Download Life history evolution in a bivoltine butterfly

Life history evolution in a bivoltine butterfly
Helena Larsdotter Mellström
Department of Zoology
Stockholm University
2012
Life history evolution in a bivoltine butterfly
Doctoral dissertation 2012
Helena Larsdotter Mellström
Department of Zoology
Stockholm University
106 91 Stockholm, Sweden
© Helena Larsdotter Mellström
ISBN 978-91-7447-592-0
Cover photo by Christer Wiklund
Printed by US-AB, Stockholm, Sweden 2012
Distributor: Department of Zoology, Stockholm University
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List of papers
I
Larsdotter Mellström H & Wiklund C. 2010. What affects mating rate? Polyandry is higher in the directly developing generation of the butterfly Pieris
napi. Animal Behaviour 80, 413-418.
II
Larsdotter Mellström H, Friberg M, Borg-Karlson AK, Murtazina R, Palm
M & Wiklund C. 2010. Seasonal polyphenism in life history traits: Time costs
of direct development in a butterfly. Behavioral Ecology and Sociobiology 64,
1377–1383.
III
Larsdotter Mellström H, Murtazina R, Borg-Karlson AK & Wiklund C.
2012. Timing of male sex pheromone biosynthesis in a butterfly – Different
dynamics under direct or diapause development. Journal of Chemical Ecology
38, 584–591.
IV
Larsdotter Mellström H & Wiklund C. 2009. Males use sex pheromone
assessment to tailor ejaculates to risk of sperm competition in a butterfly.
Behavioral Ecology 20, 1147-1151.
V
Larsdotter Mellström H & Wiklund C. Different mating expenditure in
response to sperm competition risk between generations in the bivoltine
butterfly Pieris napi. Under review, Oecologia
Paper I © The Association for the Study of Animal Behaviour
Paper II and III © Springer
Abstract
Evolution is not always straight-forward, as selection pressures may differ between different
generations of the same species. This thesis focuses on the evolution of life history of the model
species, the Green-veined White butterfly Pieris napi. In central Sweden P. napi has two generations
per year. The directly developing summer generation is short-lived and time stressed, compared to the
diapausing generation.
In paper I polyandry, defined as female mating rate, was shown to differ between generations but was
unaffected by environmental factors. In paper II both males and females of the direct developing
generation were shown to eclose more immature than the diapausing generation, indicating larval time
constraints. Consistent with this, diapausing males mated sooner than direct developers. Directly
developing females, however, mated sooner after eclosion than diapausing females, even though they
are more immature. This was shown to negatively affect fecundity, but can pay off when the season is
short.
Paper III shows that directly developing males have less sex pheromones at eclosion than diapausers,
and the differences in sex pheromone production is consistent with developmental time constraints and
the differences in mating system.
In P. napi and other polyandrous butterflies, males transfer a large, nutritious ejaculate at mating.
Large ejaculates confer advantages under sperm competition, but as they are costly, males should
adjust ejaculate size to the risk of sperm competition. In paper IV we found that males transfer on
average 20% larger spermatophores under high male competition than at low competition. The same
effect could be observed if we added male sex pheromone to the air in a mating cage without malemale competition. Paper V shows that males of the two generations respond differently to an increase
in male-male competition, with diapausing males transferring larger spermatophores than direct
developers at high male competition risk.
Key words
Bivoltine, Diapause, Lepidoptera, Life history, Mating system, Pheromone,
Polyphenism, Population density, Sexual selection, Sperm competition
Introduction
“ Nothing in Biology Makes Sense Except in the Light of Evolution ”
These eloquent words by Dobzhansky (1973) provide an excellent backdrop for this thesis. Ecology
provides the stage where evolution takes place and integrating the two is essential for a deeper
understanding of the interactions we observe around us today.
When we think about evolution what usually comes to mind is natural selection. It is even in the title
of Charles Darwin’s seminal book “On the Origin of Species by Means of Natural Selection” (1859).
By this beautiful and skilful integration of several pre-existing ideas and his own observations Darwin
suddenly made previously unexplained patterns come together into one unifying theory that still, 150
years later, holds true. Any trait that was heritable and conferred an advantage in the struggle for
existence and survival could now be explained by natural selection.
