Oecologia
DOI 10.1007/s00442-008-1206-8
PLANT-ANIMAL INTERACTIONS - ORIGINAL PAPER
Host plant preference and performance of the sibling species
of butterflies Leptidea sinapis and Leptidea reali: a test
of the trade-off hypothesis for food specialisation
Magne Friberg Æ Christer Wiklund
Received: 3 June 2008 / Accepted: 7 October 2008
Ó Springer-Verlag 2008
Abstract A large proportion of phytophagous insect
species are specialised on one or a few host plants, and
female host plant preference is predicted to be tightly linked
to high larval survival and performance on the preferred
plant(s). Specialisation is likely favoured by selection under
stable circumstances, since different host plant species are
likely to differ in suitability—a pattern usually explained
by the ‘‘trade-off hypothesis’’, which posits that increased
performance on a given plant comes at a cost of decreased
performance on other plants. Host plant specialisation is
also ascribed an important role in host shift speciation,
where different incipient species specialise on different host
plants. Hence, it is important to determine the role of host
plants when studying species divergence and niche partitioning between closely related species, such as the butterfly
species pair Leptidea sinapis and Leptidea reali. In Sweden,
Leptidea sinapis is a habitat generalist, appearing in both
forests and meadows, whereas Leptidea reali is specialised
on meadows. Here, we study the female preference and
larval survival and performance in terms of growth rate,
pupal weight and development time on the seven mostutilised host plants. Both species showed similar host plant
rank orders, and larvae survived and performed equally well
on most plants with the exceptions of two rarely utilised
forest plants. We therefore conclude that differences in
Communicated by Konrad Fiedler.
Electronic supplementary material The online version of this
article (doi:10.1007/s00442-008-1206-8) contains supplementary
material, which is available to authorized users.
M. Friberg (&) C. Wiklund
Department of Zoology, Stockholm University,
106 91 Stockholm, Sweden
e-mail: [email protected]
preference or performance on plants from the two habitats
do not drive, or maintain, niche separation, and we argue
that the results of this study do not support the trade-off
hypothesis for host plant specialisation, since the host plant
generalist Leptidea sinapis survived and performed as well
on the most preferred meadow host plant Lathyrus pratensis
as did Leptidea reali although the generalist species also
includes other plants in its host range.
Keywords Diapause/direct development Generalism/specialism Habitat Host plant shifts Species divergence
Introduction
A large proportion of the herbivorous insects are specialised
on only one or a few related hosts (Bernays and Graham
1988). It has proved very difficult to understand what factors favour a generalist (Futuyma 1979; Brooks and
McLennan 2002; C. Wiklund and M. Friberg, manuscript
submitted), whereas more studies have dealt with how a
host plant generalist species is selected to specialise on one
or a few hosts (Bernays and Graham 1988; Thompson 1988;
Feder et al. 1995; Feder and Forbes 2007; Wiklund and
Friberg 2008). Although host plant specialisation is a wellstudied and reasonably well-understood phenomenon
(Bernays and Graham 1988; Futuyma and Moreno 1988;
Thompson 1988), only a few studies have confirmed the
classic trade-off explanation for food specialisation (sensu
Fry 1996, see also Futuyma and Moreno 1988; Agrawal
2000; Futuyma 2008), which predicts that phytophagous
insects are forced to specialise, since any new mutation that
increases fitness on the primary host plant, but lowers fitness on a secondary host plant, will be favoured by
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selection. Such a mutation could, for example, be the ability
to overcome a particular plant defence, and the tendency for
egg-laying females to oviposit on the most preferred plant
would then be reinforced to an even higher extent. However, larvae often survive as well and grow as fast and as
large on other plants compared with larvae reared on the
host plant preferred by females (Wiklund 1975; Thompson
1988; Thompson and Pellmyr 1991), which suggests that
the underlying cause of host plant specialisation is not
necessarily linked to the host plants per se, but rather to
inter- and intra-specific food competition (Feder et al. 1995;
Feder and Forbes 2007, but see Lawton and Strong 1981 for
discussion about generality) or predation from host plant- or
habitat-specific predators (Bernays and Graham 1988; Wiklund and Friberg 2008).
Host plant specialisation has sometimes been shown to
lead to the formation of host plant races, where different
fractions of a population specialise into different host plant
niches, which might result in increasing reproductive isolation and eventually speciation (Ehrlich and Raven 1964;
Feder et al. 1988; Bush and Butlin 2004; Braby and Truman
2006; Janz et al. 2006). Strong assortative mating is required
for host plant races to diverge and maintain reproductive
isolation (Feder et al. 1994; Bush and Butlin 2004), and
selection for local adaptations on different host plants might
then reinforce the divergence and disfavour hybrids
(Thompson 1988; Via 1999; Forister 2004). Therefore,
when trying to reconstruct the evolutionary history of closely related, phytophagous insects, it is important to
investigate differences in both female host plant preference
and larval performance in order to determine the role of host
plant specialisation, i.e. whether it has acted as a driving
force of species divergence, or if it has developed as a secondary consequence of post-speciation processes of niche
separation and ecological character displacement.
