# Wing Color of Monarch Butterflies (Danaus plexippus) in Eastern North America across Life Stages: Migrants Are “Redder” than Breeding and Overwintering Stages

Authors: Andrew K. Davis
Journal: Psyche
 (2009)

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## Abstract

Monarch butterflies are famous among insects for their unique migration in eastern North America to overwinter sites in Mexico  and their bright orange wing color, which has an aposematic function. While capturing migrating monarchs in northeast Georgia, USA, I noticed that many appeared to have unusually deep orange wings. I initiated the current study to compare wing hues (obtained using image analysis of scanned wings) of migrants (captured in 2005 and 2008) to samples of breeding and overwintering monarchs. Consistent with initial observations, migrants had significantly lower orange hues (reflecting deeper, redder orange colors) than breeding and overwintering monarchs. There was also a difference in hue between sexes  and a relationship with wing size, such that larger monarchs had deeper, redder hues. The reasons for the color difference of migrants are not apparent, but one possibility is that the longer-lived migrant generation has denser scalation to allow for scale loss over their lifespan. Alternatively, this effect could be confined to the subpopulation of monarchs in the Southeastern United States, which may not be well represented at the Mexican overwintering sites. In any case, this discovery highlights the many questions emerging on the significance of wing color variation in this species.


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## Body

## 1. Introduction

The monarch butterfly (Danaus plexippus) is one of
the world's most well-known insects, being easily identifiable by its bright
orange and black wing colors (Figure 1). It is the posterchild for insect
conservation, and its image even graces the front cover of this journal.  One of
the reasons for this attention is certainly the amazing annual migration of the
population in eastern North America, which starts in breeding grounds in Canada
and the northern United States, and ends some 3000 km away at a select few
overwintering sites in the mountains of Central Mexico [1, 2]. There, they form massive clusters of millions of
butterflies, and wait until spring, when they remigrate northward to repopulate
the breeding grounds [3, 4]. This unique life cycle is completed in many generations.
Breeding monarchs normally live for one month, undergoing several generations
per summer. The final generation of the late summer is the one that undertakes
the migration and overwintering phase, so that these individuals live up to 9
months [5]. Over many decades, a considerable body of literature has
been developed around this unique insect, although recent discoveries are
revealing how many questions about it yet exist. For example, the functional
significance of the orange coloration of the monarch's wings has long been
known to be a warning to predators of its toxicity from the cardenolides the
larvae sequester from the host plants, which are members of the genus Asclepias [6]. However, recent work using modern image analysis
techniques has demonstrated there is considerable variation in the degree or shade of
orange color among monarchs [7]. This discovery together with the use of today's image
analysis computer programs that can easily quantify color variation on images
of butterfly wings, opens up a new body of questions regarding the significance
of this variation.Figure 1
Monarch butterfly,D. plexippus. This individual, a
male, was captured in September 2005 by AKD. Arrow indicates the cell used for
measuring color in this study.The current study was initiated because of certain casual
observations regarding monarch butterfly wing color. Specifically, I noticed
that monarchs captured during the fall migration sometimes appeared (with the
naked eye) to have more reddish-colored wings than those reared in lab
experiments [7, 8] and than those that had been captured during the summer
months. I subsequently developed the current project aimed at determining if
this difference was real, by using an image analysis approach to objectively
quantify the orange color of monarch wings, similar to that performed recently [7]. With these data, I statistically compared wing colors of
monarchs captured during migration to a sample of breeding monarchs and to a collection
of overwintering monarchs, and I report the results of this exploration here.


## 2. Methods

### 2.1. Butterfly Sources

Three sets of monarch butterfly specimens were examined in
this study. The first group (n=39) was collected during the summer of 1997 in
Minnesota and Wisconsin (hereafter called “breeding” group), between July 11
and August 30, although 85% of these were collected before August 15. These
specimens had been stored at −20°C (individually in glassine envelopes) since
capture. The second set  were
75 individuals collected at two of the Mexican overwintering sites in February
2008 (hereafter called “overwintering” group). These were netted from random
clusters and brought to the lab (with all applicable permits), where they were also
stored at −20°C. The third group consisted of monarchs captured during the fall
migration in 2005 and 2008 (n=29 and 31; hereafter called “migrant” group) and
were killed via freezing immediately after capture and stored at −20°C. All migrants
were captured while nectaring on blooming vegetation near Athens, Ga, USA (33.9°N–83.4°W), during
the periods of October 14–22, 2005 and September
11–29, 2008.