More difficult for Darwin were highly evolved and complicated features that conveyed apparently no
adaptive advantage to the organism. In his famous words from his correspondence (Darwin, 1860);
“The sight of a feather in a peacock’s tail, whenever I gaze at it, makes me sick! ”
To reconcile the theory and these apparently maladaptive traits Darwin needed to add yet another
mechanism for evolution. Sexual selection is the evolutionary process proposed by Darwin to explain
traits whose primary function appears to be ensuring an individual’s success in courtship and mating.
Or, in the words of Darwin himself in his book The Descent of Man, and Selection in Relation to Sex
(1871);
“... [sexual selection] depends, not on a struggle for existence, but on a struggle between the
males for possession of the females; the result is not death to the unsuccessful competitor, but few
or no offspring”
Different characteristics could thus be selected for if they conveyed a reproductive advantage to the
individual (Darwin, 1871). Sexual selection is therefore believed to have a strong effect on the
evolution of animal mating systems (see Wiklund, 2003) and will be an integral part of this thesis.
For studies of mating systems, insects in general and butterflies in particular are often used model
organisms. Insects display a diversity of reproductive strategies unparalleled among animals, which
has put research in this field at the forefront of the study of animal mating systems (Brown et al.
1997). Butterflies offer great model systems for evolutionary studies as they are small, fecund animals
with a reasonably short life span. They are also, at least my model species Pieris napi (the Greenveined White), easy to rear in the laboratory. This enables us to perform both artificial selection, life
time fitness measurements and behavioural studies. Using a previously well studied model species like
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P. napi also comes with the advantage of having very good background information on the species,
regarding everything from host plant use (Forsberg 1987) and courtship behaviour (Forsberg &
Wiklund 1989) to polyphenism (eg. Wiklund et al. 1991) and chemical signaling (Andersson et al.
2007), which is not very common (see Nieberding et al. 2008).
Life history evolution
Life history theory sees the scheduling of events such as growth, sexual maturation, and reproduction
as the result of strategic decisions over an organism’s life (Stearns 1992). Organisms have limited
time, energy and nutrients at their disposal. Investing time or energy into mating, for example,
decreases time and energy available for foraging and these trade-offs in allocation form the life
histories of a species. When to mature, how many eggs to lay or how much to invest in each mating
are all classical examples of life history traits and the allocation pattern that produces as many
successful offspring as possible will be selected for during evolution. Life histories thus balance tradeoffs between current and future reproduction, representing the best solution of conflicting demands on
the organism.
However, in an organism with several life cycles per year selection pressures may differ between
different generations of the same species. Multivoltine insects appear in two or more discrete
generations per year. In temperate regions the different generations will most likely experience
varying selection regimes, depending on the season. Many multivoltine butterfly species do indeed
show seasonal polyphenisms not only in appearance (e.g. wing colouration) but also in life history
traits, such as adult weight and larval development time (Nylin et al. 1989), fecundity and dispersal
(Karlsson & Johansson 2008) and female mating propensity (Friberg & Wiklund 2007). These
seasonal polyphenisms might either be the result of adaptive plasticity triggered by reliable
generation-specific environmental cues, or passively induced phenotypes caused by generationspecific environmental constraints. Therefore, the particular life history traits that are targeted by
selection or affected by developmental constraints are likely to differ between species, and relate to the
species-specific life cycle. An interesting example of this is that female Leptidea reali (sensu lato)
butterflies (Lepidoptera: Pieridae) of the time constrained directly developing summer generation
accept mating faster than the less time constrained spring generation females that have spent the winter
in pupal diapause (Friberg & Wiklund 2007). An explanation for this behavioural difference could be
that theoretical models predict a context-dependent female mate choice behaviour and relaxed
selection on female choosiness when females are time constrained (Johnstone 1997). The generality of
these life history effects, the mechanisms behind them and their effects are examined in paper I and
II.