A few decades ago, based on studies of genital preparations, Reál discovered that the wood white butterfly
(Leptidea sinapis) consisted of two species: Leptidea sinapis and Leptidea reali (Reál’s wood white) (Réal 1988;
Reissinger 1989). This rather late discovery of a Eurasian
butterfly is easily explained, since the two sister species
(Martin et al. 2003) are virtually identical in terms of
behaviour and wing colouration (Friberg et al. 2008a), and
they can at this point be distinguished only by studies of
their genitalia (Lorkovic0 1993; Mazel 2005; Schmitz
2007), by DNA-sequencing (Martin et al. 2003; Verovnik
and Glogovčan 2007; Friberg et al. 2008a, b) or by examination of small differences in coloration of diapausing
pupae (Friberg 2007). The two species are reproductively
isolated due to female choice of conspecific males (Friberg
et al. 2008a), although evidence suggests that the two species have hybridised, at least in the recent past (Verovnik
and Glogovčan 2007). The female ability to exclusively
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accept matings with conspecifics is constantly tested at
sympatric sites in nature, since males of the two species
court and attempt to mate with heterospecific females as
readily as with conspecifics (Freese and Fiedler 2002; Friberg et al. 2008a). This behaviour includes both energetic
costs as well as time costs, since unsuccessful courtships
sometimes make the female passive for as long as 30 min at
a time (Wiklund 1977a; Friberg et al. 2008a).
The two species have partitioned their niches, but the
direction of niche separation varies across Europe, creating
a geographic mosaic of niche separation (sensu Thompson
2005) with the two species shifting habitat generalist and
specialist roles. In the Czech Republic and Slovakia (Beneš
et al. 2003) Leptidea sinapis is a xerophilous habitat specialist that occurs in meadows and early succession areas,
while in Ireland (Thomas 2007) Leptidea sinapis appears
exclusively in open limestone meadows, whereas Leptidea
reali is a widespread habitat generalist also present in more
moist woodland areas in both the Czech Republic, Slovakia
and in Ireland (Beneš et al. 2003; Thomas 2007). This
pattern is reversed in Sweden (Friberg et al. 2008b, 2008c),
France (Amiet 2004) and Spain (Vila et al. 2003) where
Leptidea sinapis is a habitat generalist and Leptidea reali is
a meadow specialist (see larger review in Friberg et al.
2008b, 2008c). Hence, the niche separation between the
two species is likely to be caused by local processes in each
secondary contact zone rather than being a result of rigid
between-species differences in habitat preference (Friberg
et al. 2008b). This prediction has recently been corroborated by Friberg et al. (2008c); although Swedish Leptidea
sinapis lays eggs on both meadow and forest plants in
nature, and Leptidea reali almost exclusively oviposits on
the meadow plant Lathyrus pratensis, laboratory twochoice tests between the most often used meadow plant
Lathyrus pratensis and forest plant Lathyrus linifolius
showed that Leptidea sinapis females placed 88% and
Leptidea reali 92% of their eggs on Lathyrus pratensis
(Friberg et al. 2008c). The similar egg-laying preferences
imply that the choice of flight habitat precedes the host
plant choice, although Friberg et al. (2008c) focussed only
on the two most commonly utilised host plants in the field.
Earlier studies on female preference and its relation to
larval performance in other butterflies have shown that
larvae often survive well on a wider range of host plants
than the females choose to utilise (Wiklund 1975;
Thompson 1988; Thompson and Pellmyr 1991). Hence,
although the Leptidea females utilised only certain plants
in the field study (Friberg et al. 2008c) they may have
hidden preferences also for other plants that are used to
only a minor extent by certain populations due to, e.g. the
host plant rareness or phenology, i.e. if the host plant
suitability varies over the year and does not coincide with
the butterfly flight period at a certain field site.
Oecologia
A wider study of female preference and larval performance on different habitat-related host plants in this system
is therefore needed, both in the search for potential drivers
or co-drivers of niche separation, and to understand postspeciation processes of host plant adaptation and niche
separation between recently diverged species of phytophagous insects. The division of the sister species into a
habitat-, and thereby host plant-generalist and a habitatand host plant-specialist offers possibilities to test the
classic trade-off hypothesis for host plant specialisation,
which predicts that there are certain host-plant-related costs
of being a generalist (see above). The trade-off hypothesis
has only rarely been supported in empirical studies, and
several studies have failed to find any trade-offs of food
specialisation (for review see Futuyma 2008). Few systems
offer, however, the same possibilities as the Leptidea system, with one sister species being a generalist and the other
sister species being a specialist, with both species appearing sympatrically in nature. Hence, given that host-plantrelated trade-offs promote food specialisation, we predict
that the habitat and host plant generalist Leptidea sinapis
should perform and survive better than Leptidea reali on
the forest plants, whereas the meadow specialist Leptidea
reali will do better than Leptidea sinapis on their preferred
meadow host plants.
