Mata showing alternating layers of

electron lucent and electron dense material in the

epicuticle.

Interaction diagram constructed using information from 36

QaLijm magmas natural history. This diagram is to
be used as a starting point for path analysis.

The number of individual male Qanpjeryx maculata 39

marked on each day of the study.

Path analysis of the relationship of the predictor 44
variables (age, %fat, % water, and wavelength) with
male territorial status and mating success. This path
diagram represents an hypothesis of causation for
evaluation. Single-headed arrows represent a causal
relationship between two variables while double-headed
arrows denote correlations without causation. The
degree to which the variation in a variable is accounted
for (indicated below diagram) can be calculated by
summing the products of each path coefficient with the
respective variables’ correlation coefficient (Li, 1981).

Alternative path diagram resulting from the exclusion of 45
wavelength as a predictor variable.

vii

INTRODUCTION

The exchange of information via signals is often an integral part of
agonistic encounters (Gardner and Morris, 1989; Hasson, 1994). What
guarantees that opponents in aggressive interactions signal ‘correct’
information (Enquist, 1985)? This problem of signal reliability is a much
discussed and controversial topic (Zahavi, 1975, 1977; Enquist, 1985; Grafen,
1990; Harper, 1991).

In general, signals may be reliable if they are biologically correlated with
resource holding potential (RHP) or if they are costly. Assessment signals are
biologically correlated with RHP and therefore cannot be faked (Harper, 1991).
In guppies, color is dependent on food resources; it is therefore an assessment
signal biologically correlated with foraging ability (Kodric-Brown, 1985, 1989).
Conventional signals are not logically correlated with RHP. Color as a ‘badge
of status’ in birds is not correlated with RHP but is a reliable conventional signal
due to the costs of wearing the badge (Rohwer, 1982; Jarvi and Bakken, 1984;
Whitfield, 1987; Maynard Smith and Harper, 1988). It has been demonstrated
that conventional signals are reliable or ‘honest’ if they are costly to produce or
display (Zahavi, 1975, 1977; Enquist, 1985; Grafen, 1990) with cost being a
function of RHP.

Disputes are often settled by asymmetries between contestants
(Hammerstein and Parker, 1982; Maynard Smith, 1982; Leimar and Enquist,
1984; Huntingford and Turner, 1987). These asymmetries often reflect physical

advantages, residency, or other differences in RHP. In such contests, animals

1

2
may utilize signals to assess RHP or payoff asymmetries. Despite continued

discussion of the presence of cheating in animal signaling systems (Grafen,
1990; Dawkins and Guilford, 1991; Wagner, 1992), theory predicts that signals
used for assessment of fighting ability Should honestly advertise fighting ability
(Maynard Smith and Parker, 1976; Zahavi, 1977; Grafen, 1990). Dishonest
signals are evolutionarily unstable because they tend to be ignored or fall into
disuse if unreliable (Harper, 1991). Recent models have demonstrated that
cheating or dishonesty may persist at low levels in a system (Dawkins and
Guilford, 1991). However, as long as a signal is honest ‘on average’ it will be
reliably related to that which it predicts (Johnstone and Grafen, 1993). For
discussion of the various definitions and uses of signals see Grafen (1990) and
references therein.

Male Qalgptegx maculata are brightly colored Calopterygid damselflies
with a blue or green thorax and abdomen. Previous studies of damselfly color
have concentrated on the family Coenagrionidae. These studies have
addressed genetically determined female limited color polymorphisms (Fincke,
1987; Cordero, 1990; Forbes, 1991), temperature related physiological color
changes (May, 1976; Conrad and Pritchard, 1989) and developmental color
changes associated with maturation (Hinnekint, 1987; Ueda, 1989). No
previous studies have addressed the significance of male color differences of in
Q maculata or the use of color as a signal in agonistic encounters. This study
examined the mechanisms of color production and color change as well as the
likelihood that color conveys information as a signal in a natural population of
Q maculata. In studying agonistic encounters, it is important to estimate the
role that assessment of RHP plays in settling contests. Elucidating the

mechanisms of color production will demonstrate how color is related to the

3
biology of Q maculata and as such, will reveal what types of information can be

conveyed to opponents through color.

legptem gaming is especially well suited for the study of color as a
Signal. Adults are relatively large, slow moving damselflies and are therefore
easily observed and captured for marking. Territorial males are rather
sedentary (see below) defending the same 1-2m of stream each day.
Therefore, marked individuals can be observed throughout their lifetime.
Finally, there is variation in male color that seems to be related to age and
territorial status (Fitzstephens, unpubl), which makes color a prime candidate to
convey information about the traits important for turnover in territory ownership.

I will examine the mechanisms of color production in QM by
various physical and chemical tests to distinguish among pigment as well as
three types of structural colors. A feeding experiment will demonstrate how
color change is related to Q maculata feeding behavior and energy reserves.
In the field, individual males will be followed throughout their lifetime to
determine how color change is related to territorial and reproductive behavior.
Finally, path analysis will be used to evaluate the direct and indirect
relationships among all variables to determine the strength of the relationships
between color and the variables important for territorial defense (age and fat,
see below). This analysis will demonstrate that color is reliably related to the
measures of body condition important in winning contests and that it is the best

predictor of territorial status.

4
CALQPTERYX MACULATA NATURAL HISTORY

Mega maculata is a stream dwelling Calopterygid damselfly.

Average life span including an 11day teneral period is 16-20 days (Waage,
1972). The mating system of Q maculata has been classified as resource-
defense polygyny (Alcock, 1987). As with many animals exhibiting resource
defense polygyny, Q maculata are sexually dimorphic. Males are bright blue or
green with black wings while females are dull brown with translucent brown
wings. Males compete to attract females by defending a valuable resource
(oviposition sites). Adults are active from mid-June through mid-August; activity
peaks during July (Forsyth and Montgomerie, 1987). Adult males are territorial
and aggressively defend 1-2m of stream consisting of a perch and at least one
oviposition site. Territories are defended from approximately 10:00 to 18:00 on
sunny days, and most reproductive activity occurs during this period within 2m
of the stream (Waage, 1973). Male aggressive contests may last from 108 to
over an hour (Waage, 1988). Females mate almost exclusively with territorial
males (Waage, 1979; 1982). Mating lasts 1.0-1.5 minutes and is followed by
12-19 minutes of oviposition during which the female deposits eggs in
emergent vegetation while being guarded by the territorial male (Waage, 1979;
Forsyth and Montgomerie, 1987). Unguarded females oviposit for 1-2 minutes
before being disturbed by males.