### 2.2. Scanning and Processing Wings

All frozen stored specimens were
thawed and their forewings were removed for scanning. Scanning procedure
generally followed procedures established in prior work [7, 8]. Wings were placed face down on a standard flatbed scanner
connected to a laptop computer, and scanned at 300 dpi. The exposure settings on
the scanner were set so that the original wing color was maintained (i.e., so
that the scanner software did not manipulate the images). All forewings were
scanned in this manner (i.e., with this scanner and with these settings). When
scanned, the sex of each monarch was also recorded.When all wings were scanned, each forewing image was
imported into the image analysis software, FoveaPro  (Reindeer Graphics, Inc., NC, USA)
(http://www.reindeergraphics.com/),
which works in the Photoshop environment.  This program is ideally suited to measure color, and has
been used by the author in prior studies [7–11]. Wing color measurement generally followed procedures
established in prior work that focused on this species [7] with slight modification. Briefly, the central wing cell
of the right forewing (Figure 1) was selected using the “magic wand tool.”  I
considered this selected area to be representative of the orange color of the
entire wing. Further, choosing this location to measure provided a convenient
border for the selected area. However, since female monarchs have more black
scales over their wing veins, which makes their veins appear thicker than males,
this means that in
general, the selected area was smaller in females than in males. Next, a
color-measure routine was initiated which returned the average hue, saturation,
and brightness values for all pixels selected (usually between 5 and 10
thousand pixels). In this case, only the hue values were retained, which is in
contrast to prior work, where the saturation score was the focus [7]. The hue was more appropriate to examine in this study
since the magnitude of the color variation was thought to be large based on
initial observations of migrant monarchs (i.e., ranging from pale yellow to
orange to dull red). Hue is measured in degrees (i.e., 0–360), with 0
representing perfect red, and in this study the scores tended to be between 25
and 40 (see results). Finally, a secondary routine was run which returned the
area of the entire forewing in mm2, based on prior calibration of
the software using a scanned ruler.


### 2.3. Data Analysis

Since two sets of migrants were obtained in this study, I
initially examined these data for possible differences using a two-samplet-test,
where the wing hue was the dependent variable and the year was the independent. This test revealed
no significant variation (df=58, t=−0.811, P=.421), therefore
these data were pooled for subsequent analyses. I then used general linear
modeling procedures to examine the possible variation in monarch wing color
(hue) among life stages, that is, breeding, migrating, and overwintering. Thus,
the analysis included wing hue as the response variable, “group” as a categorical
independent, as well as gender. Finally, butterfly size (indexed by forewing
area) was included as a continuous covariate. All two-way interaction terms
were initially included but removed if found nonsignificant. All analyses were
conducted using Statistica 6.1 software
[12].


## 2.1. Butterfly Sources

Three sets of monarch butterfly specimens were examined in
this study. The first group (n=39) was collected during the summer of 1997 in
Minnesota and Wisconsin (hereafter called “breeding” group), between July 11
and August 30, although 85% of these were collected before August 15. These
specimens had been stored at −20°C (individually in glassine envelopes) since
capture. The second set  were
75 individuals collected at two of the Mexican overwintering sites in February
2008 (hereafter called “overwintering” group). These were netted from random
clusters and brought to the lab (with all applicable permits), where they were also
stored at −20°C. The third group consisted of monarchs captured during the fall
migration in 2005 and 2008 (n=29 and 31; hereafter called “migrant” group) and
were killed via freezing immediately after capture and stored at −20°C. All migrants
were captured while nectaring on blooming vegetation near Athens, Ga, USA (33.9°N–83.4°W), during
the periods of October 14–22, 2005 and September
11–29, 2008.


