Egg distribution of the Southern Festoon (Zerynthia polyxena) (Lepidoptera, Papilionidae)

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P. BATÁRY (1,2), N. ÖRVÖSSY (1), Á. KŐRÖSI (3) and L. PEREGOVITS (1), 2008. In Acta Zoologica Academiae Scientiarum Hungaricae, 54 (4) : 401-410.

• 1 Department of Zoology, Hungarian Natural History Museum H-1088 Budapest, Baross u. 13, Hungary
• 2 Current address: Agroecology, Georg-August University Waldweg 26, D-37073 Göttingen, Germany
• 3 Animal Ecology Research Group of the Hungarian Academy of Sciences and the Hungarian Natural History Museum, H-1083 Budapest, Ludovika tér 2, Hungary

SUM-UP

We investigated environmental factors groupe da long hierarchical spatial scales influencing the egg density of a monophagous papilionid, the southern festoon (Zerynthia polyxena) on its food-plant, the birth wort (Aristolochia clematitis). Two patch level variables were considered : habitat type (black locust plantation, clearing and hummock) and food-plant patchsize. We
measured several plant variables at the egg-bearing shoots. We counted the number and measured the mean heigh to birth worts within the micro-environment of the egg-bearing shoots. We also measured the height and counted the leaves of each egg-bearing shoot itself. Plant apparency was defined as the height difference between egg-bearing shoots and the surround-ingones. Twovariablesweremeasuredontheegg-bearingshoots(henceatfood-plantscale): number of leaves and position of eggs on the leaf-storeys. Habitat type affected the distribution of eggs ; black locust plantations and hummocks were preferred against clearings. At a smaller scale, neither shoot density, nor food-plant apparency affected egg distribution. At the plant level, the number of eggs increased with the number of leaves, and the position of eggs also co-varied with egg density, having fewer egg sinclusters positioned higheron shoots. We conclude that spatially correlated data on butterfly egg distribution should be analysed considering the intrinsically hierarchical structure of environmental factors.

Keywords

Zerynthia polyxena, oviposition, plant–insect interaction, Aristolochia clematitis, food-plant, spatial scale

INTRODUCTION

Egg-laying is a particularly important ecological interaction between phytophagous
insects and their food-plants (RABASA et al. 2005), therefore, oviposition
preference and larval performance are central topics in insect–plant biology (XUE
et al. 2007). Female imagoes can discriminate among sites based on characteristics
like climatic regimes, food quality and potential levels of competition and predation
(BERNARDO 1996). Variation in these aspects of offspring environments affects
offspring performance as well. In case of butterflies, egg-laying females may
exhibit biased preferences toward particular plant species, toward particular plant
individuals and even toward certain parts of the food-plant, which will determine
the physical and chemical attributes to which insects respond (THOMPSON &
PELLMYR 1991, BERNAYS&CHAPMAN 1994). Since the emerging caterpillars are
relatively immobile, the key to their survival and development is the food-plant
choice of the female (PORTER 1992).
Beside experimental studies (e.g. SINGER et al. 1993), there are two widespread
methods to study the egg-laying preferences of female butterflies. One of
them is based on tracking the female imagoes and direct observation of oviposition
(e.g. GRUNDEL et al. 1998, BERGMAN 1999, ZIMMERMANN et al. 2005, KŐRÖSI et
al. 2008). The other method takes the presence-absence, or the distribution of observable
eggs on food-plants (e.g. FLOATER&ZALUCKI 2000, ELLIS 2003, RABASA
et al. 2005). This indirect method is more flexible allowing the researcher to
plan the sampling design and to have a much larger sample size (depending on the
visibility and identification ability of eggs). However, the evident disadvantage of
the latter method compared to the first one is that it takes only the known hostplants
into account and leaves out of consideration the acceptable non-host plant
species (DE BOER & HANSON 1984).
Several studies have investigated the factors influencing egg distribution at
different spatial scales. MCKAY (1991) studied the egg-laying requirements of
Brimstone butterfly (Gonepteryx rhamni) in wet woodlands considering different
food-plant–related spatial, physical and chemical factors, and found that most eggs
were laid on juvenile host-plant trees growing in sunny sites. Further, butterflies
appeared to prefer host-trees with low concentration of secondary compounds.
Similarly, FLOATER and ZALUCKI (2000) explored the host-tree quality and
apparency preferences of an Australian moth, Ochrogaster lunifer. They found
more egg batches on high-quality trees in open homogeneous habitats, while in diverse
mixed-species habitats, more egg batches were laid on low-quality highly
apparent trees. In the case of Northern BrownArgus

