Transcript Slide 1

The Role of Cuticular Hydrocarbons in the Mate Recognition of Hybridizing Chrysochus (Coleoptera: Chrysomelidae)
Erica Davis and Danielle Juárez
Biology Department, Western Washington University, Bellingham WA 98225
Geographic Variation in Cuticular
Hydrocarbons
*
Wenatchee
Copulation Duration (min.)
Abstract
Our goal in the present study was to determine if cuticular hydrocarbons
(CHCs) of Chrysochus auratus and Chrysochus cobaltinus play a role in the
evolution of premating barriers between these beetles. To do so, we assessed
whether sex- and species-specific CHCs are involved in mate choice and whether
there is evidence of reproductive character displacement in CHCs. CHCs were
removed and isolated via a hexane wash and applied to dead C. cobaltinus
females. The perfumed females were then placed in isolated chambers with C.
cobaltinus males and the male’s copulatory behavior was observed for 30 minutes.
The cuticular hydrocarbons of Chrysochus cobaltinus are sex and species specific,
and influence the copulatory behavior of male C. cobaltinus. There is no evidence
of geographic variation in CHCs of C. cobaltinus females, but such geographic
variation does exist in C. auratus females. However, this study found no evidence
for reproductive character displacement in cuticular hydrocarbons in females of
either species. We suggest several avenues of research for determining the basis
of previous results indicating reproductive character displacement.
C. auratus only
C. cobaltinus only
Mixed populations
Source populations
Yak
ima
R
Moses Lake*
.
* Ellensburg
Introduction
displacement in cuticular hydrocarbons in females of either species.
rk
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sC
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Copulation Duration (min.)
Future Research
o For hydrocarbon removal, beetles were soaked in hexane for three
ct
o Sympatric CF extract: C.
Allopatric AF extract: C.
cobaltinus females (Yakima) with
extract from C. auratus females
(9.5km SSE Othello)
o Sympatric AF extract: C.
(17km NE Richland) with no
cobaltinus females (Yakima) with
hydrocarbon removal).
extract from C. auratus females
was provided by an REU Supplement to NSF grant DEB-0212652, awarded to M. Peterson.
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o Mating experiments were conducted within one week of collection. C.
cobaltinus males (Yakima) were paired with a female from one of the 11
treatments, and observed for 30 min. in isolated chambers (100x15mm
polystyrene petri dishes lined with filter paper). Copulation duration (total time
Figure 1. Chrysochus auratus (left) and Chrysochus cobaltinus (right) on
their host plant, Apocynum cannabinum. (Photos courtesy of Timm Beeman)
mounted with an everted aedeagus or in postcopulatory guarding (Fig. 3))
was noted for each male. Copulation durations were compared with ANOVA,
with LSD means comparisons tests.
reproductive character displacement.
research, conducted under project MORE (Minority Opportunities for Research on Evolution)
a
(25km SE Toppenish)
Mating Experiments
discrepancy between this study and previous results documenting
Kirsten Monsen and Dr. Merrill Peterson for all of their guidance and support. Funding for this
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Copulation Duration (min.)
o AF control: C. auratus females
a
tr
cobaltinus males (Yakima).
ex
hydrocarbon extract from C.
potential mates, to better understand the possible basis for the apparent
Chrysochus; Monique Brewer, Dennis Bohrer, and Dr. Jeff Young for technical advice; and Dr.
a
females (Ellensburg).
females (25km SE Toppenish)
known hydrocarbon profiles.
o Examine the effect of age on the species-specific responses of males to
and Steven Schwartz for assisting in the mating experiments; Timm Beeman for the photo of
extract from C. cobaltinus
extract from C. cobaltinus
the behavioral evidence from this study.
o Compare the responses of allopatric and sympatric males of each species to
We thank the following for their help during this research: Kati Buckingham, Maggie Brassil
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cobaltinus females (Yakima) with
removal.
females (Yakima) with
Cuticular Hydrocarbons are Species-Specific
o Allopatric CF extract: C.
cobaltinus females (Yakima) with
o Determine if hydrocarbon profiles exhibit geographic variation that matches
Acknowledgments
Richland).
