Insect Conservation and Diversity (2014) doi: 10.1111/icad.12100
Phengaris (Maculinea) alcon butterflies deposit their
eggs on tall plants with many large buds in the vicinity
of Myrmica ants
IRMA WYNHOFF, 1 RALDI B. BAKKER, 1 , 2 BAS OTEMAN, 1 , 2 , 3 PAULA
SEIXAS ARNALDO 4 and FRANK VAN LANGEVELDE 2 1Dutch Butterfly
Conservation, Wageningen, The Netherlands, 2Resource Ecology Group, Department of Environmental Sciences,
Wageningen University, Wageningen, The Netherlands, 3Spatial Ecology Department, Royal Netherlands Institute for Sea
Research (NIOZ-Yerseke), Yerseke, The Netherlands and 4CITAB - Center for Research and Technology in AgroEnvironmental and Biological Sciences, University of Tr
as-os-Montes and Alto Douro, Vila Real, Portugal
Abstract. 1. The survival of eggs and larvae is dependent on the oviposition
site selection of their mothers. In obligate myrmecophilic butterflies, both host
plant phenology and host ant presence are expected to affect the decision where
to deposit eggs. The importance of ant nest presence in the oviposition site
selection of Phengaris butterflies is, however, highly debated.
2. We studied oviposition in the largest Phengaris (Maculinea) alcon population in Portugal, exploiting Gentiana pneumonanthe as the host plant and Myrmica aloba as host ant. We collected phenological plant data and recorded the
presence and number of eggs on plants with and without Myrmica ants nearby
during the flight period of the butterfly.
3. Females oviposited on tall plants with many tall buds, while the presence
of host ant nests weakly affected oviposition on plants where the probability of
finding ants at close range was high. Moreover, larger plants with many tall
buds close to host ant nests received more eggs.
4. A density-dependent shift in oviposition was not found as the proportion
of buds not infected with eggs did not differ between plants with or without
ants, whereas plant characteristics did have an effect. Tall plants with many
large buds were associated with earlier oviposition.
5. Our results suggest that females of P. alcon in Portugal choose gentian
plants for oviposition mainly based on plant characteristics whereas the
vicinity of ants had a weak effect. Moreover, our study shows that testing the
ant-mediated oviposition hypothesis requires baiting ants more than once.
Key words. Ant-mediated oviposition, Gentiana pneumonanthe, host plant phenology, myrmecophilic butterflies, Myrmica aloba, Portugal, random oviposition.
Introduction
In most insect species females produce a high number of
offspring by depositing hundreds or even thousands of
eggs. However, females are usually very selective when
Correspondence: Irma Wynhoff, Dutch Butterfly Conservation,
PO Box 506, 6700 AM Wageningen, The Netherlands.
E-mail: Irma.Wynhoff@Vlinderstichting.nl
Ó 2014 The Royal Entomological Society
choosing oviposition sites, facilitating the access to food
resources for the hatching larvae, ensuring protection of
eggs and larval instars or finding microclimatic conditions
for optimal development (Obermaier & Zw€
olfer, 1999;
Scheirs & de Bruyn, 2002; Gullan & Cranston, 2010).
Optimal oviposition decisions support a high survival
probability during larval instars to reach the adult stage
and reproduce (Jaenike, 1978; Scheirs & de Bruyn, 2002).
Hence oviposition preferences are expected to correlate
1
2
Irma Wynhoff et al.
with host suitability for offspring development. Butterflies
typically lay eggs on or in the vicinity of plants the caterpillars feed on (Fartmann & Hermann, 2006). Some species prefer plants with high nutritional values (Baylis &
Pierce, 1991; Chen et al., 2004), a certain host plant size
(Wiklund, 1984; K€
uer & Fartmann, 2005; Nowicki et al.,
2005; Rabasa et al., 2005; Reudler Talsma et al., 2008),
suitable microclimatic conditions (Shreeve, 1986; Eilers
et al., 2013), avoidance of competition (Wiklund & Friberg, 2008; Sielezniev & Stankiewicz-Fiedurek, 2013) or
presence of specific mutualists (Pierce & Elgar, 1985).
Over 50% of lycaenid butterfly species have an antassociated lifestyle known as myrmecophily, where the
species have a neutral, mutualistic or parasitic interaction
with ants (Fiedler, 1991; Fiedler et al., 1996). The genus
Phengaris Doherty, 1891 (the senior synonym of Maculinea van Eecke, 1915) forms a specific group within this
family with an extraordinary life cycle where the caterpillars parasitise on Myrmica ants. Many of these butterfly
species have experienced severe declines over the last few
decades (Van Swaay et al., 2011). As adoption by ants is
obligate for caterpillar survival (Elmes et al., 1991; Akino
et al., 1999; Als et al., 2001), one might expect that a
Phengaris female butterfly is capable of including ant
presence into her oviposition decisions and adjust the oviposition locations accordingly, which is however highly
debated. Some studies found support of this ant-mediated
oviposition (Van Dyck et al., 2000; Wynhoff et al., 2008;
Van Langevelde & Wynhoff, 2009; Van Dyck & Regniers,
2010; Patricelli et al., 2011), whereas others did not (Thomas & Elmes, 2001; Nowicki et al., 2005; Musche et al.,
2006; F€
urst & Nash, 2009).