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In butterflies, as a general rule, incoming resources are used in preference to stored reserves (Boggs
2003). Butterfly life history is also constrained by the shift in diet between life stages. Essential amino
acids are, in most Lepidoptera, derived from larval feeding whereas sugars predominate in adult nectar
diet (Erhardt & Rusterholz 1998). This has several important effects on life history evolution. Leimar
et al. (1994) have shown that if the larval resource environment becomes more variable, male-derived
nutrients become more important for the female to ensure reproduction and thus affects mating
system. Allocation patterns (see figure 1) may also be differentially affected if larval resources are
decreased; all life history traits could be affected to the same degree or allocation of larval nutrients
could be unevenly distributed to support some important traits whereas others receive a
disproportionately small part of the resources (Boggs 2003). If reserves, measured as initial adult body
mass, are large the butterfly is able to maintain fecundity during adult food deprivation. If reserves are
small, it cannot. This shows the importance of larval feeding on adult life history patterns (Boggs
2003). In paper III we study the effect of larval and adult derived nutrients on sex pheromone
production and relate it to developmental pathway and mating system.
Figure 1. The allocation of nutrients to different life history traits.
Polyphenism
Biotic and abiotic factors vary over space and time. These variations affect an organism’s allocation of
time and nutrients to reproduction, survival, growth, storage and foraging. Allocation patterns hereby
link environmental variation with life histories and population dynamics. Allocation studies are being
carried out in a wide variety of animals; butterflies, orthopterans, lizards and plants among others
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(Boggs 2003). As mentioned above, for multivoltine insects in temperate regions the different
generations will most likely experience varying selection regimes, depending on the season. In a
seasonally changing environment active periods, such as development and reproduction, must occur
during the favourable time of year and diapause during unfavourable periods. As a consequence of this
the production of more than one generation per year involves a choice between alternative
developmental pathways, direct development or diapause. If a genotype produces individuals who are
able to complete additional generations within a given period of time, it will have a higher intrinsic
rate of increase.
In most organisms a single genotype can produce many different phenotypes and which phenotype is
expressed is dependent on the environment in which the organism develops and phenotypic plasticity
is believed to be the primitive character state for most or all traits (Nijhout 2003). Evolution of
plasticity can then move in one of two directions; stabilization of the phenotype and loss of plasticity
or exploitation of the plasticity. During evolution plasticity has been advantageous as a mechanism
that enables an organism to develop different phenotypes that can be adapted to and optimized for two
or more environments (Nijhout 2003). In insect polyphenisms the environmental variable that induces
one of the phenotypes is a stimulus that only serves as a predictor of, but is not itself, the environment
to which the polyphenism is an adaptation. The environmental stimulus changes the endocrine
mechanism of metamorphosis and the altered pattern of endocrine interactions results in the execution
of the alternative developmental pathway (Nijhout 2003).
Polyphenisms are adaptations to predictable variations in the environment. However, the inducing
environment is not the same as the environment to which the phenotype is adapted. Seasonal
polyphenism, for example, may be an adaptation to cold or food shortage but is typically induced
mainly by a change in photoperiod (Wiklund et al. 1992). The change in photoperiod is not in itself an
unfavourable environment but it is a good predictor of seasonal change. In adult polyphenisms, the
critical period typically occurs during the larval stages (Nijhout 2003).
To fully understand how these polyphenisms evolve we need to separate the effects of the
environment the two generations live in and the effect of the induced phenotype in itself. For example,
food availability may change between generations, giving different patterns of effect on life history
and allocation and hence, polyphenism. But the induced phenotypes may also have inherently different
allocation patterns. This is a common theme for all the articles in this thesis.
Mating system
Mating systems represent an array of adaptations selected to maximize the reproductive rate of
individuals of each sex (Brown et al. 1997). Life history theory tries to explain how natural selection
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has shaped traits to produce life cycles, including mating patterns, that fit the demands of the
environment. Polyphenism, physiological processes and sexual conflict are three areas that all have
direct or indirect effects on animal mating systems. The evolution of mating systems depends on many
variables, including resource and mate distribution, the presence and extent of transferable genetic and
material benefits and the degree of control exerted by each sex over events at different stages in the
mating sequence (Brown et al. 1997, see figure 2).
Figure 2. The main factors influencing animal mating systems.