Materials and methods
All butterflies tested in this study were laboratory reared
and descended from females collected during the summers of 2002–2005 in two sites containing sympatric
populations of the two species (Friberg et al. 2008a,
2008b, 2008c)—either in Riala (59°370 N, 18°290 E) or in
Kronängen (58°580 N, 17°090 E). Collected butterflies were
species determined using either genital preparations,
DNA-sequencing or both (see Friberg et al. 2008a, b, for
rationale).
We tested the female egg-laying preference on seven
potential larval host plants, on which females of at least
one of the species have been shown to oviposit in earlier
field studies (Wiklund 1977b, Friberg et al. 2008c, M.
Friberg personal observation). We also tested larval performance in terms of survival, pupal weight, development
time (egg-pupa) and growth rate (mg/day) on the same host
plants. Both female preference and larval performance
experiments were tested twice—once in late summer of
2005, and then again in spring 2006. Hence, both female
preference and larval performance were assessed on host
plants growing during seasons when these butterflies are on
the wing in nature, since both species appear in a spring
generation in May–June, and since at least Leptidea reali,
and during some years also Leptidea sinapis, appears in a
summer generation during July-August at these latitudes
(Eliasson et al. 2005; Friberg et al. 2008a).
Female host plant preference
The female host plant rank order was assessed for both
species between 21 July and 6 August 2005 and between
17 and 27 May 2006. At Riala, from where the lion’s share
of the founders of the laboratory population descends, there
are seven potential legume host plants that have been
shown to host eggs of at least one of the species. These are
the meadow plants Lathyrus pratensis, Lotus corniculatus,
and Vicia cracca, and the forest plants Lathyrus linifolius,
Lathyrus vernus, Vicia sylvatica and Vicia sepium (Wiklund 1977b, Friberg et al. 2008c; M. Friberg, unpublished
data). V. sepium was included only during the spring
experiment. Hence, we tested six host species in the summer trials, and seven host species in the spring trials.
Females were mated once with a virgin conspecific male
in the laboratory. After mating, females were transferred to
individual egg-laying cages (0.5 9 0.5 9 0.5 m) containing a nectar plant (Verbascum nigrum) on which we
sprayed 25% sugar solution twice a day throughout the
experiment. Cages were placed in a quiet egg-laying room
with a light regime of 9:15 h light/dark at room temperature. In addition to nectar plants, females were fed 25%
sugar solution once a day throughout the experiment from
soaked cotton tips. In the morning the day after a female
was mated we added two potential host plants to the cage.
All such pairs of host plants included Lathyrus pratensis,
which is the most used legume at Riala by females of both
butterfly species (Friberg et al. 2008c); the alternative host
plant was one of Lotus corniculatus, V. cracca, Lathyrus
linifolius, Lathyrus vernus, V. sylvatica and, in the spring
experiment, V. sepium.
Initially, some females were reluctant to fly around and
inspect the cage and therefore had difficulties in finding the
host plants and start egg-laying. Therefore, we visited each
female once every 30–45 min and used cotton tips to
transfer them from their current position to the host plants
by first making them climb on to the tip and then allowing
them to climb from the tip on to the host plant. Every
second visit we positioned females on Lathyrus pratensis
and the next time they were transferred to the alternative
plant. We continued this treatment throughout the experiment with all females, although virtually all females
became skilled at finding the host plants on their own after
only 1 or 2 days of egg-laying.
Eggs were counted every evening and females were
offered fresh cuttings of host plants every morning. When a
female had laid ten or more eggs on the two plants, we
replaced the test plant and introduced a new test plant
species and a new Lathyrus pratensis individual. We let
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different females start with different test plants and we
replaced the test plants following the same order as plants
are listed above, presenting new pairs of host plants to the
females. Females were tested until they had experienced all
plants or until they died.
During the summer of 2005 we tested the host plant
preferences of seven Leptidea sinapis females and 13
Leptidea reali females; during the spring of 2006, eight
females of each species participated in the experiments.