Phengaris butterflies fly for 1–2 months during summer, depositing their eggs on specific host plants. After
hatching, the caterpillars bore themselves into the flower
buds and feed on the soft internal parts where they
quickly develop to the fourth instar and leave the plant
(Akino et al., 1999; Als et al., 2001). By mimicking a
Myrmica ant larva, the caterpillar ensures adoption by
foraging Myrmica ants. The caterpillar spends around
10–11 or even 22–23 months in the ant nest, being fed
by ants or feeding on ant brood (Elmes et al., 1991; Thomas et al., 1998; Sch€
onrogge et al., 2000; Als et al., 2001;
Witek et al., 2006). To date, thirteen species of Myrmica
have been described as hosts for Phengaris in Europe
(Als et al., 2001; Steiner et al., 2003; Tartally et al., 2008;
Arnaldo et al., 2011; Witek et al., 2010). However,
depending on location, the survival rate of the caterpillars in the ant nests differs between ant species. Best survival is achieved in primary hosts, but lower survival in
secondary host ant nests may be important in years with
a high butterfly population density (Thomas et al., 2005,
2013). Myrmica ants are ground foragers, usually searching up to 2 m from the nest. The probability of a caterpillar being located by a foraging worker is directly
dependent on the density of the workers in the colony,
and proximity of the host plant to the ants nest (Elmes
et al., 1998).
The decision where to oviposit is crucial for Phengaris
with their dependence on both specific host plants as food
source for the first three instars, and their need for host
ants. Usually only a small developmental window in plant
phenology is accepted for oviposition (Thomas & Elmes,
2001; however see Van Dyck & Regniers, 2010). Since
plants grow during the flight season while ant nests
remain at the same location, it is possible that temporal
differences where butterflies oviposit can be observed following the best combination of plants as a food source
and the presence of Myrmica ants (Patricelli et al., 2011).
On the one hand, there could be a density-dependent shift
in oviposition preference when suitable plants near ants
already contain high egg loads (Van Dyck et al., 2000).
On the other hand, a female butterfly might be attracted
by plant characteristics such as the height of a plant, the
number of buds and the size of the buds, rather than the
presence of ants close to the plant (Thomas & Elmes,
2001; F€
urst & Nash, 2010; Czekes et al., 2013).
To get further insight into the relationship between
Phengaris and Myrmica ants, we studied the largest
known population of Phengaris alcon (Denis & Schifferm€
uller, 1775), commonly known as the Alcon Blue, in
Portugal (Rodrigues et al., 2010; Arnaldo et al., 2011).
The local population of P. alcon is dependent on the
marsh gentian (Gentiana pneumonanthe L.) as its single
host plant and Myrmica aloba Forel, 1909 as the single
host ant (Arnaldo et al., 2011). Our goal was to identify
the influence of plant characteristics and the presence of
Myrmica ant nests on oviposition choices over time. We
hypothesised that ant presence influences the choice of
oviposition location, in addition, to plant characteristics.
We expected to find more eggs on plants with ants in the
proximity than on plants outside the ants’ foraging range,
and that plant characteristics (i.e. plant height, flower bud
length and the number of buds) also have a positive influence on the presence and number of eggs (hypothesis 1).
We also expected that in the beginning of the season, the
eggs will be concentrated on suitable plants with ants in
the proximity until all suitable plants with ants are used
and then suitable plants without ants will be also selected
(hypothesis 2). These hypotheses were tested in a field
study, where we selected host plants with and without
ants in the proximity.
Material and methods
Set-up of field study
The study site was located near Lamas de Olo
(41°220 N, 7°480 W; 974 m AMSL) inside Alv~
ao National
Park, Portugal. The field work was conducted between 3
July and 10 October of 2012, covering the entire flight
period of P. alcon which lasted 6 weeks from mid-July till
late August. A plot inside a meadow was fenced in to
exclude large grazing animals. The plot was split into
seven rows, each of one metre wide and 12.5 m in length,
Ó 2014 The Royal Entomological Society, Insect Conservation and Diversity
Oviposition in Phengaris alcon
separated by 30 cm of path (we did not find an effect of
row in our analysis). All marsh gentians found inside this
plot were marked with a number on a plastic label placed
next to the plant. The density of marsh gentians was 7.2
plants per m2. Only the apical bud of the plant was monitored, because this is the most likely one to receive eggs
(F€
urst & Nash, 2010; Van Dyck & Regniers, 2010;
Arnaldo et al., in press). As one plant can have more than
one shoot, a string was tied around the apical bud. This
also prevented mistakes while measuring plants. To avoid
selecting shoots from the same plant, every new shoot had
to be at least 10 cm away from the already selected
plants.
Data collection
Every day during the flight period, the apical bud of
each plant was checked for new eggs, which gave us the
date that the first egg was laid on the apical bud. The field
study was designed to study caterpillar competition in the
flower buds as well (B. Oteman, R.B. Bakker, P.S.