(Yakima) with no hydrocarbon
o CM extract: C. cobaltinus
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o CM control: C. cobaltinus males
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hydrocarbons removed.
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females (Yakima), with
M
discriminating than allopatric males.
removed and reapplied.
o Stripped CF: C. cobaltinus
C
exhibit a similar result may also indicate that hybrid zone males are more
(Yakima) with hydrocarbons
C
distinguishable than allopatric females. The fact that allopatric males did not
o CF extract: C. cobaltinus females
ed
pp
et al. unpub. data). This result suggests that hybrid zone females are more
o AF extract: C. cobaltinus females
auratus females (17km NE
i
tr
female than if presented a choice of allopatric females of each species (Peterson
Treatments
hydrocarbon removal.
S
experiments to evaporate the hexane and natural volatiles.
(Yakima) with extract from C.
to be responsible for mate recognition in Chrysochus.
Figure 4. Sex-specificity of cuticular hydrocarbons as mating cues. C. cobaltinus
males had a significantly greater mean copulation duration (± SE) with conspecific
females (CF control) and conspecific females perfumed with extract from
conspecific females (CF extract) than with stripped conspecific females (stripped
CF), conspecific males (CM control), or conspecific females perfumed with extract
from conspecific males (CM extract). Different letters above bars indicate
statistically significant differences (p<0.05).
extract from a single beetle. The beetles were dried for 30 min. prior to mating
females (from Yakima) with no
ct
of potential ‘mates’ (as per the treatment) in an amount approximating the
o CF control: C. cobaltinus
a
tr
extract, which was then evaporated to a crust.
o For perfuming, the crust was reconstituted in hexane and applied to the elytra
ex
C. cobaltinus from the hybrid zone are more likely to mate with the conspecific
o Isolate and determine the chemical profiles of cuticular hydrocarbon thought
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consecutive 20 min. intervals. Vacuum filtration was used to concentrate the
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studies have shown that, if given a choice of females from the hybrid zone, male
t
females. However, this study found no evidence for reproductive character
o All beetles used as potential ‘mates’ for C. cobaltinus males were flash frozen
C
assortative mating (Peterson et al. unpub. data). Furthermore, mate choice
c
ra
C. cobaltinus females, but such geographic variation does exist in C. auratus
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the lab and the field. In both the lab and the field, the two species exhibit positive
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showing positive assortative mating.
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daily (Dickinson 1995), making their mating behaviors unusually easy to study in
AF
in male response, and may provide an explanation for previous results
o There is no evidence of geographic variation in the cuticular hydrocarbons of
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completely sterile or nearly so (Peterson et al. in review). These beetles mate
ct
tra
ex
o Cuticular hydrocarbons are also species-specific, as evidenced by differences
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a
Dobler & Farrell 1999). The adults of both species (Fig. 1) spend the majority of
75km-wide hybrid zone (Fig. 2), where hybrids are common but are either
AF
influence the copulatory behavior of male C. cobaltinus.
at -80o C for 2h, then thawed for 15 min.
a mating and egg-laying site. In central Washington State, the species form a
c
tri
pa
Conclusions
o The cuticular hydrocarbons of Chrysochus cobaltinus are sex specific, and
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their lives on the leaves of their host plants, which serve as their food in addition to
ct
b
Hydrocarbon Isolation and Application
found to feed on plants in the Asclepiadaceae (Williams 1991; Dickinson 1995;
ct
tra
ex
cuttings.