## 2.2. Scanning and Processing Wings

All frozen stored specimens were
thawed and their forewings were removed for scanning. Scanning procedure
generally followed procedures established in prior work [7, 8]. Wings were placed face down on a standard flatbed scanner
connected to a laptop computer, and scanned at 300 dpi. The exposure settings on
the scanner were set so that the original wing color was maintained (i.e., so
that the scanner software did not manipulate the images). All forewings were
scanned in this manner (i.e., with this scanner and with these settings). When
scanned, the sex of each monarch was also recorded.When all wings were scanned, each forewing image was
imported into the image analysis software, FoveaPro  (Reindeer Graphics, Inc., NC, USA)
(http://www.reindeergraphics.com/),
which works in the Photoshop environment.  This program is ideally suited to measure color, and has
been used by the author in prior studies [7–11]. Wing color measurement generally followed procedures
established in prior work that focused on this species [7] with slight modification. Briefly, the central wing cell
of the right forewing (Figure 1) was selected using the “magic wand tool.”  I
considered this selected area to be representative of the orange color of the
entire wing. Further, choosing this location to measure provided a convenient
border for the selected area. However, since female monarchs have more black
scales over their wing veins, which makes their veins appear thicker than males,
this means that in
general, the selected area was smaller in females than in males. Next, a
color-measure routine was initiated which returned the average hue, saturation,
and brightness values for all pixels selected (usually between 5 and 10
thousand pixels). In this case, only the hue values were retained, which is in
contrast to prior work, where the saturation score was the focus [7]. The hue was more appropriate to examine in this study
since the magnitude of the color variation was thought to be large based on
initial observations of migrant monarchs (i.e., ranging from pale yellow to
orange to dull red). Hue is measured in degrees (i.e., 0–360), with 0
representing perfect red, and in this study the scores tended to be between 25
and 40 (see results). Finally, a secondary routine was run which returned the
area of the entire forewing in mm2, based on prior calibration of
the software using a scanned ruler.


## 2.3. Data Analysis

Since two sets of migrants were obtained in this study, I
initially examined these data for possible differences using a two-samplet-test,
where the wing hue was the dependent variable and the year was the independent. This test revealed
no significant variation (df=58, t=−0.811, P=.421), therefore
these data were pooled for subsequent analyses. I then used general linear
modeling procedures to examine the possible variation in monarch wing color
(hue) among life stages, that is, breeding, migrating, and overwintering. Thus,
the analysis included wing hue as the response variable, “group” as a categorical
independent, as well as gender. Finally, butterfly size (indexed by forewing
area) was included as a continuous covariate. All two-way interaction terms
were initially included but removed if found nonsignificant. All analyses were
conducted using Statistica 6.1 software
[12].


## 3. Results

Across all monarchs examined in this study (n=174), there
was considerable variation in wing hue scores, which ranged from 25.3 to 40.1 degrees
(Figure 2). Lower hue scores reflect deeper orange colors, almost nearing red,
while the higher scores indicate generally paler, more yellowed wings. In the
statistical analysis of wing hue, there was a significant effect of group (P<.0001;
Table 1) in that migrants had significantly lower hues than all other life
stages (Tukey's pos-hoc test; Figure 3). The mean hue of migrants was 30.5 and
the hues of breeding and overwintering monarchs were 35.2 and 35.6,
respectively. To visually depict this large difference between migrants and
other groups, Figure 4 shows selected forewings from each group. Note the color
of the orange wing cells in each group. Furthermore, the distribution of
migrant monarchs is graphed separately from other nonmigrant groups in Figure 2
to highlight the large difference in wing color of migrants.Table 1
Summary of general linear model examining factors influencing wing hue of
monarch butterflies in this study. Groups were “breeding,” “overwintering,”
and “migrating.” Nonsignificant interaction terms (i.e.,
whereP>.05) were removed.
VariabledfMSFP-valueSex1475.93187.47<.0001Group2391.56154.23<.0001Wing area112.354.87.0288Sex*group210.994.33.0147Error1672.54——Total173———Figure 2
Frequency distribution of wing color (hue score, measured in degrees)
observed among all migrating (n=60) and nonmigrating (n=114) monarchs. Colored
scale included which shows the hue scores of each category.Figure 3
Average wing hue score (in degrees) across all groups for male (n=107)
and female (n=67) monarchs in this study. Error bars represent 95% CI.Figure 4
Forewings of selected migrant, breeding, and overwintering monarch
butterflies. Note the difference in orange color between migrants and all others.
All wings were scanned with the same scanner using the same settings. Males (M)
and females (F) are indicated.In addition to the differences among stages, I also
discovered that males differ in wing hue from females (P<.0001; Table 1). In all three life stages, males had significantly lower hue scores than
females (Figure 3). In other words, the orange color on male monarch wings
tends to be deeper, or more reddish, while female monarchs have a paler orange
color. However, the magnitude of this gender difference differed among groups,
as evidenced by the significant interaction term of sex*group in the final
model (P=.0147; Table 1). This interaction effect can also be seen in
Figure 3, where the overall difference between male and female hue scores was the
greatest in the migrant group (4.6 degrees difference), versus 3.3 degrees for
breeding and 3.1 degrees for overwintering monarchs.Finally, there was a surprising effect of wing size on hue
score (P=.0288; Table 1). This effect was negative, such that large
wings tended to have lower hue scores. Put another way, larger monarchs tended
to be more red-colored. The interaction terms of wing area*sex and wing
area*group were not significant, meaning that this relationship held for both
sexes and across all life stages.