Arnaldo, I. Wynhoff and F. van Langevelde, unpubl.
data). As it takes approximately 7 days for a P. alcon egg
to hatch (Bos et al., 2006), a hood made of fine meshed
gauze (approximately 5 by 10 cm in size) was placed over
the apical bud 1–6 days after a plant received its first apical egg. The gaze was closed with a noose made of plasticised wire to ensure caterpillars could not leave their
hostplant. More ovipositions after day 6 were therefore
impossible. For this study, we analysed only the number
of eggs on those buds which were encapsuled after exactly
6 days (n = 147, this represented 52% of the encapsuled
buds). Plant characteristics (i.e. plant height in cm, number of buds and length of the apical bud in cm) were
recorded weekly. The means of plant height, length of the
apical bud and the number of buds were calculated by
interpolation using the data collected closest to the day
the very first egg was laid and the data closest to the day
the very last egg was laid, thus including the entire oviposition period of P. alcon.
At the base of each selected plant, ants were baited
three times during the field period, in the middle of July,
the beginning of August and the beginning of September.
Ant baiting took place in the morning around 10:00 am
and evening around 18.00 pm when ants are most active
(Elmes et al., 1991; F€
urst & Nash, 2010). A small paper
cup with a sugar cube inside was placed within five centimetres of the stem of each plant in the early morning.
The sugar baits were checked every 2 hours starting at
10:00 am. We kept the attracted ants in 70% ethanol for
later identification in the laboratory. Baits without ants
were left in the field and were checked regularly every
2 hours until the end of the day. Ants were later identified
using a binocular microscope and literature (Radchenko
& Elmes, 2010). Plants were considered to grow inside the
foraging range of a Myrmica ant nest when ants were
found in one or more of the ant capturing events. In this
3
study, the only Myrmica species we found was Myrmica
aloba. Furthermore, the spatial distribution of ant presence was tested by calculating the distance from each
plant to the closest plant with ants and the closest plant
without ants.
Statistical analyses
We tested the effects of the presence of Myrmica, the
plant characteristics and their interaction due to the presence and absence of eggs as a binary response variable,
with a Generalised Linear Model (GZLM) with a logit
link function based on a binomial distribution (hypothesis
1). This analysis was followed by a GZLM where we only
included plants where at least twice ants were found and
plants where we did not find ants. With this analysis we
could evaluate oviposition on plants for which we were
more sure that they are located nearby ant nests. Then we
replaced in each model the presence of Myrmica by the
distance to the closest plant with ants to test the effect of
the spatial distribution of ant presence. We removed the
non-significant interaction effects to reduce the number of
parameters in the final models. As we had many independent variables, we applied the False Discovery Rate
approach (Benjamini & Hochberg, 1995) by calculating
the expected proportion of rejected null hypotheses that
are falsely rejected. This False Discovery Rate approach
can assign a lower P-value threshold a than the often used
a = 0.05, based on the number of independent variables
and their P-value. Secondly, we tested the effects of the
presence of Myrmica, the plant characteristics and their
interaction on the number of eggs, using a GZLM with a
log-linear link function based on a Poisson distribution.
Again, we replaced the presence of Myrmica by the distance to the closest plant with ants.
Based on the results of the GZLMs, a regression-based
path analysis was conducted to examine the direct and
indirect effects of the independent variables on both the
presence and the number of eggs (Jaccard & Wan, 1996).
A recursive conceptual path model was constructed
(Fig. 1a). The final parsimonious path models were constructed based on the W-statistic and v2 tests (Epstein
et al., 1994). Path coefficients were calculated using a fully
standardised (logistic) regression coefficient to compare
the strength of the effects on continuous and binary
endogenous variables (Menard, 2004). Finally, we tested
the spatial autocorrelation of the residuals of the GZLMs
(De Knegt et al., 2011) using SAM: Spatial Analysis in
Macroecology (Rangel et al., 2010).
An independent samples t-test was used to test the difference between the date of first oviposition on plants
with and without the presence of Myrmica. To show the
time it took plants to receive eggs depending on whether
they were growing inside or outside the range of ants
(hypothesis 2), we used Cox regression tests, where we
also included plant characteristics as independent variables. A Cox regression model produces a function that
Ó 2014 The Royal Entomological Society, Insect Conservation and Diversity
4
Irma Wynhoff et al.
(a)
(b)
(c)
(d)
(e)
Fig. 1. The recursive conceptual path model for the effects of the plant and ant characteristics on the presence or number of eggs (a), the
results of the analysis explaining the presence of eggs for all data (b), the presence of eggs for the plants without ants and the plants for
which at least two times ants were found (c) and the number of eggs per bud for all data (d), and the number of eggs for the plants without ants and the plants for which at least two times ants were found (e). The values in (b–e) represent fully standardised regression coefficients. ***P < 0.001, **P < 0.01, *P < 0.05, ns, non-significant.
Ó 2014 The Royal Entomological Society, Insect Conservation and Diversity
Oviposition in Phengaris alcon
predicts the time to oviposition of the first egg as a
function of the independent variables. We only included
plants that received eggs and related the timing of this
event as the dependent variable to the presence of Myrmica ants and the plant characteristics measured at the
moment of oviposition as independent variables. Apart
from the spatial autocorrelation, all statistical analyses
were performed using IBM SPSS statistics version 20
(IBM Corp., 2011).