The presence of non-territorial, reproductively active males has been
documented in several studies of Q maculata (Waage, 1972; 1973; Forsyth and
Montgomerie, 1987). Nonterritorial or sneaker males do not defend a specific
area. Instead, they pursue and attempt to mate with ovipositing females
opportunistically and do not guard females after mating (Forsyth and

Montgomerie, 1987). Nonterritorial males cover up to 100m of stream per day

5
and are decidedly surreptitious in their behavior around territorial males.

Females often terminate encounters with nonterritorial males by dislodging
them with wing flips (Waage, 1973) or evasive flights typically used as mate
rejection responses (Waage, 1979; Forsyth and Montgomerie, 1987). The daily
copulation rate for territorial males is higher (3.9 matings/male/day) than that for
nonterritorial males (0.14-2.8 matings/male/day; Forsyth and Montgomerie,

1987)

CHAPTER 1

A MULTILAYER INTERFERENCE REFLECTOR IN THE EPICUTICLE OF MALE
CALOPTERYX MACULATA

INTRODUCTION

Colors may have a physical (structural) rather than a chemical (pigment)
basis (Mason, 1926). Pigment colors result from absorption of selective
constituents of white light. Physical colors depend on the structure of the cuticle
and may be due to scattering, reflection, refraction, or diffraction of incident light
(Neville, 1975; Hinton, 1976). See examples below.

Cromartie (1959) provides an extensive review documenting the
occurrence of carotenoid, anthraquinone, aphin, anthroxanthin, anthrocyanin
and pterin pigments in the following insect families: Orthoptera, Lepidoptera,
Coleoptera, Phylloterida and Aphididae.

Mason (1926) established general criteria by which physical or structural
colors can be differentiated from pigment colors. Physical colors can be altered
or destroyed by pressure, distortion, swelling, shrinking, and immersion in a
medium of the same refractive index as the structure. Unlike pigment colors,
structural colors are unaffected by bleaching or chemical reagents unless the
tissue is altered. Chemical digestion of the integument of insects exhibiting
structural colors releases only black or brown pigments.

The most commonly discussed types of structural colors include
scattering by particles (Tyndall colors), diffraction grating, and interference by
thin films. Tyndall colors arise by scattering of shorter wavelength light and
transmission of longer wavelengths when light penetrates a system of randomly
dispersed particles (Mason, 1926; Neville, 1975). The resulting color depends
on particle size. If white light penetrates a heterogeneous system composed of
bodies embedded in a medium, the refractive index of which is different from
that of the bodies, and the particle diameter is small compared to the

wavelength of light, the scattered light will be blue (Onslow, 1921). The

8
smallest particles reflect violet, larger ones reflect deep to pale blue, and the

largest ones reflect white. The most extensive evidence of Tyndall colors in
insects was found in Coenagrionid damselflies (Mason, 1926; Charles and
Robinson, 1981) and the following dragonflies: An_ax, EMhemis, and Libellula
(Mason, 1926).

Structural colors can occur in insects having a cuticle with suitably
spaced surface ridges (Anderson and Richards, 1942). These systems behave
as diffraction gratings. Diffraction colors are produced by a series of evenly
spaced parallel groves or ridges on the epicuticle. White light is scattered from
the lines and the resulting waves either reinforce or cancel each other. In
diffuse light, the resulting spectra overlap so much that the original color is
significantly dimmer or reduced to dull black. Diffraction colors differ from other
structural colors in that they can only be seen from certain directions relative to
the striations (Hinton, 1976; Hinton and Gibbs, 1971). Diffraction colors as the
major source of color have been reported in beetles of the following families:
Scarabaeidae, Phalacridae, Cerambycidae, Gyrinidae, Carabidae,
Staphylinidae, Torridincolidae, and Silphidae (for review see Neville, 1975).

The most widely cited system of structural color production in insects is
that of constructive interference colors produced by thin films or lamellae in the
epicuticle (Richards, 1951; Fox and Ververs, 1960; Simon, 1971; Schultz and
Rankin, 1985). Differences in color correspond to differences in uniformity or
thickness of the layers. Constructive interference of waves occurs between light
reflected at successive interfaces. Diffuse illumination does not cause
interference colors to disappear (Mason, 1927). As the angle of viewing is
increased from grazing incidence to a direction normal to the cuticle surface,
color produced by multilayer interference changes from shorter to longer

wavelengths (Onslow, 1921).

9
Swelling and pressure also alter the appearance of interference colors in

predictable ways (Mason, 1927). Swelling (with water for example) increases
lamellar spacing and shifts color to longer wavelengths. Increasing pressure
decreases lamellar spacing, shifting in color to shorter wavelengths. These
color shifts are reversed upon removal of the swelling agent or release of the
pressure.

Males of the damselfly Qaloptegg maculata are conspicuous having
black wings and a brightly colored blue or green thorax and abdomen.
Females are more cryptic, having translucent brown/gray wings and a brown
thorax and abdomen. Preliminary observations suggested that color in male Q
maculata may be structural in nature and change over an individual’s lifetime
(Fitzstephens, pers obs). The objective of this study was to determine the
nature of male Q maculata coloration and to reveal the microstructure of the
color producing components of the cuticle. Determining the nature of color
production and how it is related to the biology of Q maculata is the first step in

the determination of what information is provided by color in this species.

MATERIALS AND METHODS

| assessed the relationship between age and color of male Q maculata,
from 15 June to 15 August 1991 at Augusta Creek, approximately three miles
from the Kellogg Biological Station in Southwestern Michigan. I performed field
comparisons of the color of young versus old males by netting and marking
them using a Testor’s © paint marker with a unique number on the hindwing
(Hinnekint, 1974). Male age refers to the number of days elapsed since an
individual was first marked at the stream (Forsyth and Montgomerie, 1987).

This categorization does not include the 11 day teneral period which males

10
spend away from streamside (Waage, 1972). I performed pairwise

comparisons for color between young and old males by viewing the males side
by side at grazing incidence to the cuticle surface and assessing their color as
either ‘blue’ or ’green’. Grazing incidence refers to a view parallel to or at 0° to
the cuticle surface. A more quantitative assessment of color (see next section
and results) determined that males subjectively labeled as ‘blue’ or ‘green’ in
fact reflected different wavelengths. I selected males for comparison by walking
the stream and capturing the first male encountered that fit one of the two age
categories. I then captured the next male encountered of the opposite age
category. For these tests, young males were those marked fewer than five days
prior to the test, while old males were those marked more than 10 days prior to
the test (Marden and Wage, 1990).

I also performed pairwise comparisons of the age of ‘blue’ versus ‘green’
males by netting one blue and one green male and recording the number of
days elapsed since that individual was marked. I obtained pairs by capturing
the first male encountered of one color category (‘blue’ or ‘green’) and then
capturing the first male encountered of the other color category. I used each
male in only one field comparison. The color of male Q maculata did not
change with time of day or with sunny versus shady locations.