Larval performance
Larval performance on the same six host plants used in the
preference experiments was tested with the offspring of the
females in the preference experiments. Plants that had been
oviposited on were transferred to climate cabinets (Termaks Series KB8000L; Termaks, Bergen, Norway) that were
programmed to keep a constant temperature of 23°C and a
22:2 h light dark regime. On the morning when the first
instar larvae had hatched from the eggs they were transferred to fresh cuttings of the different host plants. Larvae
were reared in the same four climate cabinets two by two in
0.5 L jars with their host plant supplied ad libitum
throughout their development. As far as possible we tried
to rear larvae on the same host plant species where they
were laid as eggs, but as females preferred to lay eggs on
some plants over others, and since the host plant Lathyrus
pratensis was present constantly during female egg laying
(see above), some first instar larvae had to be transferred
from one host plant species to another. This procedure is,
however, unlikely to have biassed the results of the performance study, since all larvae were transferred to a new
host plant individual immediately after hatching from the
eggs, and since the transfer took place at such an early
stage that the majority of larvae had not yet started to feed
on the host plant on which they hatched. Due to the variation in the number of eggs present on each plant, the
sample sizes differ somewhat between plants and between
seasons (spring/summer), but at the experiment start at
least 30 larvae of each species were placed on each of the
six different host plants in 2005 and on the seven different
plants in 2006. Individual females were allowed to contribute only a maximum of five eggs to each host plant
treatment. The jars were inspected each day of the experiment, and newly formed pupae were individualised on
detection. All pupae were weighed 2 days after pupation
when their cuticle had hardened enough to allow handling.
The development time was noted as well as whether the
pupa was set for diapause, i.e. remaining in the pupal stage
throughout winter and not eclosing as an adult until next
spring, or whether it was set for direct development, i.e.
remaining in the pupal stage for only about a week, before
eclosing as an adult. Adults of pupae that developed
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directly were sexed upon eclosion, whereas diapausing
pupae were sexed as pupae.
Statistical analysis
All statistical tests were conducted using the statistical
software R 2.6.0 (R Development Core Team 2007). The
female preference for ovipositing on Lathyrus pratensis
was indexed to 1 by dividing the number of eggs laid by
each female on each test plant by the number of eggs laid
by the same female on Lathyrus pratensis in that contrast.
Hence, if a female in a certain contrast laid ten eggs on
Lathyrus pratensis and five eggs on Lotus corniculatus, her
preference for Lathyrus pratensis in that contrast was given
the index value of 1 and her preference for Lotus corniculatus was assigned the relative value of 0.5. The species’
preferences were then calculated by averaging the individual female preferences for all plants and the preference
rank orders of the two species were compared using nonparametric Spearman Rank correlations.
The binomially distributed response variables survivorship (death during development = 0, survival to
pupae = 1) and larval pathway strategy (diapause development = 0, direct development = 1) were tested using
logistic regressions with logit as link factor, and with the
categorical factors of season, host plant species and butterfly species.
Diapausing pupae are often larger, whereas direct
developers are more prone to finish growth fast, since
they have more to gain from reaching the pupal stage,
and eventually adulthood, sooner (Wiklund et al. 1991;
Friberg and Wiklund 2007), and, similarly, females typically grow larger, whereas males in turn reach the pupal
stage sooner (Wiklund et al. 1991). For the above reasons,
and due to the innate between-sex and between-species
differences in entering direct development (Friberg et al.
2008b) and the different sensitivity to host plant in the
larval decision to enter direct or diapause development
(Friberg et al. 2008b; this study), the dataset was predestined to be unbalanced. Therefore, we calculated the
average value of each performance variable (pupal
weight, development time, growth rate) for each lowest
common unit, for example, for female Leptidea sinapis
that entered direct development on Lathyrus pratensis in
the spring experiment or for male Leptidea reali that
entered diapause on Lathyrus linifolius in the summer
experiment. In total, the dataset included 52 such potential unique groups of each species, and of these we had
data from at least one individual of both Leptidea sinapis
and Leptidea reali in 33 cases (Table 1 in S1). We then
calculated a new response variable for each performance
variable, i.e. for growth rate, pupal weight and development time, respectively, as the difference between each
Oecologia
Table 1 Results of a logistic regression (binomial, logit) testing the
impact of three different factors on the propensity to enter direct
development
v2
Season (S)
Butterfly species (BS)
Host plant species (HPS)
S 9 BS
S 9 HPS
df
0.13
104.8
36.56
0.9
P
1
0.72
1
\0.001
4
1
\0.001
0.34
6.25
4
0.18
BS 9 HPS
18.66
4
\0.001
S 9 BS 9 HPS
15.06
4
0.005
Leptidea sinapis group and the corresponding Leptidea
reali group. Hence, for pupal weight, a positive value
means that Leptidea sinapis, on average, grew larger than
Leptidea reali on a certain plant, whereas a positive
development time difference means that Leptidea reali
grew slower on that plant (see ‘‘Results’’). These data
rearrangements allowed testing of the new performance
variables in linear models with the categorical factors
season, host plant, larval pathway and sex. The new
sample sizes (4–7 data points/host plant) did not allow
tests of interactions between factors. Hence, in a study of
this magnitude with several predictors and a suite of host
plants, it is only possible to detect large-scale differences
in performance between butterflies reared on the different
plants. Such large-scale differences should on the other
hand be expected to be present between closely related
species, if diverging host-plant adaptations are important
drivers of niche-separation.