Results
In our field study, we monitored 447 plants that had at
least one bud. From the selected plants, 165 (36.9%) were
found to grow within the foraging range of Myrmica
aloba while 282 (63.1%) plants grew outside the foraging
range. These two groups did not differ in the mean plant
height (with Myrmica 21.7 cm SE 0.5, n1 = 282, without Myrmica 21.3 cm SE 0.4, n2 = 165; t-test,
t = 0.592, d.f. = 445, P = 0.554), mean bud length (with
Myrmica 1.65 cm SE 0.07, n1 = 282, without Myrmica
1.54 cm SE 0.05, n2 = 165; t-test, t = 1.239,
d.f. = 445, P = 0.216) or mean number of buds (with
Myrmica 2.9 SE 0.2, n1 = 282, without Myrmica
2.7 SE 0.1, n2 = 165; t-test, t = 1.162, d.f. = 445,
P = 0.246). In three baiting sessions, we collected ants one
time underneath 125 plants (76%), two times underneath
35 plants (21%) and three times underneath 5 plants
(3%).
We found that the mean distance from plants where
ants were captured to other plants with ants was shorter
than the mean distance to plants without ants (distance to
(a)
5
plants with ants 29.7 cm SE 1.3, n1 = 222, distance to
plants without ants 35.0 cm SE 1.5 n2 = 222; t-test,
t = 2.65, d.f. = 434, P = 0.008). The mean distance from
plants without ants to plants where ants were captured
was longer than the mean distance to plants without ants
(distance to plants with ants 41.3 cm SE 1.2, distance
to plants without ants 20.6 cm SE 0.8: t-test,
t = 14.49, d.f. = 700.1, P < 0.001). The frequency distributions show that, in general, distances are short (Fig. 2).
Moreover, this analysis reveals that plants with ants occur
clustered.
In total, we found that 1735 eggs have been deposited
on 282 plants from a total of 447 plants, which were monitored during our study. For these 282 plants, 170
(60.3%) plants had no Myrmica ants in the proximity and
112 (39.7%) plants did. We found 948 eggs (54.6%) on
plants without ants, whereas 787 eggs (45.4%) were found
on plants with ants. Again there were no differences in
the mean plant height (with Myrmica 23.5 cm SE 0.5,
n1 = 170, without Myrmica 23.3 cm SE 0.4, n2 = 112;
t-test, t = 0.297, d.f. = 280, P = 0.767), bud length (with
Myrmica 1.70 cm SE 0.08, n1 = 170, without Myrmica
1.69 cm SE 0.06, n2 = 112; t-test, t = 0.104,
d.f. = 280, P = 0.917) or number of buds (with Myrmica
3.4 SE 0.2, n1 = 170, without Myrmica 3.3 SE 0.2,
n2 = 112; t-test, t = 0.470, d.f. = 280, P = 0.638)
between these two groups. Using a GZLM with a logit
link function, we found that plant height, the number of
buds and bud length had a positive effect on the probability that eggs were oviposited on the host plant (Table 1a).
The presence of Myrmica did not have an effect on
whether a plant would receive an egg or not. The same
was found for the effect of the distance to the closest
(b)
Fig. 2. Frequency distribution of the distance to the closest Gentiana pneumonanthe plant with Myrmica aloba ants to gentian plants without (a) or with (b) Myrmica ants in their proximity.
Ó 2014 The Royal Entomological Society, Insect Conservation and Diversity
6
Irma Wynhoff et al.
Table 1. Results of the effect of Myrmica presence, the distance
to the closest plant with ants, the plant characteristics and their
interaction on the presence or absence of eggs using a Generalised Linear Model with a logit link function based on a binomial
distribution. The coefficient, its standard error, the Wald statistic
and associated P -value are given. We removed the non-significant interaction effects to reduce the number of parameters in the
models. The first analysis was done using all data (a: n = 447),
whereas the second analysis was done using the plants without
ants and the plants for which at least two times ants were found
(b: n = 322 from which ants were found at least twice underneath
40 plants). The False Discovery Rate approach assigned the Pvalue threshold a = 0.039.
Independent variables
Coefficient (SE)
Wald
P-value
(a) Models based on all data
Myrmica (=0)
0.272 (0.216)
1.595
0.207
No. of buds
0.372 (0.070)
28.429 <0.001
Distance
0.008 (0.004)
3.686
0.055
Nr of buds
0.379 (0.070)
29.609 <0.001
Myrmica (=0)
0.296 (0.210)
1.998
0.157
Bud length
0.042 (0.012)
12.177 <0.001
Distance
0.007 (0.004)
3.095
0.079
Bud length
0.043 (0.012)
12.882 <0.001
Myrmica (=0)
0.329 (0.230)
2.060
0.151
Plant height
0.019 (0.002)
68.671 <0.001
Distance
0.012 (0.005)
6.369
0.012
Plant height
0.020 (0.002)
71.240 <0.001
(b) Models based on data for the plants without ants and the plants
for which at least two times ants were found. Plants where ants
have been found only once are excluded
Myrmica (=0)
2.193 (1.418)
2.390
0.122
Nr of buds
2.280 (0.912)
8.595
0.003
Myrmica (=0) 9 Nr of buds 1.873 (0.917)
4.177
0.041*
Distance
0.006 (0.008)
0.675
0.411
Nr of buds
1.078 (0.213)
25.485 <0.001
Distance 9 Nr of buds
0.010 (0.003)
13.617 <0.001
Myrmica (=0)
0.965 (0.421)
5.259
0.022
Bud length
0.052 (0.015)
12.443 <0.001
Distance
0.013 (0.005)
6.890
0.009
Bud length
0.056 (0.015)
13.870 <0.001
Myrmica (=0)
0.808 (0.442)
3.338
0.068
Plant height
0.019 (0.003)
47.326 <0.001
Distance
0.018 (0.006)
9.968
0.002
Plant height
0.020 (0.003)
52.031 <0.001
characteristics. Adding the variable distance to the closest
plant with ants to the model would cause the variable bud
length to change sign and was therefore omitted. For
these models, the spatial autocorrelation was found to be
low (0.019 < Moran’s I < 0.015 for the first 10 lags).