(Adapted from Brown et al. 1997).
The common view was, for a long time, that male fitness increased through copulating with multiple
partners, whereas female fitness only increased with the quality of, and care for, offspring. However,
several studies have now shown that females receive direct fitness benefits, i.e. increased offspring
production, from mating polyandrously (e.g. Wiklund et al. 1993, Kaitala & Wiklund 1994, Karlsson
1998).
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Whether an individual maintains monandry or polyandry could be dependent on a number of factors.
Sexual selection affects both male and female life history traits. Sexual conflict also has an effect on
the optimal mating frequency and different generations may experience different selection regimes,
leading to a seasonal polyphenism in mating system. Välimäki et al. (2006) have shown that
monandrous females perform better than polyandrous during unpredictable conditions and when
pressed for time. Consistent with these findings, Välimäki et al. (2008) have found that females with a
low mating frequency are more likely to produce an additional summer generation in seasonal
environments than the females with a high degree of polyandry. This contributes to explaining the low
mating frequencies in P. napi and shows that life history, polyphenism and mating system are closely
intertwined. Comparing life histories of females with varying degree of polyandry Välimäki & Kaitala
(2007) concluded that polyandrous females developed at a faster rate that monandrous females, in both
direct and diapausing cohorts.
If females mate more than once during a reproductive period, it has profound effects on females’ role
in sexual selection. It could generate sperm competition (Parker 1970) which in turn could lead to
several evolutionary responses in males, females and features of the ejaculate. It could, for example,
create the possibility for females to, via cryptic female choice, choose which male’s sperm will
fertilize the eggs (Eberhard 1996). Polyandry, and mixed paternity in offspring, have been recorded in
organisms as different as snails, honeybees, mites, spiders, fish, frogs, lizards, snakes, birds and
mammals (Birkhead & Møller 1998). In polyandrous species the last male to mate with a female
generally enjoys higher fertilization success than previous males (Wiklund 2003). Male size has also
been shown to be associated with success in sperm competition because large males transfer more
sperm (Wiklund 2003).
Sexual selection
Natural selection favours traits that increase survival, but an individual’s fitness is also dependent on
how many offspring that are produced. Sexual selection theory therefore predicts, as mentioned
earlier, that a trait that enhances reproductive success will be favoured and spread in the population.
If a female mates more than once, as in P. napi, this confers that sperm from different males will
compete for fertilizations (Parker 1990a, b, 1998, Parker et al. 1997, Parker & Ball 2005). Sperm
competition has been recognized as a particularly powerful force that can lead to adaptations in male
behaviour, morphology and physiology that contribute to competitive fertilization success (e.g. Parker
1970, Birkhead & Møller 1998). Game theory has been used to develop predictions about how male
ejaculation strategies should be influenced by variation in sperm competition risk (Parker 1990a, b,
1998, Parker et al. 1997, Parker & Ball 2005). Sperm competition game theory assumes that (1)
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ejaculates are costly - meaning that expenditure on any fertilization must be traded against that of
gaining future fertilizations, and (2) that fertilization success is proportional to the number of sperm
transferred to females at mating (Parker 1998) - like the tickets in a lottery. The models predict that
increased risk of sperm competition should favour the evolution of increased expenditure on
ejaculates, and there is now evidence suggesting such an effect both between and within species (Gage
& Baker 1991, Gage 1991, 1994, Harcourt et al. 1995, Hosken 1997, Stockley et al. 1997, Byrne et al.
2002, Pitcher et al. 2005, Simmons et al. 2007). Although this phenomenon is widespread in insects
and other taxa, the mechanisms that allow males to assess sperm competition has only recently been
investigated with the results that pheromones are important for assessing sperm competition risk in the
beetle Tenebrio molitor (Carazo et al. 2007), the fruit fly Drosophila melanogaster (Friberg 2006), the
cricket Teleogryllus oceanicus (Thomas & Simmons 2009) and the meadow vole Microtus
pennsylvanicus (delBarco-Trillo & Ferkin 2004, 2007).