From June through August 1992 and 1993, I quantified the color of ‘blue’
and ‘green’ male Q maculata. During this time, I assessed the color of each
male at normal incidence by comparison to Munsell Color Chips (Munsell,
1976a). Normal incidence refers to a view perpendicular or at 90° to the cuticle
surface. Comparison at normal incidence provided a more reliable color
assessment than comparison of color at grazing incidence (pers obs). Munsell

color notation was later translated into a wavelength (Munsell, 1976b).

11
To determine whether the colors were produced by pigments or some

type of structural colors, I collected 20 blue and 20 green male Q maculata and
subjected them to a variety of physical and chemical tests. I first viewed each
cuticle in diffuse light to determine whether the color changed with lighting
conditions, as would be expected for structural colors. To examine positional
color change, I viewed each cuticle from a direction at grazing incidence to the
surface and then rotated the cuticle for viewing normal to the surface, noting the
progression of color change. To distinguish between epicuticle and exocuticle
as the source of color, I immersed the cuticles of five blue and five green males
in 8% KOH at 60 °C and monitored color change every 30 minutes (Schultz
and Rankin, 1985). Immersion in dilute KOH dissolves the epicuticle,
separating it from the rest of the cuticle. Separation of the epicuticle will reveal
whether the color is produced in the epicuticle or the remainder of the cuticle. If
the epicuticle is the source of the color, the blue/green color will disappear and
the resulting cuticle will be black. I bleached the cuticle of five blue and five
green males by immersion in 10% hydrogen peroxide at 26 °C and monitored
color change every 30 minutes (Mason, 1927). Hydrogen peroxide will bleach
pigment colors and alter interference colors if melanin is a component of the
interference reflector (Schultz and Rankin, 1985). I immersed five blue and five
green cuticles in water to swell the exoskeleton. After noting any color change, I
allowed the cuticles to dry to determine if the initial color could be restored. l
subjected the remaining five blue and five green males to shrinking by
compressing the cuticle with a dissecting needle and noting any color change
(Mason, 1927).

I collected an additional five blue and five green males and obtained
transmission electron micrographs (T EM’s) of their cuticle for microstructural

analysis of the presumed color producing layer. TEM’S were obtained from the

12
same abdominal segment of each male. Transmission electron microscopy

was performed by the Center for Electron Optics at Michigan State University.

RESULTS

In 48 of 52 pairwise color comparisons of young versus old male Q
magnate, the color of the younger male was judged blue while that of the older
male was green ()_(Z = 81.7, df = 2, P < 0.0001). Only one older male was
judged blue while its younger counterpart was judged green. In three cases the
color of both the young and the old male was judged blue.

In 49 pairwise comparisons for age (days marked) between blue versus
green males, the mean age Lt SD) of the blue males was 3.0 days i 3.2 SD
while that of green males was 12.1 days i 7.3 SD (Wilcoxin signed ranks test,
T5 = 7, N = 49, P < 0.001)

Viewing in diffuse light did not reduce the intensity or alter the color of
any of the 20 blue or 20 green cuticles. This means that the colors are not
produced by diffraction. When viewed at grazing incidence to the surface, all 20
blue cuticles were deep to medium blue while all 20 green cuticles were blue-
green to green in color. When the angle was increased to normal incidence,
the 20 blue cuticles gradually progressed to bright green while that of the 20
green cuticles progressed to yellow-green. These color changes are
suggestive of constructive interference colors.

Figure 1 reveals the Munsell Chip-quantified colors of 617 ‘blue’ and 314
‘green’ male Q maculata. The resolution of the Munsell Chips used for color
assessment is 0.5-5.0nm. The ability of Q maculata to distinguish among these
colors is being investigated. The subjective color assessment of whether a male

was ‘blue’ or ‘green’ was made upon viewing flying or perching animals which

13
approximates a view at grazing incidence to the cuticle surface. The Munsell

color assessments were made from a view normal to the cuticle surface. At
normal incidence, ‘blue’ males look bright green and ‘green’ males look yellow-
green. Translation of Munsell color notation into wavelengths indicated that
blue males exhibited short normal incidence wavelengths of 509nm or 525.5nm
while green males exhibited long normal incidence wavelengths of 550nm or
558nm (Figure 1). Two males reflected a normal incidence wavelength of
559nm. No color matches were found for the color chips corresponding to the
intermediate wavelengths. The mean wavelength of 'blue' males (520.1nm i
7.7SD) was significantly shorter than that of 'green' males (550.7nm i 2.3SD;
Mann-Whitney Q = 0.00, P < 0.0001).

Chemical treatments of cuticle produced the same results for both blue
and green cuticle. Portions of Q maculata cuticle treated with 8% KOH
gradually lost their color. During the first 30 minutes, the color of both green
and blue cuticles lengthened to red/orange. After one hour, all cuticle
fragments were devoid of color and became a flat black/brown. The solution of
dissolved epicuticle was brown. This suggests that the epicuticle is the source
of the bright blue/green male colors. Fragments of cuticle bleached in 10%
hydrogen peroxide changed to longer wavelength green during the first 30
minutes. After three hours the intense green color began to dull and continued
to dull slowly over the next eight hours. This suggests that the color is structural
rather than chemical in nature.

Assessments of the physical tests were made while viewing the cuticles
at normal incidence in the field. Therefore ‘blue’ cuticles initially looked bright
green and ‘green’ cuticles initially looked yellow-green. Swelling the cuticles
with water resulted in the five ‘blue’ cuticles turning a lighter green while the five

‘green’ cuticles turned pale yellow-green. The color change was reversed upon

14

 

 

 

 

an P-
am ”"
am F
2
an r-
1m —
I 'Groon' rnoloo
0 l ; 'Bluo' males
525.5 590.0 553.0 559.0

Wovolongth (nm)

Figure 1. The wavelengths of color reflected by male Qalgpteryx maculata
categorized as ‘blue’ or ‘green’. (N = sample size)

1 5
drying. Compressing the cuticles resulted in changes to slightly shorter

wavelength colors. The five initially bright green ‘blue’ cuticles turned blue-
green while the five initially yellow-green ‘green’ cuticles turned bright green.
The color change was reversed upon release of the pressure. These changes
associated with position, pressure, and swelling are suggestive of constructive
interference colors.

TEM’s of both blue and green cuticles revealed a series of electron
dense bands separated by electron lucent bands in the epicuticle (Figure 2),
providing direct evidence of a multilayer interference reflector. The cuticles of
blue and green male Q maculata did not differ with respect to the number of
electron lucent or electron dense bands. Cuticles of blue males had thinner
electron lucent bands as well as thinner electron dense bands than cuticles of

green males (T able1; Figure 2).

16

Table 1. Mean number and spacing of electron lucent and electron dense
lamellae in the epicuticle of blue versus green male Caloptegg maculata.