When selecting the plants without ants and the plants
for which at least on two occasions ants were found
(n = 322 from which ants were found underneath 40
plants), we found a similar effect of the plant characteristics (Table 1b). However, in most of the cases, the presence of ants or a short distance to the closest plant with
ants increased the probability that eggs are deposited. The
interaction between the number of buds and the distance
to the closest plant with ants implies that when the plant
has a high number of buds, the probability of finding eggs
increases when it is also growing close to plants with ants
(Fig. 3). The path analysis showed that indeed distance to
the closest plant with ants had a negative direct effect on
the presence of eggs, but the effect was not very strong,
given the relatively low standardised coefficient, whereas
the positive direct effect of plant height was larger
(Fig. 1c). We again found a very low spatial autocorrelation for these models (0.02 < Moran’s I < 0.026 for the
first 10 lags).
In total, 890 eggs were distributed over 131 plants
(Fig. 4), with on average 6.8 eggs on the apical bud (median 5.0, SE 0.54). Again there were no differences in the
mean plant height (with Myrmica 24.6 cm SE 0.8,
n1 = 81, without Myrmica 24.0 cm SE 0.6, n2 = 50;
t-test, t = 0.658, d.f. = 129, P = 0.512), bud length (with
Myrmica 1.84 cm SE 0.09, n1 = 81, without Myrmica
1.78 cm SE 0.09, n2 = 50; t-test, t = 0.395, d.f. = 129,
P = 0.693) or number of buds (with Myrmica 3.9 SE
0.4, n1 = 81, without Myrmica 3.5 SE 0.3, n2 = 50;
*Not significant given the threshold a = 0.039 based on the False
Discovery Rate approach.
plant with ants, except for the model with plant height:
here a negative effect of distance to the closest plant with
ants was found, suggesting that the probability to find
eggs increases when ants are near.
The path analysis confirmed the GZLM results for
explaining egg presence (Fig. 1b): plant height appeared
to be the most important independent variable that has a
direct, positive effect on egg presence (total effect is 0.45).
Ant presence had no significant effect on egg presence.
The non-significant effect of the number of buds and bud
length is due to the high correlation between the plant
Fig. 3. Predicted relationships between bud length and the probability that eggs were oviposited in relation to the distance to the
closest Gentiana pneumonanthe plant with Myrmica aloba ants in
the proximity, for different number of buds per plant (see text
for statistics).
Ó 2014 The Royal Entomological Society, Insect Conservation and Diversity
Oviposition in Phengaris alcon
7
Table 2. Results of the effect of Myrmica presence, the distance
to the closest plant with ants, the plant characteristics and their
interaction on the number of eggs deposited until day 6 after the
first egg was found, using a Generalised Linear Model with a log
link function based on a Poisson distribution. Buds encapsuled
between day 1 and 5 after the first egg was removed from the
analysis. The coefficient, its standard error, the Wald statistic and
associated P-value are given. We removed the non-significant
interaction effects to reduce the number of parameters in the
models. The first analysis was done using all data (a: n = 131),
whereas the second analysis was done using the plants without
ants and the plants for which at least two times ants were found
(b: n = 94 from which ants were found at least twice underneath
13 plants). The False Discovery Rate approach assigned the Pvalue threshold a = 0.039.
Independent variables
Fig. 4. Frequency distribution of the number of eggs laid by
Phengaris alcon females per apical gentian flower bud within
6 days after the first egg was laid.
t-test, t = 0.747, d.f. = 129, P = 0.457) between plants
with and without Myrmica ants. Using GZLM with a
log-linear link function, both plant characteristics and the
presence of Myrmica ants as well as the distance to the
closest plant with ants significantly affected the number of
eggs (Table 2a). The interactions between the plant characteristics and the distance to the closest plant with ants
imply that when the value of a plant characteristic is high,
the predicted number of eggs increases when it is also
growing close to plants with ants. The path analysis
showed that the number of buds had the largest effect
(direct effect size of 0.02), meaning that plants with more
buds would have more eggs (Fig. 1d). Besides, the distance to the closest plant with ants had a negative effect
(a small direct effect size of 0.01), and plant height only
indirectly positively affected the number of eggs (through
the number of buds: a small, indirect effect size of 0.004).