In P. napi males, as in most butterflies, cannot enforce copulations on females, and therefore have
two, mutually non-exclusive, options to increase fertilization success: to manipulate the female into
delaying her next mating or to increase the number of sperm transferred to the female. Both of these
options are mediated by a large spermatophore. In contrast, under low risk of sperm competition males
should benefit from saving resources which can be allocated to the next mating. How males from both
developmental pathways allocate their resources under male-male competition for matings, and if they
use the amount of sex pheromone as a cue for mating investment is examined in paper IV and V.
The male donation of nutrients must be regarded both as paternal investment, increasing the number
and quality of the male’s own offspring up to the time the female remates, and as mating effort,
delaying female remating (Wiklund & Kaitala 1995). This kind of mating system leads to a
fundamental asymmetry between males and females. The male’s ability to invest in reproduction is
dependent on the resources he can accumulate as a larva, capital investment, whereas females can
obtain resources for reproductive investment both as larva and from nuptial gifts, a combination of
capital and income investment.
Sexual conflict
Conflict arises whenever the outcome of an interaction yields differing optima for different individuals
or groups/classes of individuals (Brown et al. 1997). Each individual will then be selected to
manipulate the interaction in ways that bring the outcome closer to its optimum. Conflict of interest
between the sexes is expected at each stage between pair formation until the end of parental care, and
in all mating systems (Brown et al. 1997) and sexual conflict is a potent force in male-female
coevolution (Chapman et al. 2003).
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Sexual selection can be divided into two classes; in indirect models the female preference evolves as it
becomes genetically associated with genes that confer either sexy sons and/or high viability offspring.
In direct models the female preference itself is under natural selection. Females could for example
gain resources or greater parental care. The models of sexually antagonistic coevolution also fall into
this class. There are two main mechanisms for sexual conflict (Chapman et al. 2003). The intralocus
conflict where the fitness optimum for a trait expressed in both sexes is different for males and
females. The interlocus conflict occurs when there is conflict over the outcome of male-female
interactions – the optimal outcome is different for the two sexes. Examples of this are conflicts over
mating frequency, parental effort and so on. Both sexes are then expected to evolve suites of sexually
antagonistic adaptations that bias the outcome towards their own respective interests. The result is
sexually antagonistic coevolution between interacting traits in males and females. Because of sexual
conflict, the selection pressures on males and females are different and represent an important factor in
shaping the evolution of mating systems and sexual dimorphism (Rutowski 1997).
Even though females receive direct benefits by mating multiply, only two out of twenty-three
investigated butterfly species, showed an average lifetime number of matings exceeding three in the
wild (Wiklund & Forsberg 1991). So far we have seen that polyandrous females obtain direct benefits
from mating multiply. So why then aren’t female butterflies in all species polyandrous?
The reason why females do not mate more could be that there is limited space for spermatophores in
the female bursa and that they take time to break down (Oberhauser 1992). This fact indicates a
conflict between the sexes in that males would profit from a slow rate of degradation which delays
female remating, whereas females would benefit from fast degradation to make possible the reception
of a new nutritious spermatophore (Boggs 1981).
In P. napi, females obtain direct fitness benefits from mating multiply. Multiply mated females of
naturally polyandrous P. napi have been shown to lay larger eggs, live longer and have larger life time
fecundity then singly mated females (Wiklund et al. 1993) since males transfer a large nutritious gift
to the females that the females use to increase both their fecundity and lifespan (Arnqvist & Nilsson
2000). Despite this positive relationship between fitness and number of matings, there is a large
variation in female mating frequency and 12% of old females are monandrous (Bergström et al. 2002).
Monandry in females is not coupled to lack of mates (Bergström et al. 2002, Bergström & Wiklund
2005, Välimäki & Kaitala 2006). It could instead be discussed in terms of sexually antagonistic
coevolution and whether environmental conditions influence optimal mating frequency (Bergström et
al. 2002). The reason for the low mating frequency could either be that males have evolved the ability
to manipulate females to mate at a suboptimal rate as a measure of protection against sperm
competition, or as suggested by Bergström & Wiklund (2005), that female mating is suppressed by
some costs.