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butterfly’s (Aricia artaxerxes)
oviposition, ELLIS (2003) described the effects of different food-plant characteristics,
microenvironmental factors, such as food-plant versus bare ground cover, and
sward height. The eggs of this butterfly were more common on younger and larger
food-plant leaves and on unmanaged vs managed sites (shorter vegetation), while
food-plant density and bare-ground cover did not affect oviposition. Furthermore,
FARTMANN (2006) studied the effects of food-plant, microenvironment and microclimate
on the egg distribution of the Duke of Burgundy Fritillary (Hamearis lucina).
He found a lower deposition height of eggs on the host-plant (Primula veris)
and that majority of egg clutches situated at sites that receive direct insolation between
09:00–17:00, and which have more than 60% herb cover. DENNIS (1996)
studied the oviposition in Zerynthia cretica in relation to food-plant leaves, shoots and patches. He found that females laid more eggs on large plant patches with large
leaves, typically at the plant patch margin.
We found only two studies that focused on monophagous butterflies and
used the presence-absence data of butterfly eggs in relation to factors at different
spatial scales in hierarchical nested models. KÉRY et al. (2001) studied the presence
of Maculinea rebeli eggs across Gentiana cruciata fruits, genets and populations,
and found that the factors measured at the genet level were more important
than those measured at population level. RABASA et al. (2005) used a similar nested
design to investigate the egg presence of Iolana iolas on the Colutea hispanica
shrubs at fruit, plant and patch levels. They showed important factors influencing
the egg presence in each level.
In the present study we aimed to investigate hierarchically structured environmental
factors influencing the density and distribution of Z. polyxena eggs in a
black locust – poplar plantation complex, where the food-plant of this butterfly
was abundant.
MATERIALS AND METHODS
Study species
The southern festoon (Zerynthia polyxena DENIS et SCHIFFERMÜLLER, 1775) is a papilionid
species that reaches its northern range in Central Europe (TOLMAN 1997). The species is protected by
law in Hungary. It is a monophagous species in Hungary feeding on a herbaceous plant, the birthwort
(Aristolochia clematitis LINNEAUS, 1753, Aristolochiaceae). This food-plant is common on disturbed
habitats, like flood plains, orchards, roadsides or black locust (Robinia pseudoacacia LINNEAUS,
1753, Fabaceae) and hybrid poplar (Populus × euramericana, Salicaceae) plantations. The flight period
of the butterfly starts from mid/end April and lasts until the mid/end of May. The females oviposit
on the abaxial surface of the food-plant leaves, laying either a single egg, or a smaller or larger cluster
of eggs. Contrary to Zerynthia rumina LINNEAUS, 1758 which has two food-plant species (Aristolochia
baetica LINNEAUS, 1753, Aristolochiaceae and Aristolochia longa LINNEAUS, 1753, Aristolochiaceae),
the Z. polyxena is monophagous at least in Hungary; both eggs and larvae were observed
only on A. clematitis (ROTHSCHILD et al. 1972, JORDANO&GOMARIZ 1994,ÖRVÖSSY et al. unpubl.)
Study area and sampling design
The study area was situated on the Hungarian Great Plain near Csévharaszt (Central Hungary,
47°18’N, 19°26’E), in a landscape comprising of tree plantations, mainly black locust and poplar
cultivar plantations interrupted by clearings. The 0.02–0.03 km2 large plots were separated by hummocks
originating from the last harvest of the plantations and consisted of stumps and roots covered
by soil, providing an ideal place for the birthwort. A clump of food-plants consisting of at least five
shoots per m2 and separated by at least 10 m from other food-plants was considered as a patch. We
chose 4–4 food-plant patches for sampling in the three available habitat types, i.e. in black locust plantations, in clearings and in hummocks. In our earlier study on the same study area, we found that
the imagoes avoided the poplar plantations (ÖRVÖSSY et al. 2005). Each of these food-plant patches
were covered by several thousands of birthwort shoots. We randomly selected 10 points within each
patch, where we checked food-plant shoots for eggs in a 5-meter radius circle. Since relatively few
food-plant shoots had leaves loaded by eggs, we stopped further searching after finding the first shoot
with eggs. These circles were covered by an average of 775 food-plant shoots. From the total of 120
randomly chosen circles, 98 contained eggs, and circles without eggs were excluded from the analy-
ses. The flight period (26 April to 15 May) ended before the egg searching (17–23 May), thus new
ovipositions during the sampling period could not cause a bias. No larvae were found during the egg
survey.
During the egg-survey several environmental variables were measured in the close proximity
of the egg-bearing shoots. We grouped these variables according to spatial scales. The habitat type of
a given food-plant shoot (black locust plantation, clearing or hummock) was interpreted as a patch