Again, the non-significant effects of plant height and bud
length were due to the high correlation between the plant
characteristics. The spatial autocorrelation of the models
was low (0.062 < Moran’s I < 0.039 for the first 10
lags).
When only selecting the plants without ants and the
plants for which at least two times ants were found (n
= 94 from which ants were found underneath 13 plants),
we found similar effects of the plant characteristics and
ant presence as well as the distance to the closest plant
with ants (Table 2b). The interaction effects should be
interpreted as for Table 2a. The path analysis showed that
again the positive direct effect of plant height was the
most important for explaining the number of eggs,
whereas the presence of ants had a small, direct positive
effect and the distance to the closest plant with ants had a
Coefficient (SE)
Wald
P -value
(a) Models based on all data
Myrmica (=0)
0.127 (0.120)
1.125
0.289
Nr of buds
0.097 (0.017)
30.803 <0.001
Myrmica (=0) 9
0.067 (0.023)
8.622
0.003
Nr of buds
Distance
0.001 (0.003)
0.271
0.603
Nr of buds
0.103 (0.029)
27.779 <0.001
Distance 9 Nr of buds
0001 (0.0005)
6.695
0.010
Myrmica (=0)
0.517 (0.170)
9.288
0.002
Bud length
0.032 (0.006)
11.449
0.001
Myrmica (=0) 9
0.036 (0.008)
19.925 <0.001
Bud length
Distance
0.012 (0.004)
11.478
0.001
Bud length
0.047 (0.008)
35.795 <0.001
Distance 9 Bud length
0.001 (0.0002)
23.454 <0.001
Myrmica (=0)
0.169 (0.068)
6.197
0.013
Plant height
0.002 (0.0006)
7.102
0.008
Distance
0.023 (0.006)
12.640
<.001
Plant height
0.006 (0.001)
27.838 <0.001
Distance x Plant height
0.0001 (2.4 9 10-5)
18.300 <0.001
(b) Models based on data for the plants without ants and the plants
for which at least two times ants were found. Plants where ants have
been found only once are excluded
Myrmica (=0)
0.015 (0.212)
0.514
0.473
Nr of buds
0.113 (0.034)
14.981 <0.001
Myrmica (=0) 9
0.083 (0.037)
5.047
0.025
Nr of buds
Distance
0.004 (0.003)
1.544
0.214
Nr of buds
0.093 (0.024)
15.243 <0.001
Distance 9 Nr of buds
0.001 (0.0006)
5.053
0.025
Myrmica (=0)
0.687 (0.358)
3.675
0.055
Bud length
0.037 (0.013)
5.241
0.022
Myrmica (=0) 9
0.042 (0.014)
8.359
0.004
Bud length
Distance
0.012 (0.005)
6.648
0.010
Bud length
0.033 (0.010)
10.353
0.001
Distance 9 Bud length
0.001 (0.0002)
9.581
0.002
Myrmica (=0)
1.544 (0.543)
8.072
0.004
Plant height
0.008 (0.002)
22.684 <0.001
Myrmica (=0) 9
0.007 (0.002)
11.864
0.001
Plant height
Distance
0.032 (0.008)
17.360 <0.001
Plant height
0.008 (0.002)
33.334 <0.001
Distance 9 plant height 0.0001 (3.0 9 105) 20.675 <0.001
Ó 2014 The Royal Entomological Society, Insect Conservation and Diversity
8
Irma Wynhoff et al.
negative indirect effect (through the ant presence; Fig. 1e).
Again, the level of spatial autocorrelation of the models
was low (-0.081 < Moran’s I < 0.074 for the first 10 lags).
The 282 plants received the first eggs between the 22nd
of July and the 27th of August (Fig. 5). No significant
difference between the date of first oviposition for plants
with (33.1 days SE 0.63, n1 = 170) and without the
presence of Myrmica ants (34.4 days SE 0.49, n2 = 112)
was detected (t-test, t = 1.643, d.f. = 280, P = 0.110).
Using the Cox regression test, we did not find a difference
in time of first oviposition between plants with or without
ants in the proximity, whereas the plant characteristics
did have an effect (Table 3; Fig. 5). Tall plants with many
Fig. 5. Proportion of buds not infected with eggs over time for
Gentiana pneumonanthe plants inside (grey line) and outside
(black line) the home range of Myrmica aloba ants (Day 1 = 1
July), plotted for the mean plant height (21.45 cm). The plot is
based on the Cox regression with presence/absence of Myrmica
aloba ants and plant height as independent variables (Table 3).
Table 3. Results of the effect of Myrmica presence and the plant
characteristics on the timing of depositing the first egg (n = 282)
using a Cox regression model. The coefficient, its standard error,
the Wald statistic and associated P-value are given. A positive
coefficient means that higher values of independent variables is
associated with later oviposition.