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Studies have shown that there is genetic variation in female mating rate (Wedell et al. 2002, Välimäki
et al. 2006) and that female mating rate and fecundity are under genetic control and show high
heritability. Even though polyandrous female P. napi have higher lifetime fecundity than monandrous
females, some females simply abstain from remating irrespective of the number of times they have
been courted. Explaining this genetic variation in mating rate requires that monandrous females
perform better than polyandrous ones under some conditions.
Using high and low intrinsic mating rate females Välimäki et al. (2006) showed that during the first
days of reproduction, females with a low mating rate produced more eggs than females with a high
mating rate. This could lead to an evolutionary advantage for the monandrous females if the time at
hand for reproduction is limited. The study also shows that unpredictable weather favours monandry,
as hypothesized in Bergström et al. (2002). Since mating takes several hours (Bergström et al. 2002),
there is an apparent short-term conflict between mating and egg-laying. Under extended poor weather
conditions when there are few opportunities to lay eggs, optimal mating frequency may be lower than
in good weather. So the explanation for the apparent suboptimal mating rate in females could be that
there are costs associated with mating in the wild that have been underestimated and that optimal
mating frequency covaries with environmental factors, in which case females do not in fact mate
below the optimal frequency. A combination of life-history cost and unpredictability of fitness may
explain the maintenance of monandry in the wild and the increasing frequency of monandry with
latitude.
Study species
The butterflies used in these studies were the offspring of wild-caught Pieris napi (Green-veined
white; Lepidoptera: Pieridae) from the Stockholm area. In central Sweden, P. napi has two generations
per year, both of which are generally widespread and common. Larvae feed on a variety of crucifers,
including several cultivated plants e.g. cabbages and rapeseed. The diapausing generation ecloses in
spring after having spent winter in the pupal stage. The offspring of this generation undergoes direct
development, eclosing during summer, and their offspring in turn pupate and diapause during winter.
There are potentially very different selection regimes acting on the two generations. Individuals from
the diapausing generation have plenty of time to develop physiologically in the pupal stage during
winter and are not time constrained in the adult stage as they eclose in spring. In central Sweden the
flight period of the direct developing generation, on the other hand, is approximately two months later
than that of the diapausing generation, yielding a correspondingly higher time stress in the adult stage,
as the offspring must reach pupation before the onset of winter (Abrams et al. 1996, Gotthard et al.
1999) and the direct development also imposes shorter time in the pupal stage.
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At mating male P. napi transfer an ejaculate that, on average, corresponds to 15 % of male body mass
(Svärd & Wiklund 1989). The ejaculate contains both fertilizing and non-fertilizing sperm as well as
significant amounts of nutrients and female fitness increases with number of matings (Wiklund et al.
1993, Karlsson 1998). In the wild females mate between 1 and 5 times during their lifetime, with an
average of 2.7 times (Bergström et al. 2002). P. napi females usually mate on an interval of 3-5 days
(Wiklund et al., 1993), the duration of the refractory period being positively correlated with ejaculate
mass. As in most butterflies, females control mating (Bergström & Wiklund 2005) i.e. males cannot
mate without a female accepting him. Male P. napi emit a sex- and species specific pheromone, citral,
which makes females accept mating with a courting male and both males and females have receptors
that are sensitive to the male sex pheromone (Andersson et al. 2007). Citral is a citrus smelling 1:1
combination of the volatile isomers geranial and neral, with the molecular formula C10 H16 O. The
adult scent composition consists to around 95% of citral.
Methods
By manipulating temperature and light conditions we can obtain both generations in the laboratory
during the same period and could thereby exclude effects of phenotypic plasticity induced during the
adult stage. Experiments were carried out at the Department of Zoology, Stockholm University, using
mating cages measuring approximately 0.8x0.8x0.5m. Cages were housed in a room where the
daylight regime could be controlled. All pheromone measurements where performed at KTH - Royal
Institute of Technology, Stockholm, in collaboration with Prof. Borg-Karlson and her ecological
chemistry group. With the P. napi butterfly system we can collect the sex pheromone either by Solid
Phase Micro Extraction (SPME) of live emissions of volatiles or by extracting the volatiles from the
wings of euthanized males and then identify the components by gas chromatography. For more
detailed studies on biosynthesis, stable carbon isotopes were used. By this procedure, we can describe
citral production by studying time allowed for feeding on labeled glucose together with the appearance
of labeled pheromone components. This then can be compared with known geranial and neral
biosynthetic pathways in plants (Iijima et al. 2006, Schilmiller et al. 2009) to investigate whether P.
napi uses similar or different biosynthetic pathways.