 

Number of lamellae (iSD) Width of lamellae (mm :80)

 

 

Male Color N lucent dense lucent dense
Blue 5 6.3 :I: 1.0 6.3 _+__1.0 96.1 i 4.6 56.0 :t 2.6
Green 5 6.4 i 1.0 6.4 :t 0.9 103.6 i 2.3 60.0 i 1.9
Statistic df=8 t=0.24ns t=0.24ns t=3.28* t=3.28*

 

*P<005

 

 

 

 

 

 

Figure 2. Transmission electron micrographs of the outer exoskeleton of blue
(top) and green (bottom) male Calgptem maculata showing alternating layers
of electron lucent and electron dense material in the epicuticle.

18
DISCUSSION

The most likely cause of color in male Q maculata is constructive
interference by thin films or lamellae in the epicuticle. When the angle of
viewing the cuticle is changed from grazing to normal incidence the color
progressed from shorter wavelength blues to longer wavelength greens as
predicted (Onslow, 1921; Mason, 1927). Swelling the cuticle lengthened
wavelengths while the converse was true for compressed cuticle. The direction
of these color changes in addition to their reversibility is also indicative of
constructive interference colors (Mason, 1927).

It is unlikely that Q maculata color is the result of Tyndall effects (color
produced via scattering of light by particles), as the positional color changes are
uncharacteristic of Tyndall blues. Also, the green color would require the
presence of a yellow pigment (Neville, 1975) which was not detected in the
chemical tests. Diffraction gratings are also unlikely to be the major source of
color in male Q maculata, Since the color does not dim in diffuse light nor
disappear at certain angles of viewing (Hinton, 1976).

The source of the structural colors in male Q maculata is the Iamellate
epicuticle. Abdominal fragments treated with 8% KOH to remove the epicuticle
exhibited a loss of color. Bleaching in 10% H202 did not alter the hue but
reduced the intensity of color. This dulling of color with bleaching likely
indicates the presence of melanin as an absorbent background to the
interference reflector (Mason, 1927; Schultz and Rankin, 1985).

The alternate layering of electron lucent and electron dense materials in
the epicuticle as indicated by the TEM’S is characteristic of a multilayer
interference reflector. Other studies of multilayer interference reflectors in

insects suggest that the system is composed of electron dense melanin layers

1 9
separated by chitin (Durrer and Villiger, 1972; Schultz and Rankin, 1985). This

may also be the case for Q. maculata; however, it is difficult to detect small
amounts of melanin. The brown-stained solution from dissolving the epicuticle
in dilute KOH may indicate the presence of melanin in the epicuticular layers,
however, melanin may also have been leached from the exocuticle (Richards,
1967; Schultz and Rankin, 1985).

If we assume that the multilayer interference reflector consists of layers of
chitin and melanin, with refractive indices of 1.5 and 2.0 respectively, the
wavelength of peak reflectance such a system can be calculated with the
formula:

11 max = 2 (nLdL + nDdD)

where A max is the wavelength of peak reflectance, nL and no refer to the
refractive indices of the electron lucent and electron dense layers respectively
while d|_ and dD refer to the thicknesses of the electron lucent and electron
dense layers respectively (Neville, 1975; Schultz and Rankin, 1985).
Calculating the wavelength using refractive indices of 1.5 and 2.0 and mean
thickness for the electron lucent and electron dense layers of blue and green
cuticles respectively yielded wavelengths of 512nm for blue males and 552nm
for green males. These numbers closely correspond to the wavelengths
reflected by these males as indicated by the field comparisons with Munsell
Chips. In the field, ‘blue’ males reflected normal incidence wavelengths of
520.1nm i 7.7 SD (range: 509nm to 525.5nm) while ‘green’ males reflected
normal incidence wavelengths of 550.7nm i 2380 (range: 550nm or 558nm).
According to color quantification with Munsell Chips, blue males had
shorter wavelength coloration that green males. Due to positional color

changes characteristic of interference colors described earlier, future work will

20
refer to ‘blue’ Q maculata as short wavelength males and ‘green’ Q maculata

as long wavelength males.

The present study is the first to address color production and color
change in a Calopterygid damselfly. This study also presents the first indication
of a multilayer constructive interference reflector in a damselfly epicuticle.
Tyndall effects are responsible for the blue colors of Coenagrionid damselflies
(Mason, 1926; Charles and Robinson, 1981) as well as the colors of the

following dragonflies: Anax, EMhemis, and Libellula (Mason, 1926). The

 

current field evidence indicates that male Q maculata change color from blue to
green as they age, suggestive of swelling of the epicuticular lamellae
responsible for color production. The exact mechanism and significance of

color change with age in this species will be discussed in subsequent chapters.

CHAPTER 2

DECREASING FAT LEVELS WITH AGE RESULT IN COLOR CHANGE IN
MALE CALOPTERYX MACULATA

21

22
INTRODUCTION

Males of the damselfly Calopteyx maculata appear bright blue or green
as a result of constructive interference by thin epicuticular lamellae (see
Chapter 1). Transmission electron microscopy indicates that short wavelength
colors (512nm) are produced by epicuticle with thin lamellae while longer
wavelength colors (552nm) are produced by thicker lamellae in the epicuticle.
In the field, males with shorter wavelength coloration (509-525nm) are younger
than males with longer wavelength coloration (550-559nm).

Young male Q maculata have higher fat levels and are more likely to win
territorial contests than are old male Q maculata (Forsyth and Montgomerie,
1987; Marden and Wage, 1990). Fixed body size at emergence, combined
with the energetic demands of territoriality and reduced foraging time of
territorial males compared to nonterritorial males results in energy depletion
and declining resource holding potential with age (Forsyth and Montgomerie,
1987)

The relationship between age and color, and age and fat or energy
reserves suggests that declining fat levels with age may result in the observed
change in the wavelength of male coloration. The objective of this study was to
examine the relationship between fat levels and male color and to discern the
relative importance of fat versus age for color change. I met this objective by
manipulating the fat reserves of same age/color males and monitored the rate

and degree of color change.

23
MATERIALS AND METHODS

From 15 July to 15 August 1993, I collected male Q maculata from a
stretch of Augusta Creek in the Kellogg Forest approximately six miles from the
Kellogg Biological Station in Southwestern Michigan. I collected only those
males with minimal wing wear and that were Similar in size and color. After
netting, I measured each male’s forewing length and assessed its color by
comparison with Munsell Color Chips at normal incidence to the cuticle surface
(Munsell 1976a). Damselflies were maintained in the laboratory facilities at the
Kellogg Biological Station. I housed males individually in quart-sized glass
jars, placed on their sides, and with fine mesh screen covering the mouth of the
jar. The bottom of each jar was covered with thick wettened filter paper. I
randomly assigned males to one of two treatment groups to determine the
effects of high or low food consumption on color change. I fed each male
individually two times per day by presenting live Drosophila melanogaster to
them with a forceps (Hinnekint, 1987). I fed males in the low food treatment two
Q melanggaster per feeding (4/day) and males in the high food treatment , five
Q melanggaster per feeding (10/day). Four Q melanogaster is the lowest
number of prey that will maintain Q maculata in the laboratory for at least 10
days (Fitzstephens, unpubl.). Five Q melanogaster is the largest number of
prey that Q maculata can comfortably consume at one feeding without force
feeding (Fitzstephens, unpubl.).