Independent variables
Coefficient (SE)
Myrmica (=0)
Nr of buds
Myrmica (=0)
Bud length
Myrmica (=0)
Plant height
0.069
0.112
0.119
0.031
0.109
0.005
(0.124)
(0.022)
(0.123)
(0.008)
(0.123)
(0.001)
Wald
P-value
0.311
25.529
0.933
16.048
0.788
23.859
0.577
<0.001
0.334
<0.001
0.375
<0.001
large buds were associated with shorter oviposition time
(see coefficients of Table 3: a high value of a positive
coefficient implies that the drop in the proportion of notinfected buds is fast). Restricting the data to the plants
without ants and the plants where at least two times the
ants were found, did not change the results of the Cox
regression (data not shown).
Discussion
The female Phengaris alcon butterflies are expected to
make optimal decisions when depositing eggs to support
high survival of their offspring. We found that tall plants
with many tall buds are most likely to be chosen for oviposition (hypothesis 1). This is consistent with earlier
studies (Dolek et al., 1998; K€
uer & Fartmann, 2005;
Nowicki et al., 2005; Arnyas
et al., 2006, 2009; Van Dyck
& Regniers, 2010). Arnyas
et al. (2009) also showed that
females avoid plants infested with aphids while they do
not mind rust. When the caterpillars hatch and bore
themselves into the flower buds, they find a high quantity
of food as resource during their first three larval instars.
Sheltered and hidden from enemies inside the flower bud,
the only competition by conspecifics or other organisms
using the same host plant, such as the micromoth Nemophora violellus, could reduce the probability of reaching
the final larval instar, and subsequently leave the host
plant and spend the winter in a Myrmica nest (Br€
au et al.,
2006; Arnaldo et al., in press). Females are known to
occasionally deposit many eggs on one single host plant,
with a high number of eggs per bud resulting in competition which negatively affects the condition of the caterpillars (Br€
au et al., 2006). The chance of being successfully
adopted by worker ants and surviving until leaving the
ants’ nest as an adult is dependent on the caterpillar’s
body condition (Nash et al., 2011).
An effect of the presence of Myrmica ants close to
plants on the probability of finding eggs of P. alcon was
found only for the plants for which we were sure that ants
are in the proximity, although the effect was weak
(hypothesis 1). Our results show that females in search of
an oviposition site were mainly attracted by the size of
the plants (plant height is the most explaining variable in
the path analyses). Indeed, Van Dyck and Regniers (2010)
suggested that tall plants represent a visually attractive
target to the female. Moreover, tall host plants might provide high quantities of food for the caterpillars as they
have many large buds. Even though the amount of food
consumed in the period of time needed to reach the L4
instar is undoubtedly small, effects of competition are well
known. The survival rate of the caterpillars is mostly
between 40% and 60%, depending on the length of the
bud and the phenological stage (Br€
au et al., 2006;
Arnaldo et al., in press). The question remains how it is
possible that female butterflies do not respond to ant
presence or absence at a certain host plant individual, but
then have a higher likelihood to lay eggs on plants if ants
Ó 2014 The Royal Entomological Society, Insect Conservation and Diversity
Oviposition in Phengaris alcon
are nearby on neighbouring plants although the effect size
is small given the relatively low standardised coefficient.
Our finding that more eggs are found on tall plants
with many large buds is consistent with several other
studies that showed that P. alcon would only select for
the developmental stage of the flower (bud) for oviposition (K€
uer & Fartmann, 2005; Nowicki et al., 2005; F€
urst
& Nash, 2010). In contrast, we found that more eggs were
laid on plants that had Myrmica ants present or were
growing close to plants with Myrmica than on plants
where the ants were absent, which indicates that the presence of ants influences the egg load of P. alcon. This is
consistent with previous research of Patricelli et al. (2011)
and Wynhoff et al. (2008), who also found more eggs
being deposited by females of P. arion, P. teleius and
P. nausithous where ants were present. We found an effect
of Myrmica ants on egg load, even though egg load was
limited to only 6 days due to our experimental design.
While females of P. arion spread their eggs over plants in
the home ranges of all present Myrmica species, P. teleius
and P. nausithous preferred host plants within the home
ranges of their specific host ants, M. scabrinodis or M. rubra respectively. It is often assumed that an effect of ant
presence on oviposition can be expected when the host
plant is situated in the home range of an ants’ nest. In the
case of Myrmica ants, workers move up to a distance of
about 2 m from the nest site to collect food. Given the
host plant density and the spatial distribution of host
plants with ants on the baits, all host plants are expected
to grow within the home ranges of the host ants’ nests at
the study site. However, only for 165 out of 447 plants
ants were found and, moreover, only for 40 out of 165
plants ants were captured more than once. If females
would be able to detect ants based on the assumption that
ant cues are present in the whole home range of their
nest, no effect of ants would be expected. However, we
found the effects of ant presence on the presence and
amount of eggs the females deposit per bud. Apparently,
when selecting a bud for oviposition, visual cues (likely
plant height) are most important and decisive, as was
observed by Van Dyck and Regniers (2010). Only after
the plants with low probability of ants’ nests nearby were
removed from the analysis, effects were found. Later,
probably after alighting or only at a very close distance to
host plants, chemical cues maybe involved as well, resulting in longer oviposition bouts under favourable conditions. The distances involved are much shorter than the
home range size of the Myrmica ants. While an effect of
ants on egg load could be shown, the nature of this effect
is unknown. A direct effect is possible; in this case, it is
expected to be caused by the ant colony itself rather than
markings of the worker ants in the home ranges, because
distance to the nearest neighbours with ants are rather
short. It is also possible that a plant-derived cue affects
the behaviour of the females. Gentian plants growing
close to Myrmica ant nests could react to this presence by
emitting certain herbivore-induced plant volatiles (Dicke
et al., 2009) which are detectable by the females. Root lice
9
which are often visited and tended by Myrmica ants might
induce such a chemical defense reaction.