Objectives
Paper I Whether an individual maintains monandry or polyandry could be dependent on a number of
factors. Sexual selection affects both male and female life history traits. Sexual conflict also has an
effect on the optimal mating frequency and different generations may experience different selection
regimes, leading to a seasonal polyphenism in mating system. Polyandrous females have higher
10
lifetime fecundity compared to monandrous females (Wiklund et al. 1993). Nevertheless, 12% remain
monandrous throughout their life (Bergström et al. 2002).
We investigated which factors affect polyandry in a bivoltine central Swedish population of P. napi.
Our objective was to examine: (1) the effect of environmental factors on the level of polyandry; and
(2) the effect of developmental pathway on the level of polyandry. We did this by staging experiments
in which larval host plant, adult activity level, nectar and mate availability, and daylength were
manipulated, and by assessing lifetime number of matings of diapause and directly developing
generation females, controlling for possible variation between families in the level of polyandry.
Paper II The aim of this study was to test whether mating propensity differs between generations, i.e.
do males and females of the two generations differ in time from eclosion to first mating? Along with
this we also tested several physiological parameters to establish whether males and females of the time
stressed direct developing generation eclose as adults less mature than the diapausing generation and if
this correlates to mating propensity.
To determine if there are any differences in maturity at eclosion between the generations we first
looked at female daily and lifetime fecundity. Females were allowed to mate either within the first 12
hours after eclosion, or after 13-84 hours after eclosion. Eggs were then counted daily for the duration
of their life. For males we assessed male sexual maturity by their emission of sex pheromone.
Paper III The time constrained directly developing generation males take, on average, more than 6 hr
longer to mate for the first time after eclosion than do diapausing males (23 vs. 29,5 hours) (Paper II).
To study the differences in physiology between generations that experience different levels of time
stress and explore the dynamics in male sex pheromone production we first set out to assess
pheromone biosynthesis in P. napi, and to compare sexual maturity at the time of adult eclosion,
between the two generations. We hypothesized that the directly-developing males would take longer to
reach adult scent composition than diapausing males. Second, we also more specifically studied
whether the sex pheromone components are biosynthesized from larval and/or adult-derived material
by using stable carbon isotopes.
Paper IV In this study we investigate the reproductive behavior of male P. napi under different
population densities to test if males 1) tailor their ejaculate expenditure to the degree of competition
and 2) use the male sex pheromone citral as a cue when assessing the risk of sperm competition,
predicting that males mating in high male competition treatments or citral treatment will transfer larger
ejaculates.
Virgin butterflies (from the direct developing generation), 1-3 days old, were put in mating cages to
test the impact of population density on male ejaculate expenditure, with the number of males (M) to
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females (F) in each cage as follows, 1M:2F, 2M:2F, 10M:20F, 20M:20F, 1M:30F or 30M:1F. For the
pheromone experiments large plastic jars were used as mating cages. In each of these a pair of a virgin
male and a virgin female was allowed to mate. Citral solution was added to a filter paper at the bottom
of the jar. After mating the ejaculate was dissected out and weighed.
Paper V In this study we investigate the relative mating investment in response to sperm competition
risk, by male P. napi from the two different generations. To test whether developmental time
constraints affect male ejaculate expenditure, we staged mating experiments in the two generations,
with equal sex ratio and in which the number of partaking males varied from 1 to 25. The ejaculates
were then dissected out and weighed as in paper IV.
Conclusions
Paper I Results showed that (1) females did not compensate for bad environmental conditions (such
as larval host plant or nectar availability) by mating more often to procure nutrients; and (2) the level
of polyandry was higher in the directly developing generation than in the diapausing generation. The
results also indicate that there is a correlation between the time to first mating and subsequent lifetime
number of matings. This could be an indication that the degree of polyandry is contingent on mating
propensity and that whether an individual is monandrous or polyandrous might not be a true
dichotomy but rather depend on a continuum of mating propensities.