During the first feeding bout of each day, I assessed each male’s color by
comparison with Munsell Chips at normal incidence to the cuticle surface.
Munsell color notation was later translated to a wavelength (Munsell 1976b).
After 10 days of feeding, l dissected the males for assessment of fat and water

content. I determined fat content by subtracting lean dry body mass from dry

24
body mass, dividing by dry body mass and multiplying by 100%. Lean dry body

mass was determined following fat extraction for four hours with refluxing

chloroform (Marden 1989; Marden and Waage 1990). A measure of percent
water that was independent of fat content was determined by subtracting dry
mass from fresh mass, dividing by lean fresh mass and multiplying by 100%

(Marden and Chai, 1991).

RESULTS

A total of 55 male Q maculata were captured and maintained in the
laboratory as described above. At the start of the experiment, the 25 males
assigned to the high-food treatment and the 30 males assigned to the low-food
treatment did not differ with respect to size or the wavelength of color (Table 2).
Over the 10 days on their respective diets, low-food males experienced a
significantly greater positive change in the wavelength of color reflected than
high-food males (Table 3). This resulted in the low-food males exhibiting
Significantly longer wavelength coloration than high-food males at the end of
the study (Table 3). Low-food males also changed color faster than high-food
males. High-food males retained their initial color almost twice as long as low-
food males (Table 3).

High-food males had a significantly higher fat but not water content than
did low-food males (Table 4). The values of percent fat content reported here
for high-food and low-food males are similar to those reported elsewhere for
short wavelength males (6.7% : 2.0SD) and long wavelength males (4.4% i

1.63D) respectively (see Chapter 3).

25

Table 2. Mean wing length and initial wavelength of color for high food and low
food male Qaloptemx maculata.

 

 

Male N Wing Length (mm 1. SD) Initial Wavelength (nm at SD)
High food 25 29.8 i 1.1 517 J: 8.4
Low food 30 29.6 _+_ 0.7 517 .t 8.4

 

Statistic df=53 t=0.91 ns t=0.14ns

 

26

Table 3. Mean latency to change color, final and change in wavelength for
high food and low food male Qaloptem maculata.

 

 

 

Latency (days 1 SD) Wavelength (nm i SD)
Male N to change Final Change
High food 25 6.9 i 2.6 526 i 15.5 9 i 11.0
Low food 30 3.5 i 2.1 539 i 12.4 22 :t 6.5
Statistic df=53 t=5.31*** t=3.54** t=5.73***

 

** P < 0.001, *** P < 0.0001

27

Table 4. Mean fat and water content for high food and low
food male Caloptegx maculata.

 

 

 

Male N %Fat (i SD) %Water (i SD)
High food 25 7.3 i 2.2 7.5 i 2.0
Low food 30 4.1 i 2.7 7.4 _+_ 2.0
Statistic df=53 t=4.68*** t=0.18ns

 

*** P < 0.0001

28
DISCUSSION

The relationship between diet and color has been studied in other
odonates. McVey (1985) demonstrated that in males of the dragonfly EMhemis
simplicicollis, decreasing food consumption decreased the rate of male color
maturation. McVey’s study examined color change from juvenile to mature
color. The present study, which examines color change from young mature
male Q maculata to old males, indicates an increasing rate of color change with
decreasing food consumption.

Fat levels are more important than age for determining the wavelength of
color reflected by male Q maculata. Males that were maintained on a high food
diet had a higher percent body fat than males maintained on a low food diet.
These males with higher percent body fat experienced a significantly smaller
color change than similar age males with lower percent body fat. The high fat
males retained their initial color almost twice as long as did low body fat males.

The decrease in percent body fat must increase the lamellar spacing in
the epicuticle to result in the observed color change from blue to green. The
initial high percent body fat may compress the lamellae thereby resulting in the
reflection of shorter wavelength blues. A decrease in body fat releases that
pressure causing the lamellar spacing to widen and reflect the longer
wavelength green color. Qaloptega maculata exoskeleton is malleable
compared to other insects exhibiting constructive interference colors and is
likely to be susceptible to these slight changes in internal pressure (Schultz,
pers. com.)

Marden and Waage (1990) also concluded that fat or energy is more
important than age for determining the winners of aggressive contests in male

Q maculata. They demonstrated that differences in fat or energy reserves are

29
an important asymmetry between male Q maculata engaging in wars or attrition

or escalated territorial contests. Young territorial males had more body fat than
old territorial males and winners of fights had Significantly more body fat
remaining at the end of contests than did losers of fights (Marden and Waage,
1990). Marden and Waage concluded that the ability of relative age to predict
relative energy was less than the observed ability of relative energy to predict
winners of contests. The authors did not; however, find evidence of assessment
of fat reserves in male Q maculata. The relationship presented here between
fat reserves and the wavelength of color reflected by male Q maculata suggests
that color might provide an index which males may use to assess each others

fat levels.

CHAPTER 3

COLOR RELIABLY SIGNALS FIGHTING ABILITY IN MALE
CALOPTERYX MACULATA

30

31
INTRODUCTION

This chapter investigates the role of color as a signal of male resource
holding potential (RHP) in a natural population of Caloptegcx maculata.

The presence of territorial and nonterritorial males has been well
documented in Q magtLaLa populations (Waage, 1972; Forsyth and
Montgomerie, 1987). Nonterritorial, sneaker, males are previously successful
territorial males that have adopted an alternative strategy later in their lifetime
as their resource holding potential decreases (Forsyth and Montgomerie, 1987).
Male Q maculata often engage each other in aerial contests, a primary
mechanism for turnover in territory ownership (Marden and Waage, 1990).
Marden and Waage (1990) established that in Q W populations,
winners of contests have significantly more fat (energy reserves) than do losers
and that young territorial males have higher fat levels than old territorial males.
They conclude that fat content or energy reserves were more often correlated
with winning contests than size or physical attributes related to fighting ability.
Forsyth and Montgomerie (1987) hypothesized that fixed body size at
emergence, combined with the physical and energetic demands of territoriality
and the reduced foraging time of territorial males compared to nonterritorial
males results in energy depletion and declining resource holding potential with
age.