In our study, no clear indication was found that oviposition on plants without ant nests changed over time
(hypothesis 2). Van Dyck et al. (2000) did find that the
egg load for gentians without the ants present increased
over time. We expected a similar change in oviposition,
because at the beginning of the flight period of P. alcon,
only green buds were available and butterflies would have
no choice but to oviposit on them, leaving only the presence or absence of ants as a valuable decision for the
females to make. Later in the season, all possible phenological stages of buds are present, giving females more
choice to ensure the brood has enough food to develop
into the fourth instar (Nowicki et al., 2005). When P. alcon would select plants where ants are present early in the
season, egg density build-up could be a reason that butterflies would later choose plants further away from ants
to avoid competition inside the flower buds. However, no
temporal change in oviposition preference was found. It
could mean that in our case there was no densitydependent shift and butterflies did not need to oviposit
more on plants where ants were absent, which is
consistent with the study of Wynhoff et al. (2008) where a
density-dependent shift only occurred in years with high
butterfly densities. In our study year, the butterfly density
seemed to be much lower than in the years before.
Van Dyck et al. (2000) found a density-dependent shift,
but they considered a gentian in the home range of the
ant nests when a nest was found within a 3 m radius
around each gentian. We captured ants using sugar cubes
as bait, and used the capturing of ants next to the plant
as an indication of ant presence or the distance to the
closest plant with ants was used. These differences in
interpreting ant presence could account for differences in
results since an assumed radius is not necessarily the ants’
actual range or covers the spatial dimension in which cues
are expected to occur. Moreover, an effect of ants was
found to be stronger when the plants where ants have
been found only once were removed from the analysis,
showing that repeatedly capturing ants is of importance
too. Our study shows that testing the ant-mediated oviposition hypothesis requires baiting ants more than once.
When reducing the spatial scale by taking the distance to
the closest plant with ants into account, effects were even
stronger and more consistent. In addition, at our study
site, the density of gentians of 7.2 plants per m2 is much
higher than reported from the other sites in Europe,
where it is usually below 0.5 plants per m2 (Maes et al.,
2004; WallisdeVries, 2004; K€
uer & Fartmann, 2005; Nowicki et al., 2005; Br€
au et al., 2006; Van Dyck & Regniers,
2010; Czekes et al., 2013). Fewer suitable plants in the
area for ovipositing females might have caused the density-dependent shift in oviposition which was not seen in
our study site.
When caterpillars are dependent on being found by
worker ants which are active only within a limited distance from their colony, their adoption chance is related
Ó 2014 The Royal Entomological Society, Insect Conservation and Diversity
10
Irma Wynhoff et al.
to the distance between the host plant and the closest ant
nest. Given that P. alcon in the majority of its geographical range encounters low gentian density, selection for
ant-mediated oviposition is less likely to occur in areas
with relatively low plant density than in areas with high
plant density where many more plants might be sinks for
the caterpillars, assuming that ant nest density is similar
in these areas (which is often not measured). In populations with high host plant densities, the probability of a
caterpillar to survive until the L4 larval instar might be
high. However, later many caterpillars will be taken into
a limited number of ant nests. Once adopted and taken
into the nest, caterpillars experience contest competition
with high mortality rates in the first weeks in the nest
(Thomas et al., 1993).
If host plant density is low and host ant nest densities
high, selecting oviposition sites only in response to plant
characteristics without considering the ants still yields a
high probability that caterpillars will be found by worker
ants. If so, then this could explain the variation in antmediated oviposition in P. alcon from undetectable to
clearly present, whereas it supports ant-mediated oviposition in P. teleius and P. nausithous (Wynhoff et al., 2008)
and in closed populations of M. arion (Patricelli et al.,
2011) that encounter high host plant densities. Given the
very high density of gentians with only half of them found
by Myrmica aloba ants, ant-mediated oviposition was
found in the investigated population, especially for plants
where ants were frequently found. With these findings,
this study contributes to the discussion about ant-mediated oviposition versus random oviposition because we
found that oviposition choice of P. alcon is determined by
both plant characteristics and ants but in different ways.
Acknowledgements
The research was supported in part by CITAB and funding from the Portuguese Government through the ON.2/
QREN PROGRAMM and the project PROTEGER E
CONHECER. We are also thankful for Peter Boer who
shared his knowledge on ant identification to determine
the species we found in the field. Lastly many thanks to
Maria da Conceicß~ao Rodrigues for her assistance in the
field. Finally, we are most grateful to three anonymous
reviewers for many comments that significantly improved
the paper.
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Accepted 10 October 2014
Editor: Karsten Schonrogge
Associate editor: Francis Gilbert
Ó 2014 The Royal Entomological Society, Insect Conservation and Diversity
Irma Wynhoff