Results
Female host plant preference
The 8 Leptidea reali females laid 481 eggs, and the 8
Leptidea sinapis females laid 370 eggs during the spring
experiment, and the 13 Leptidea reali females and 7 Leptidea sinapis females of the summer experiment laid 816
and 311 eggs, respectively. Both species showed similar
host plant preferences in spring, preferring to oviposit on
Lathyrus pratensis, although Leptidea sinapis showed a
slightly higher propensity than Leptidea reali to also
include other plants in its host plant range (Fig. 1a,b). In
the summer experiment, the Leptidea sinapis females
showed stronger preference for both Lotus corniculatus and
V. cracca than for Lathyrus pratensis, whereas the Leptidea
reali females still preferred Lathyrus pratensis, although
they seemed more prone to oviposit also on other
host plants compared with the conspecific females of the
spring experiment (Fig. 1a). Very few eggs were laid on
V. sylvatica, Lathyrus vernus and V. sepium (present only
during spring), and the only forest plant that was oviposited
on to a fairly high extent was Lathyrus linifolius, although
this plant was ranked as host species three and four during
spring by the two butterfly species, as the second most
preferred plant by Leptidea reali females and as the fourth
most preferred plant by Leptidea sinapis in summer
(Fig. 1b). Hence, both species showed a strong preference
for meadow plants in both seasons as predicted by Friberg
et al. (2008c).
In summary, the host plant rank orders (V. sepium
excluded) of Leptidea sinapis and Leptidea reali were
strongly correlated in spring (nplants = 6, S = 2, r = 0.94,
P = 0.017; Fig. 1a), but with this small sample size, it was
not possible to detect a significant correlation in summer
(nplants = 6, S = 18, r = 0.48, P = 0.36; Fig. 1b). Within
species, neither the rank orders of spring and summer
females of Leptidea reali, nor of Leptidea sinapis females
in the two seasons, were significantly correlated
(nplants = 6, SL.reali = 8, r = 0.77, P = 0.10, Fig. 1a;
SL.sinapis = 10, r = 0.71, P = 0.14, Fig. 1b), although both
species ranked the same plants as the top four choices in
both seasons, i.e. the meadow plants Lathyrus pratensis,
Lotus corniculatus and V. cracca and the forest plant
Lathyrus linifolius (Fig. 1a,b).
Larval performance
The survival rates on the different host plants were unaffected by season (v21 ¼ 0:44, P = 0.51), but the
survivorship of Leptidea sinapis and Leptidea reali varied
on the six tested host plants [host plant species (HPS):
v26 ¼ 46:4, P \ 0.001; butterfly species (BS); v21 ¼ 2:2,
P = 0.14; HPS 9 BS: v26 ¼ 80:3, P \ 0.001; Fig. 2).
Leptidea sinapis suffered significantly higher mortality on
Lotus corniculatus compared with Leptidea reali, whereas
only 6 of 66 and 0 of 30 Leptidea reali larvae survived on
the V. sylvatica and V. sepium—two rarely utilised forest
plants (Friberg et al. 2008c; Fig. 1) on which Leptidea
sinapis did not suffer any decreased survival compared
with the more commonly used host plants (Fig. 2). The
survival rates did not differ significantly between the butterflies on any of the other plants, and survivorship varied
typically between 50 and 70% on most host plants (Fig. 2).
The propensity to develop directly differed between the
butterfly species, but also between larvae reared on the
different host plants, and between Leptidea sinapis larvae
in the two seasons when reared on Lathyrus vernus
(Table 1; Fig. 3). The four Leptidea reali survivors on
V. sylvatica in spring all entered direct development and
the two survivors in summer both entered diapause, but
survival was too low to detect significant seasonal differences in the propensity to enter direct development
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Fig. 1 Leptidea sinapis and
Leptidea reali host plant
preferences in the spring (a) and
summer (b) experiment. Note
that the preferences to oviposit
have been indexed to 1 within
each species, and that the
preferences to oviposit on the
other plants have been
calculated in relation to the
index preference (see
‘‘Materials and methods’’)
Fig. 2 The proportional survival of Leptidea sinapis and Leptidea
reali (compiled over the two seasons) on the seven different host
plants. Note that survival on V. sepium was only tested in spring. Bars
95% confidence intervals
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(Fig. 3). Leptidea reali entered direct development to a
higher extent than Leptidea sinapis on all six host plants on
which they survived, and larvae of both species entered
direct development in a higher proportion on the meadow
host plants compared with the forest host plants (Friberg
et al. 2008b). Especially Leptidea sinapis showed a strong
host-plant-related propensity whether to enter direct or
diapause development in both the summer and the spring
experiment, whereas Leptidea reali larvae reared on
Lathyrus linifolius, Lathyrus vernus, and V. sylvatica in
summer were less prone to enter direct development than
those reared on the same plants in spring (Fig. 3).