Male Q maculata exhibit constructive interference colors on their thorax
and abdomen that change from blue to green with age (see Chapter 1).
Munsell color assessment indicates that ‘blue’ males exhibit short normal
incidence wavelengths that range from 509nm to 525nm while ‘green’ males
exhibit longer normal incidence wavelengths that range from 550nm to 558nm.

To avoid confusion, all color assessments will refer to normal incidence and

32
‘blue’ males will be referred to as ‘short-wavelength’ males while ‘green’ males

will be referred to as ‘long-wavelength’ males. Transmission electron
micrographs of the cuticle of male Q maculata indicate that the increase in
wavelength results from an increase in epicuticular lamellar spacing which may
be related to fat content or energy reserves. Male Q maculata maintained in
the laboratory on a high food diet had significantly higher percent fat and
exhibited a significantly smaller color change than males on a low food diet
(see Chapter 2). High-food males also retained their initial color almost twice
as long as low-food males.

In this study, I investigated the relationships among male Q maculata
age, size, energy reserves and color and how these variables affected male
territorial behavior and mating success in the field. I then use this information to
evaluate the potential for color to act as a useful signal for assessment of male

fighting ability in this species.

MATERIALS AND METHODS

The study site consisted of a 100m stretch of Augusta Creek
approximately three miles from the Kellogg Biological Station in Southwestern
Michigan, USA. From 16 June through 20 August of 1992 and 1993, I collected
data on sunny or partly cloudy days by netting male Q. maculata, measuring
their forewing length (Southwood 1968), and individually marking them. I
censused the population daily at 10:00, 13:00, and 16:00 by walking the length
of the study site and capturing male Q maculata. During each census, l marked
all animals not previously seen on the study Site and mapped the location of all
territorial and nonterritorial males. As described in Chapter 1, male age refers

to the number of days elapsed since an individual was first marked at the

33
stream (Forsyth and Montgomerie, 1987). Territorial males were those that

perched on or near floating oviposition material and defended these sites by
chasing away intruding males. Nonterritorial males were those that were highly
mobile, visiting locations on the stream up to 100m apart on a single day.
Nonterritorial males did not defend specific locations along the stream and were
often chased away from oviposition sites by territorial males (Forsyth and
Montgomerie, 1987).

Every day, I captured males once for assessment of color at normal
incidence by comparison with Munsell Color Chips (Munsell, 1976a). Munsell
color notation was later translated into a wavelength (Munsell, 1976b). Other

data collected ad libitum between census times included the location, outcome

 

and identity of males observed in copulatory attempts. During copulation, the
male mounts the female from above forming the tandem position. Copulation
typically lasts one to two minutes after which the female initiates oviposition
(Waage, 1973). I only recorded as successful copulations those interactions in
which the complete sequence of events occurred. I recorded an unsuccessful
copulation when females were successful in actively refusing males using wing
flaps or evasive flights.

I collected a sample of 30 short-wavelength and 30 long-wavelength
males for analysis of fat and water content. After assessing each male’s color
with Munsell Chips, l determined percent body fat and percent water as
described in Chapter 2.

Finally, I utilized path analysis (Li, 1981) to investigate hypothetical
causal relationships among the variables. Path analysis is essentially a series
of multiple regressions and correlations structured by prior knowledge of the
biological system and can be used to predict important interactions (Wootton,

1994). The relationships among variables in a system are represented by a

34
path diagram based on knowledge of the biology of the system (Li, 1981;

Kingsolver and Schemske, 1991). In a path diagram, single-headed arrows
reflect a causal relationship between two variables while double-headed
arrows represent correlations without causation. The path coefficient indicates
the strength of association of two variables by providing a relative measure of
the amount of variation explained by each causal variable and the sign of the
interaction. Path coefficients are estimated as the partial regression coefficient
standardized by the ratio of the standard deviations of the independent and
dependent variables (Li, 1991; Wootton, 1994). Path coefficients represent the
direct contribution of the predictor variables to the predicted variables, holding
other variables constant. The degree to which the variation in a variable is
accounted for can be calculated by summing the products of each path
coefficient with the respective variables’ correlation coefficient (Li, 1981).

A single path diagram representing causal relationships among
variables can be analyzed to estimate the relative strengths of interactions
among variables. Path analysis also distinguishes direct from indirect effects.
Conclusions based on this type of analysis are to be treated as predictions that
indicate experiments to be conducted rather than as conclusions in themselves
(Wootton, 1994).

Prior to this section of the study, relationships among variables are
discussed in terms of pairwise comparisons. These relationships can be
organized into a coherent account of Q maculata territorial and mating behavior
using path analysis. This will allow for the partitioning of direct versus indirect
interactions while eliminating from the analysis variables with no significant
direct effects on other variables. In this chapter, path analysis will be used to
determine the relative strengths of the biological interactions depicted in Figure

3. Figure 3 represents the positive and negative relationships among variables

35
important for Q maculata territorial and mating behavior as discussed prior to

my study. I will incorporate my data into a path analysis of these interactions to
factor out insignificant variables and to determine the relative strengths of the
remaining interactions for predicting male Q maculata territorial and mating
success. I used information of Q maculata natural history to construct the
interaction diagram as a starting point for path analysis. I included paths in the
path diagram only if the partial regression in the underlying multiple regression
was statistically different from zero. The path analysis will demonstrate the
relationship between color and the measures of body condition important in Q
maculata contests as well as the degree to which color contributes to our

understanding of territorial behavior.

36

Fat
Content
+
- Fighting + Territorial + I Mating
Ability . Status Success
4,.
Age

Figure 3. Interaction diagram constructed using information from Caloptem
maculata natural history. This diagram is to be used as a starting point for path
analysis.

37
RESULTS

l marked a total of 932 individual male Q. maculata during the two year
study for a total of 2008 animal observations (Figure 4). The size of individuals
at first capture, as indicated by forewing length, declined throughout the season
(Kendall’s tau = -0.22, P < 0.0001). This negative relationship between size at
marking and day of season is common in odonate studies (Harvey and Walsh,
1993; Tsubaki and Ono, 1987; Banks and Thompson, 1985; Harvey and Corbet,
1985). This seasonal effect accounted for a small percentage of the variation in
size (r2 = 11.6%). Since the size of Q maculata is fixed at emergence (Forsyth
and Montgomerie, 1987), the seasonal change in emergence size sets up a
pattern of older males (those that emerged early in the season) being larger
than younger males (those that emerged later in the season). Therefore, size
(the physical dimensions of the damselfly) should not be confused with fat
content which is variable and changes throughout an individuals lifetime. In
addition to size and fat content, fresh body mass will be reported in this study.
However, the results involving body mass should be interpreted with caution as
body mass incorporates both body size (which is static) and body fat (which is
dynamic). Since the statistical analyses yielded similar results for each year
analyzed separately as well as combined, we report the results for 1992 and
1993 combined. Territorial males were significantly smaller in body size and
younger than nonterritorial males (Table 5). Despite the statistical significance,
the differences in size as measured by wing length are probably not biologically
meaningful. The sample sizes were very large thus exaggerating the
significance of the very small differences in wing length. Also, the size
differences are most likely the result of the seasonal decline in emergence size.