## 4. Discussion

The results of this exploratory study corroborate the
initial observations made of the wings of migrating monarchs. That is, migrating
monarch butterflies appear to have significantly deeper orange wings than they
do during the summer. Further, I observed this phenomenon in two separate
migrations (2005 and 2008), and in both cases the migrants' wing colors were
similarly deep orange or nearly red (and were statistically similar). Thus,
this phenomenon is not random but appears real. This discovery then represents
the first morphological trait shown to be different among the final summer
migratory generation and the mid-summer breeding generations. The reason for
this difference is not clear, although one could speculate that the deeper
orange color may help to absorb solar energy, which would enable flight at
lower temperatures. Alternatively, the deep orange could reflect some
physiological shift by larvae of the final generation (i.e., allocating
resources from other processes to pigment production).Besides the differences in wing color found between
migrating and breeding monarchs, a more surprising result of this study was
that the wing color ofmigrating monarchs did not resemble that of overwintering monarchs. The overwintering monarchs all appeared to have the same pale-orange
color of breeding monarchs. How then, is this possible if they are all cohorts
of the same generation? Does the migrant wing color fade over time while at the
overwintering locales? While there is little research on this subject, the
answer to this question would probably be yes. This generation is certainly the
longest lived by far, and it may make sense that the migratory generation
produces heavily pigmented wings to account for the eventual scale loss over
time.A second explanation may be that this phenomenon (of
deeper-orange migrants) is unique to the population of monarchs in the
southeastern United States, and that monarchs from this area are not well represented
at the Mexican overwintering site. In support of this idea, recent work
examining migration routes of monarchs indicated that monarchs migrating along
the eastern seaboard have a reduced chance of reaching the overwintering sites
than those in the interior United States [13]. Furthermore, migrating monarchs captured and tagged in
the Florida Panhandle have only a 1 in 4000 chance of being recovered at Mexico overwintering
sites  (cited in [13]). In contrast, the normal Mexican recovery
rate for tagged monarchs from the eastern population as a whole is 1 in 250 [14]. Thus, monarchs migrating through Georgia may not be well represented
at the Mexican overwintering site. Clearly, future work should examine wing
colors of migrating monarchs from other regions to determine how widespread
this phenomenon is. Only then, will we know if this phenomenon is unique to the
southeast, or if all migrants display this trait. If the latter is true, then
the discrepancy between migrant and overwintering wing color would become even
more perplexing.There were two other surprising findings in this study.
First, I found that males and females differ statistically in wing hue, with
males tending to have a deeper orange color than females. This species was
already known for another sexually dimorphic trait, in that females have a
greater degree of black pigmentation [8], see Figure 4, but it would seem that the sexes differ in other features
as well. Secondly, there was an unexpected relationship found (although wing
size was included as a covariate in the analysis to account for this possibility)
between wing size and hue, such that larger individuals tended to have a deeper
orange to dull-red color. Like most butterflies, large body size in monarchs is
generally considered an indicator of fitness [15, 16], so deeper orange wing color may be a sign of more robust
individuals. However, a greater understanding is needed of the causal
mechanisms behind wing color formation before this idea can be fleshed out
further.Clearly, there are a number of findings from this work that
open up a large number of additional questions on the significance of monarch
butterfly wing color, and which will take much more study to address. However,
since even these simple comparisons of wings between breeding, migrating, and
overwintering monarchs here proved fruitful, the possibility for further
exciting discoveries in this research avenue is high. This is especially true
since the discoveries made here only serve to emphasize the large gaps that
remain in our knowledge of the biology of this unique and charismatic insect.



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*Source: 705780-2009-02-05.xml*