There was no host-plant-dependent size difference
between the two species (Fig. 4a), hence no species
became unusually small on a non-preferred host plant
(Table 1 in S1). There was also no size difference between
sexes, between different developmental larval pathways or
Oecologia
Fig. 3 Proportional propensity to enter direct development in the
spring (filled squares) and summer (open squares) experiments
(means ± 95% confidence intervals), and the average propensity to
enter direct development for Leptidea sinapis (white bars) and
Leptidea reali (grey bars)
between seasons (Table 2, Fig. 4a). Interestingly, however,
there were significant host plant effects on the measured
difference in development time between the two species.
Leptidea sinapis had slightly shorter development times on
the meadow plant Lotus corniculatus and the forest plant
Lathyrus linifolius, and also reached pupation much sooner
on V. sylvatica, on which the few Leptidea reali survivors
had extremely long development times (40 to 60 days). A
significant difference remained in a model that excluded
data obtained from V. sylvatica (F4,23 = 3.03, P = 0.038).
The difference in development times between the species
was more pronounced in the direct development pathway
(Table 2), but this difference was dependent on including
V. sylvatica in the model, since the significance disappeared when V. sylvatica was excluded (F1,23 = 3.44,
P = 0.076). Both species grew as large on each host plant,
but Leptidea sinapis did so faster than Leptidea reali on
several host plants, hence Leptidea sinapis did maintain a
higher growth rate on at least some plant species (Table 2;
Fig. 4c). The model was no longer significant after the
removal of V. sylvatica (F4,24 = 1.93, P = 0.14).
Discussion
This study has shown a lack of potential between-species
differences in female host plant preference or larval survival and performance that could have explained the
division into Leptidea sinapis being a habitat generalist,
and Leptidea reali having adopted a habitat specialist
lifestyle. Interestingly there were larger differences in
female preference between seasons than between species.
Both species showed similar host plant rank orders in
spring, preferring to oviposit on Lathyrus pratensis, and
both species were more prone to expand the number of
utilised host plants during summer. Leptidea reali did,
however, still prefer to oviposit on Lathyrus pratensis,
while Leptidea sinapis showed a higher preference to lay
eggs on the other meadow plants Lotus corniculatus and V.
cracca than on Lathyrus pratensis in the summer experiment. Hence, both the meadow specialist Leptidea reali,
and the habitat generalist Leptidea sinapis preferred to
oviposit on the three meadow plants; the only forest plant
that appears to be at least partially preferred by both species is Lathyrus linifolius, which has also been shown to be
the host plant most commonly used by Leptidea sinapis in
the forest habitat in nature (Friberg et al. 2008c).
The difference in preference between spring and summer is likely explained by seasonal differences in host plant
quality, and although we offered only as fresh host plant
specimens as possible to the females, the different plant
species appear to decrease in suitability to different extents
over the season. As an example, Lotus corniculatus and
Lathyrus linifolius produce new, single shoots throughout
summer and appear rather similar throughout the growth
season, whereas most summer individuals of Lathyrus
pratensis are bushy, dry and stiff plants that look very
different from the moist specimens that are present during
spring. Several butterfly species, including Leptidea sinapis, have been shown to also choose between individual
host plant specimens in the wild (Damman and Feeny
1988; Doak et al. 2006; Friberg et al. 2008c), and an
average Leptidea sinapis female only oviposits on 27% of
the inspected host plants in the field (Friberg et al. 2008c).
Hence, if the quality difference between a spring and a
summer individual of Lathyrus pratensis is larger than the
quality difference between seasonal morphs of other host
plants this could explain the wider host plant preference in
the summer experiment.
In the summer experiment, Leptidea sinapis females
preferred to oviposit on Lotus corniculatus, which was the
host plant with the lowest survival of Leptidea sinapis
larvae, and the only host plant on which Leptidea reali
survived significantly better than Leptidea sinapis.
Whereas less than 10% of the Leptidea reali larvae survived on V. sylvatica, and no Leptidea reali larvae reached
the pupal stage on V. sepium, the Leptidea sinapis survivorship was never below 40% in this study. Hence, the
meadow specialist Leptidea reali survived poorly on two of
the four forest plants, whereas larvae of the habitat generalist Leptidea sinapis survived in similar proportions on
all plants regardless of habitat affiliation (Fig. 2).
The host plant dependent propensity to enter direct
development has been thoroughly discussed from a broader
123
Oecologia
Fig. 4 Difference in a pupal weight (mg), b development time (days) c
and c growth rate (mg/day) between Leptidea sinapis and Leptidea
reali on six different host plants. The response variable in each figure
is calculated as the difference between the average Leptidea sinapis
and Leptidea reali value in each unique group (Table 1 in S1). Hence,
in cases where the confidence intervals do not overlap the dotted line
that crosses zero, the two species make different growth decisions, or
have different possibilities of rapid growth or reaching a large pupal
size on that plant
habitat perspective in an earlier study (Friberg et al.