Paper II From our results we conclude that neither males nor females seem to be physiologically
mature when they eclose from direct development, probably due to larval time constraints. Directly
developing males take longer to synthesise the sex pheromone and longer to mate for the first time.
When comparing females mated before 12h of age, the direct developing generation has significantly
lower fecundity than diapausing females. There was however no difference in fecundity between the
generations when comparing all age classes. This is explained by the older females of the direct
generation laying significantly more eggs than the young during the first day of egg laying. So, the
females of the direct developing generation do not have lower fecundity per se but cannot realise their
full fecundity if they are mated when they are less than 12 hours old.
Interestingly though, directly developing females mate sooner after eclosion than diapausing females,
even though this reduces their fecundity. The results may seem contradictory, as these females were
shown to be less mature than the diapausing females and loose in fecundity by mating early. However,
laying fewer eggs but laying them early will pay of if the season is short and adult time stress
substantial, and we contend that this can explain the result. The results are also in agreement with
results by Friberg & Wiklund (2007) and consistent with the theory that females should reduce
choosiness when time stressed (Johnstone 1997). Females can get mating out of the way as quickly as
12
possible, even if they are not fully reproductively mature. For males, on the other hand, mating is
tightly linked to physiological maturation. Females seem to only accept mating if the male emits the
species-specific sex pheromone citral (Andersson et al. 2007) and the males are therefore constrained
by their physiological development.
Paper III The two generations are shown to have significantly different scent composition early in
life. The directly developing males, who have shorter time for pupal development, need the first 24
hours after eclosion to synthesise the sex pheromone whereas the diapausing generation males eclose
with adult scent composition. We could also show that the sex pheromone components can be
synthesised from both larval and adult derived nutrients. In summary, paper III shows that time-stress
changes the timing of biosynthesis of the sex pheromone components between generations and
underpins the importance of understanding resource allocation and physiology and their effect on life
histories.
Paper IV The results show that male P. napi tailored their reproductive investment in response to the
risk of sperm competition; ejaculates transferred by males in the high male density treatments were on
average 23 % larger than ejaculates transferred at low male densities. The results also show that citral
is the cue used by males to determine how much they will invest in the ejaculate; ejaculates transferred
by males in presence of added citral were 19 % larger than ejaculates transferred in absence of added
citral.
This shows that male P. napi have not only evolved increasingly larger spermatophores in response to
sexual selection, but also an ability to accurately determine the level of intra-sexual competition and
adjust reproductive expenditure accordingly. The results also show that the sex pheromone has
assumed dual functions – (1) facilitating female acceptance when dispensed by courting males and (2)
allowing males to assess the degree of male competition for matings.
Paper V The results show that male P. napi, as in paper IV and in accordance with sperm competition
theory, tailor their reproductive investment to the risk of sperm competition. Ejaculates transferred by
males under high sperm competition risk were larger than those transferred by males without
competition. Moreover, there was a significant difference between generations in response to malemale competition; in the absence of competition males invest equally, but under high sperm
competition risk directly developing males increase spermatophore size by 42% and diapausing males
by 67%. In conclusion, males from the two developmental pathways differ in mating investment in
response to sperm competition risk and so may influence life history evolution and potentially also
geographic differences in mating patterns.
13
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Acknowledgements
First and foremost, I am indebted to my supervisor Christer Wiklund, who has co-authored all of the
papers in this thesis and been an inspiration throughout my PhD. All his hard work in the lab,
expertise in writing and great support in all aspects of my PhD studies have been invaluable.
Thanks also to my co-supervisor Bengt Karlsson, who has always taken the time to help with
statistical issues, manuscript input or teaching questions. I have been very lucky to have two such
competent, kind and devoted supervisors.
I am also grateful to my co-authors who have contributed to the articles in this thesis.
Thanks also to all of the people at Zootis who contribute to making it a true privilege to come to work
every day.
And last but not least - thanks to my friends who have supported me both in- and outside academia,
and a big hug to my family; Niklas and Lin.
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