Territorial males also reflected shorter wavelength colors than did nonterritorial

38
males (Table 5). Of the 143 males exhibiting both territorial and nonterritorial

behavior during their lifetime, all began as territorial males and later switched to
nonterritorial.

I observed a total of 275 mating attempts involving marked males during
the study. Significantly more mating attempts by territorial males were
successful (203 of 210) than were mating attempts by nonterritorial males (1 of
65, _)_(_Z = 229.6, df = 1, P < 0.001). Successful males were smaller and younger
than unsuccessful males (Table 6). Again, the relationship with size is most
likely the result of the size/emergence day pattern. Successful males also
reflected shorter wavelength coloration than unsuccessful males (Table 6).

Wavelength was positively correlated with damselfly size (Kendall’s tau =
0.10, P < 0.01) as well as with age (Kendall’s tau = 0.29, P < 0.0001). As
damselflies aged, their color changed from short wavelength to longer
wavelength. Since size is fixed at emergence, the positive relationship
between size and color is due to the fact that older (and therefore larger) males
reflect longer wavelengths. l analyzed a sample of 30 short wavelength males
and 30 long wavelength males for fat and water content. The mean body mass
of short wavelength males (65.24mg i 5.258D) was slightly but Significantly
smaller than that of long wavelength males (69.03mg i 5.94SD; Mann-Whitney
Q = 291, P < 0.05). The mean fat content of short wavelength males (6.7% i 2.0
SD.) was significantly higher than that of long wavelength males (4.4% j; 1.6
8.0., Mann-Whitney Q = 764, P < 0.0001). Short wavelength males also had a
significantly higher water content (7.5% i 2.1 8.0.) than did long wavelength
males (7.3 % i 1.4 SD, Mann-Whitney Q = 710, P < 0.001). Short wavelength
males have a smaller body Size and lower body mass than long wavelength
males. The relationship between wavelength and body size reflects the

age/Size relationship established by the trend of decreasing emergence size

39

 

 

 

 

 

 

 

 

 

 

50 _
1992
40 -I
.3. 30 -
fl
2
'5 20 I
3 ..
E
3
2 1o-
0 III‘I‘I‘I TITAMGA [Till-ITTIIII IIIIII IIIIII IIITIIXTAI I II
o 9 I4 21 23 It 42 49 55 I53

Day (1 =June 16)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

40 1
1993
30 .-
fl
2
s I
*5 2° " . N
g i
S ' .
z 10 -
o llllllllllllll IITIIIIITITIIIIIIIITIIII ITTTIITIIIIIIIITI
ID 7 14 21 28 35 42 49 56

Day (1 =June 16)

Figure 4. The number of individual male Calopteryx maculata marked on each
day of the study.

40

Table 5. Mean (1 SD) size (forewing length), age, and wavelength of territorial
versus nonterritorial male Calopteryx maculata.

 

 

 

Male Size (mm) Age (days) Wavelength (nm)
Territorial (N=618) 29.1 11.1 1.5 i 2.8 520.2 i 8.1
Nonterritorial (N=171) 29.4 _+_ 1.1 5.0 i 6.5 550.5 i 3.0
Mann-Whitney U 1.1x105 ** 1.3x105 *** 1.9x105***

 

** P < 0.001, *** P < 0.0001

41

Table 6. Mean (: SD) size (forewing length), age, and wavelength for
successfully mated versus unsuccessful male Calopteryx maculata.

 

 

 

Male Size (mm) Age (days) Wavelength (nm)
Successful (N=210) 29.4 i 0.9 1.9 i 3.6 523.6 _t 10.8
Unsuccessful (N=65) 29.6 ;I-_ 1.0 6.0 i 7.1 548.2 _-I; 11.2
Mann-Whitney _U 8.4x103 * 10.0x103 ** 1.3x104 **

 

* P < 0.05, ** P < 0.001

42
with day of season. Body mass is also confounded by this relationship since it

incorporates body size as well as fat content.

Path analysis was used to create a model depicting the hypothesized
causal relationships among all variables relative to mating success. Figure 5
represents the diagram that best explains the variation in territorial and mating
behavior. This diagram represents a 20% increase in explanatory power over
the many alternative path diagrams created from the data. This path diagram
will also be used to determine the relative strengths of the interactions depicted
in Figure 3. The hypothesis predicts that mating success is determined
primarily by male territorial status. No other variables measured accounted for
a significant portion of the variation in mating success according to this path
diagram. Male territorial status was determined by wavelength. Age, fat and
water content directly contributed to male color; wavelength increased as age
increases and as fat and water decrease. Table 7 shows the correlations and
path coefficients used for determining the direct contributions of variables. The
total contribution of fat and water content and age toward wavelength can be
calculated by multiplying each path coefficient with the respective variable’s
correlation with wavelength and summing over all three variables (Li, 1981).
This calculation indicated that age and fat and water content accounted for
54.5% of the variation in the wavelength of male color. By the same argument,
wavelength accounted for 81.0% of the variation in male territorial behavior
which in turn accounted for 84.6% of the variation in mating success (Figure 5).
If we omit wavelength as a variable, the resulting path coefficients from age, fat
and water content to territorial status are much lower than the paths between
age, fat and water content to wavelength in the previous path diagram (Figure
6). These three variables directly contribute to only 39.1% of the variation in

male territorial status compared to their 54.5% determination of wavelength and

43
to the 81.0% determination of territorial status by wavelength alone (Figure 6).

Therefore, path analysis indicates that age, fat and water content are better
predictors of color or wavelength than they are of territorial status directly. Color
or wavelength is a better indicator of male territorial status than are age, fat and
water content combined. The variables ‘day of season’, ‘year’, ‘body mass’ and
‘forewing length’ are not represented on the diagram because they had no
significant effects on the other variables (i.e. the partial regression in the

underlying multiple regression was not significantly different from zero).

44

%Fat
-0.46
0.35

-0.32 -0.90 Territorial 0.92 Mating
%Water -—> Wavelength ——> Status —> Success

0.36

 

 

 

54.5% 81.0% 846%

Figure 5. Path analysis of the relationship of the predictor variables (age, %fat,
% water, and wavelength) with male territorial status and mating success. This
path diagram represents an hypothesis of causation for evaluation. Single-
headed arrows represent a causal relationship between two variables while
double-headed arrows denote correlations without causation. The degree to
which the variation in a variable is accounted for (indicated below diagram) can
be calculated by summing the products of each path coefficient with the
respective variables’ correlation coefficient (Li, 1981).