lo
Al
night (16:8h cycle) with a constant supply of fresh Apocynum cannabinum
tra
o Beetles were collected in the field, and kept in an incubator (22o C day, 16o C
ex
Collection and Maintenance
AF
Materials and Methods
reinforcing selection is operating in this hybrid zone. Our goal in the present study
feeds exclusively on plants in the Apocynaceae, while C. cobaltinus has also been
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which exist strong postzygotic barriers. Behavioral evidence for reproductive
F
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the chrysomelid beetles, Chrysochus auratus and Chrysochus cobaltinus, between
(Apocynaceae) and the closely related milkweeds (Asclepiadaceae). C. auratus
tri
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fitness. Recently, Peterson et al. (2001) discovered such a hybrid zone between
pa
ct
tra
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character displacement (Peterson et al. unpub. data) supports the hypothesis that
m
Sy
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Figure 6. Geographic variation in species-specific cuticular hydrocarbon mating
cues. C. cobaltinus males from an allopatric source population exhibited similar
responses (mean copulation duration ±SE) to conspecific females perfumed with
conspecific female extract from three populations (two allopatric (CF extract,
Allopatric CF extract), and one hybrid zone (Sympatric CF extract)). In contrast,
their responses to conspecific females perfumed with heterospecific female (AF)
extract depended on the source population used for the extract. Responses of
males to sympatric species-specific extracts were not more divergent than their
responses to allopatric species-specific extracts. Different letters above bars
indicate statistically significant differences (p<0.05).
Cuticular Hydrocarbons are Sex-Specific
Figure 2. Map of the Chrysochus hybrid zone in the Yakima River valley of southcentral Washington. This region contains the only known sympatric populations of
C. auratus and C. cobaltinus (Peterson et al. 2001). Evidence suggests that this
hybrid zone has been formed as a result of secondary contact (Dobler and Farrell
1999, Peterson et al. 2001). Populations sampled for this experiment (two
allopatric populations of each species and one sympatric population) are indicated
in red.
zones in which reinforcing selection (Dobzhansky 1940) results from low hybrid
c
Figure 3. The copulatory behavior of Chysochus cobaltinus males exhibited during
the experiment. Arrows indicate the everted aedeagi.
*
Richland
whether CHCs are under sexual selection or natural selection is often difficult to
selection may play an important role in the evolution of divergent CHCs is in hybrid
tri
Prosser
is under selection (Higgie et al. 2000, Blows 2002, Howard et al. 2003), though
Throughout their ranges, these beetles have host associations with dogbane
C
ct
*
50km
determine (Coyne 1992, Panhuis et al. 2001). One situation in which natural
pa
tra
*
hybridizing Drosophila indicates that CHCs may be altered when mate recognition
eastern North America and C. cobaltinus restricted to western North America.
c
tri
pa
Evidence of reproductive character displacement in male and female CHCs of
Chrysochus auratus and C. cobaltinus are sister species (Dobler & Farrell
d
ex
2000), it is important to understand the process by which these mating cues evolve.
1999) with largely allopatric distributions, with C. auratus primarily occupying
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F
C
Toppenish
of divergent CHCs may play an important role in insect speciation (Higgie et al.
Natural History of Chrysochus auratus and Chrysochus cobaltinus
c
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Yakima*
Blomquist et al. 1998, Akino et al. 2001, Ginzel et al. 2003). Because the evolution
displacement in CHCs.
bc
m
Sy
Othello
*
Columbia R.
mate recognition (Howard 1993, Nelson & Blomquist 1995, Stoffolano et al. 1997,
involved in mate choice and whether there is evidence of reproductive character
ab
ab
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Cuticular hydrocarbons (CHCs) are the predominant cuticular lipid in most
between these beetles, by assessing whether sex- and species-specific CHCs are
a
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0
insects, and are often used for intra- and interspecific communication, particularly
was to determine if CHCs may play a role in the evolution of premating barriers
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Figure 5. Species-specificity of cuticular hydrocarbons as mating cues. C.
cobaltinus males had a significantly greater mean copulation duration (± SE) with
conspecific females (CF control) than with heterospecific females (AF control).
Their response to perfumed conspecific females (CF extract, AF extract) depended
on the species from which the extract was obtained and matched their responses
to controls for the two species. Different letters above bars indicate statistically
significant differences (p<0.05).
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