2008b). Focussing only on the two most commonly used
host plants in the field—the meadow plant Lathyrus pratensis and the forest plant Lathyrus linifolius (Friberg et al.
2008c)—Leptidea sinapis entered direct development in
higher proportions on the meadow plant in both summer
and spring, whereas Leptidea reali larvae in the spring
experiment entered direct development in as high proportion on Lathyrus linifolius as on Lathyrus pratensis
(Fig. 3). The variation in propensity to enter direct development on the different plants could result from differences
in how well-adapted the butterflies are to the different
plants, i.e. if the larvae can develop faster on certain host
plants, and therefore have a higher possibility of entering
direct development on these plants. This effect is studied
only indirectly here, but the observation that Leptidea reali
had a higher propensity to develop directly on all host
plants, but showed longer development times on three of
the six host plants tested, stands in direct contrast to the
prediction that the host plant dependent pathway strategy is
caused by differences in the levels of adaptations to different host plants. Likewise, it appears unlikely that the
host plant dependent propensity to enter direct development is caused by differences in levels of nutrients between
forest and meadow plants, which appears to be the case in
other systems where different host plants induce different
larval pathway strategies (Hunter and McNeil 1997; Wedell et al. 1997). This is because Lathyrus linifolius and
Lathyrus pratensis are quite closely related congeners, and
the variation in performance characters such as butterfly
growth rate or development time between the different
plants is small (Fig. 4a–c; Table 1 in S1).
Alternatively, the variation in the propensity to enter
direct development on different host plants could be an
adaptive strategy if larvae receive information from the
host plant about their location in a spatially heterogeneous
environment (cf. Friberg et al. 2008b). Being habitat generalists, larvae of the Leptidea sinapis butterfly in south
central Sweden must be able to develop under quite different circumstances, both in the homogeneous sunexposed meadows, and in the darker, colder and more
heterogeneous forest habitat (cf. Friberg et al. 2008b, c);
Leptidea sinapis larvae also appear more sensitive to larval
host plant in their decision to enter direct development or
diapause than larvae of Leptidea reali. Leptidea reali, the
123
meadow specialist, shows a higher overall propensity to
enter direct development, and a higher relative propensity
to enter direct development on the forest host plant
Lathyrus linifolius than Leptidea sinapis although Leptidea
reali only rarely oviposits on this plant in nature (Friberg
Oecologia
Table 2 ANOVA tables showing the results from three linear
models, with four factors. The response variables denote differences
in pupal weight, development time and growth rate, respectively,
between the two butterfly species (Leptidea sinapis value-Leptidea
reali value) in each unique group (see ‘‘Materials and methods’’).
df
Hence, a significant result (highlighted in italics) means that the two
butterfly species developed differently on e.g. different host plants
(Fig. 4a–c) or within the different pathway strategies (diapause/direct
development)
Pupal weight
SS
Development time
MS
F
P
SS
MS
F
Growth rate
P
SS
MS
F
P
Season
1
77.02
77.02
1.44
0.24
17.39
17.39
1.56
0.22
0.029
0.029
0.32
0.58
Host plant species
5
77.98
15.6
0.29
0.91
456.45
91.29
8.21
\0.001
1.52
0.30
3.42
0.018
Pathway
1
0.00000021
1.0
55.37
55.37
Sex
1
10.44
10.44
0.19
0.66
0.47
0.47
24
1,285.29
53.55
Residuals
0.000011
0.000011
et al. 2008c). Future studies of the host-plant-dependent
developmental larval pathway decision in different daylengths and temperatures are needed in order to fully
determine whether the host plant effect on the propensity to
enter direct development is a side-effect of the nutritional
uptake from different host plants or if it is an adaptive
strategy of particular importance for the habitat generalist
Leptidea sinapis.
Interestingly, there were no host-plant-related differences in pupal weight between the two butterfly species.
Hence, although Leptidea sinapis developed faster than
Leptidea reali on the meadow plant Lotus corniculatus, and
on the two forest plants Lathyrus linifolius and V. sylvatica,
the pupal weights were similar between the two species on
all plants, and Leptidea sinapis had, hence, a slightly
higher growth rate on Lotus corniculatus and Lathyrus linifolius, and a much faster growth rate on V. sylvatica than
Leptidea reali. On the latter plant, only 6 of 66 Leptidea
reali larvae survived, and neither Leptidea reali nor Leptidea sinapis includes V. sylvatica to more than a marginal
extent in their host plant diet in the field (Friberg et al.