45

 

 

%Fat
0.37
0.35
0.28 Territorial 0.92 . Mating
%Water —> Status Success
-0.23
Age
I 84.6%
39.1%

Figure 6. Alternative path diagram resulting from the exclusion of wavelength
as a predictor variable.

46

Table 7. Correlation and path coefficients for calculation of the contributions of
predictor variables to predicted variables in Figures 5 and 6.

 

 

Path Correlation Path Coefficient
Territorial Status to Mating Success 0.92 0.92
Wavelength to Territorial Status 090 -0.90
%Fat to Wavelength -0.57 -0.46
%Water to Wavelength -0.48 -0.32
Age to Wavelength 0.36 0.36
%Fat to Territorial Status 0.51 0.37
%Water to Territorial Status 0.44 0.27

Age to Territorial Status -0.36 -0.23

 

47
DISCUSSION

Territorial male Q. maculata were smaller in terms of both size and body
mass, younger and more successful during mating attempts than nonterritorial
males. Marden and Wage (1990) concluded that size and mass are not
important in damselfly aggressive interactions. Path analysis of the data
supported this conclusion. That territorial males were smaller than nonterritorial
males is simply be an artifact of the negative relationship between day of
emergence and hence, age, with size. That territorial males are younger than
nonterritorial males, and that the 143 males exhibiting both territorial and
nonterritorial behavior began as territorial and switched to nonterritorial
behavior, is Similar to other examples of age related behavior patterns in
odonates (T subaki and Ono, 1987; Campenella and Wolf, 1974). Forsyth and
Montgomerie (1987) also demonstrate that in Q. maculata the probability of
adopting an alternative reproductive behavior such as sneaking increases with
male age.

Successfully mated male Q. maculata are younger and smaller than
unsuccessful males. This is most likely because females mate almost
exclusively with territorial males, which tend to be younger and smaller than
nonterritorial males. Territorial male Q. maculata are able to control female
access to oviposition sites, exchange matings for guarding (Waage, 1979;
1982) and exclude nonterritorial males.

This study demonstrates an age-related color change that is also related
to territorial behavior in male Q. maculata. Territorial males exhibited
significantly Shorter wavelength coloration than nonterritorial males.
Wavelength was also shown to increase with both Size, body mass and age.

Again, the relationship with size is most likely an artifact of the relationship

48
between size and day of marking resulting in older males being larger than

younger males. Since size is a component of mass, body mass is also
confounded by the Size/emergence clay relationship. Therefore, as Q. maculata
males age, their color changes from short to long wavelength and many switch
from territorial to nonterritorial behavior.

The importance of fat or energy reserves in male Q. maculata aggressive
interactions has been suggested in a number of studies (Forsyth and
Montgomerie, 1987; Marden and Waage, 1990). Marden and Waage (1990)
show that winners of escalated contests among male Q. maculata have
significantly more fat or energy reserves than losers. They also show that
young territorial males have significantly higher fat levels than old territorial
males. The present study indicates that the wavelength of male color is also
related to fat, as well as water content in this species. Short wavelength males
had a significantly higher fat and water content per unit body mass than long
wavelength males.

Path analysis of the hypothesized causal relationships among the
variables predicts that male Q. maculata mating success is affected most
directly by male territorial status (Waage, 1973, 1979). Male territorial status is
affected most directly by male wavelength. The variables age, fat and water,
which have previously been shown to vary with male territorial and aggressive
behavior (Forsyth and Montgomerie, 1987; Marden and Waage, 1990), make
their greatest direct contribution to male wavelength variation rather than to
male territorial behavior directly. The variables day of season, male size
(forewing length), body mass, and year do not make a significant direct
contribution to any other variables. These variables may affect the predicted
variables but only through their relationships with the other more important

predictor variables of age, fat and water.

49
Path analysis predicted that age, fat and water content are better

indicators of color or wavelength than they are of territorial status directly. Color
or wavelength is a better indicator of male territorial status than are age, fat and
water content combined. Path analysis has organized the many pairwise
comparisons of this and previous studies into a coherent picture of male Q
maculata territorial and mating behavior. Path analysis has indicated the
relative importance of variables having significant direct effects on territorial and
mating behavior (age, fat and water content) while eliminating from the analysis
the variables whose affects occur only through their interaction with other more
important variables (wing length, body mass, day of season, year). Color is
reliably related to the measures of body condition (energy reserves) which have
been shown to affect the outcome of male Q maculata interactions. Age and
energy reserves account for 54.5% of the variation in wavelength which in turn
accounts for 81.0% of the variation in territorial status. Without wavelength, our
accounting of the contributions to territorial status is only 39.1%. Thus,
wavelength greatly increases the ability to predict male territorial status. The
high predictability of territorial status from wavelength alone (81.0%) suggests
that color is a reliable signal or index of fighting ability. Color may be used to
assess energy reserves while the significant positive relationship between
wavelength and fat content maintains a level of reliability or honesty in the
signal. Future research must focus on testing whether or not Q maculata make

use of the information provided by color.

SUMMARY AND CONCLUSIONS

The exchange of information via signals is often a means by which
opponents in agonistic encounters can assess each other’s resource holding
potential or fighting ability. I investigated the role of color as a signal of fighting
ability in males of the territorial damselfly Qeloptem mgchata. l accomplished
this by elucidating the mechanism of color production and color change to
reveal the kinds of information provided by color. I also conducted field
observations to establish the relationship of color with territorial and mating
behavior.

In my study, I was able to demonstrate that the bright blue and green
colors of male Q maculata are caused by constructive interference by thin
lamellae in the epicuticle. The lamellae consist of alternating layers of electron
lucent chitin and electron dense melanin. Male Q maculata change from blue
to green as they age. Fat levels; however, are more important than age for
determining the wavelength of color reflected. This is the first study to
demonstrate a multilayer interference reflector in an odonate cuticle. It is also
the first study to address the role of color and color change in a natural
population of Q maculata.

Field data and path analysis indicate that territorial male Q maculata are
younger, have higher percent body fat, and are more successful at mating than
nonterritorial males. These results are similar to those reported in other studies

of Q magglata. What is new and exciting about the present study is that color,

50

51
a variable previously unmeasured in Q maculata populations is revealed to be

the best predictor of territorial behavior, with territorial males reflecting shorter
wavelength color than nonterritorial males. Furthermore, color is shown to be
reliably related to the measures of body condition (fat or energy reserves)
known to affect the outcome of Q maculata aggressive interactions.

These results indicate that color provides reliable information by which
male Q maculata can assess each other’s fat levels or energy reserves as they
indicate fighting ability. What remains to be shown is the extent to which males

utilize the information provided by color.

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