2008c). Hence, there are no clear-cut differences in larval
performance between Leptidea sinapis and Leptidea reali
on the four most preferred host plants in the laboratory
study. Furthermore, Leptidea sinapis did not survive better
than Leptidea reali on the forest host plant Lathyrus linifolius, although Leptidea sinapis lays more than seven
times more eggs than Leptidea reali on Lathyrus linifolius
in the field (Friberg et al. 2008c). The habitat niche separation into habitat generalist and habitat specialist is thus
unlikely to have been driven by differences in female
preferences to oviposit on certain plants, and it is also
unlikely that differences in larval survival or performance
between habitats is a major factor for maintenance of niche
partitioning. Instead, the ability of Leptidea sinapis to
survive on the two plants on which Leptidea reali mortality
was extremely high is probably a result of post-separation,
habitat-specific selection on being able to survive also on
these plants, which, in this part of Europe, Leptidea reali
267
11.12
5.0
0.035
0.059
0.059
0.66
0.43
0.043
0.84
0.057
0.057
0.65
0.43
2.13
0.089
virtually never experiences and hence has not been selected
to tolerate. Butterfly larvae of several species have been
shown to maintain the ability to survive on ancient host
plants outside their current host plant range (Janz et al.
2001), and the high survival of Leptidea sinapis compared
with the low survival of Leptidea reali on the non-preferred
forest host plants implies that Leptidea sinapis is more
likely to have expanded its host plant range rather than
Leptidea reali having evolved towards specialisation. Thus,
both species survive in similar numbers on meadow plants,
and there are no differences in performance that could
imply that Leptidea reali is more specialised than Leptidea
sinapis on meadow plants in general and the most commonly used plant Lathyrus pratensis in particular.
Furthermore, Swedish females of the habitat generalist
Leptidea sinapis reach their optimal flight temperature at
lower temperatures than females of the habitat specialist
Leptidea reali (Friberg et al. 2008c). These different temperature adaptations might have driven niche partitioning,
although they are perhaps also more likely a result of postseparation selection in the different habitats. Future studies
of temperature reaction norms of Leptidea butterflies
descending from different parts of Europe are warranted in
order to further investigate the role of flight temperature in
niche separation in this system.
The finding that Leptidea sinapis did not perform any
worse than Leptidea reali on the most preferred meadow
plant Lathyrus pratensis, although Leptidea sinapis also
incorporates other host plants, and in particular the forest
host plant Lathyrus linifolius, in its realised host plant
range implies that host plant generalisation is not necessary
associated with a cost, and the findings in this study do not
support the trade-off hypothesis (Futuyma and Moreno
1988; Agrawal 2000) for host plant specialisation. The
female host plant preference, which in many systems has
been shown to be much smaller than the larval tolerance
spectrum of host plants (cf. Wiklund 1975, 1982), were in
this study not correlated to either survival, or performance
on the different plants, with the exception of the reluctance
123
Oecologia
of both species to oviposit on V. sylvatica and V. sepium,
on which Leptidea reali but not Leptidea sinapis had difficulty in surviving. Hence, Leptidea sinapis appears to
have incorporated the forest legumes into its host plant
range, without having to carry any costs in terms of survival or growth rate, and the apparent lack of correlation
between female preference and larval performance on the
most-used host plants is in concordance with several other
studies that also have failed to discover any tight connection between female preference and larval performance (for
review, see Futuyma 2008). Instead, studies where the
connection is the tightest between preference and performance are often performed in the field (Damman and
Feeny 1988; Doak et al. 2006; Wiklund and Friberg 2008),
in the presence of natural enemies, and female preference
is then more a case of selecting an enemy-free space than
choosing the host plant that allows the fastest larval growth
rate (Bernays and Graham 1988; Murphy 2004; Doak et al.
2006; Wiklund and Friberg 2008). Hence, enemy-free
space as an explanation for food specialisation also
deserves to be investigated in this system, and especially
the potential for sexual competition as a driver of niche
partitioning needs to be studied further. Male courtship
imposes time-limitations on females (unpublished data)
and the male inability to distinguish between con- and
hetero-specific females (Friberg et al. 2008a) is likely to be
costly for both males and females, and especially those of
the least common species that have to put up with a disproportionally high courtship pressure due to the interest of
heterospecific males. This risk of either courting, or being
courted by heterospecifics might then favour aggregation
and habitat specialisation or even a habitat switch to a
novel habitat. This, in turn, would also lead to ecological
host plant specialisation in this system, and future studies
should deal with the potential for heterospecific sexual
competition as a driver of niche separation and as an
explanatory model for the geographic mosaic of niche
separation between Leptidea sinapis and Leptidea reali
throughout Europe.
Acknowledgements We thank Martin Bergman and Martin Olofsson for assistance during the laboratory experiments, and for
discussion and input on an earlier draft of this manuscript, and Helena
Larsdotter Mellström and Didrik Vanhoenacker for useful comments
on earlier drafts of this manuscript. This study complies with the
current laws of Sweden and was financed by a grant from the Swedish
Research Council to C.W.
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