level variable, and incorporated into the model as a factor. To characterise the habitat types we
counted the food-plant shoot density on 20 randomly selected 2×2 m squares in each food-plant
patches. We measured the size of patches. To characterise the microenvironment of the selected
food-plant shoots, we counted the number of shoots and measured the mean shoot height in a 1×1 m
square. We also measured the height and counted the leaves of egg-bearing shoots. We expressed
food-plant apparency as the height difference between an egg-bearing shoot and the mean of sur-
rounding shoots in the 1×1 m square. On the egg-bearing shoots two variables were measured; the
number of food-plant leaves and the position of the egg or the egg cluster (hence we call the scale of
these variables as food-plant level to differentiate from the scale of variables measured in the direct
proximity of the egg-bearing shoots, i.e. microenvironment level). The latter one was expressed as
the number of leaves below the leaf bearing the egg(s) divided by the total number of leaves.
Statistical analysis
The effects of above mentioned environmental variables on egg distribution were analysed in
general linear mixed-effects models with the Restricted Maximum Likelihood method. The normal-
ity of the distribution of eggs per leaf was assessed using normal quantile plots. Log-transformation
was applied to handle non-normal distribution. The following non-correlated variables were consid-
ered in the statistical model: 1) habitat type as a factor and patch size as a co-variable at patch level; 2)
at the microenvironment level, the food-plant shoot density around the egg-bearing shoot and
apparency; 3) the number of food-plant leaves and the position of eggs at food-plant level. Since
food-plants were nested within food-plant patches, the latter was used as a random factor. Although
food-plant leaves were also nested within food-plants, we could not include food-plant as a random
factor in the model, because it would have had too large effect relative to the residual. Altogether
there were only five food-plant shoots with eggs on two leaves and one food-plant shoot with eggs on
three leaves. Leaves without eggs (1036) were excluded from the analysis. Furthermore, we com-
pared models with and without the inclusion of food-plant as a random factor. We found that the two
models were significantly different (L-ratio = 59.8; P < 0.0001), and the model without food-plant
had smaller AIC value, indicating that this model was more supported. By right of these, we decided
to apply the latter model. The calculations were made using R (version 2.2.1; R DEVELOPMENT CORE
TEAM 2006) and the nlme package for R (version 3.1, PINHEIRO et al. 2007).
404 BATÁRY, P., ÖRVÖSSY, N., KŐRÖSI, Á. & PEREGOVITS, L.
Acta zool. hung. 54, 2008
RESULTS
We registered 597 eggs of Z. polyxena laid either singly, or in small (2–8
eggs) or large (10–99 eggs) clusters on the underside of food-plant leaves. During
the survey we did not find any larvae nor hatched eggshells. The habitat type sig-
nificantly affected the distribution of eggs (Table 1). When we compared the
food-plant shoot density between habitat types with a one-way ANOVA, we found
significant difference between them (F = 23.207, P < 0.001, N = 240, Fig. 1, Tukey
HSD post-hoc tests showed that all habitat types differed significantly from each
other regarding food-plant shoot density). There were significantly more eggs on
food-plants in black locust plantations and hummocks than in clearings (Fig. 2). At the microenvironment level, neither food-plant shoot density, nor food-plant apparency affected the egg distribution (Table 1). At the food-plant level, the number of
eggs increased significantly with the number of food-plant leaves/shoot (Table 1).
Finally, the position of eggs also affected significantly the number of eggs; there
were more eggs on the lower part of the food-plants than upward (Fig. 3).

Table 1. Linear mixed models for testing the effects determining the egg-laying preference of Z. polyxena at different levels. Bold p values indicate significant effects.

Fig. 1. Mean (±SE) number of food-plant shoots per m2 in the three habitat types. The different letters indicate significant differences at P < 0.05

Fig. 2. Mean (±SE) number of eggs of Z. polyxena per food-plant leaf in the three habitat types. The different letters indicate significant differences at P < 0.05

Fig. 3. Egg position on the plant’s leaf-storeys (the number of leaves below the leaf bearing the egg(s)
divided by the total number of leaves) is plotted against log number of eggs. Smaller value of egg position
indicates that the eggs are on lower leaves. The solid line represents the fix effect of egg position
of the fitted model

DISCUSSION
The behaviour leading to oviposition, i.e. selection of oviposition site, is a
complex process, because successful oviposition greatly contributes to individual
fitness (SCHOWALTER 2006). However, females do not always select the most appropriate
host and newly hatched larvae may reject the plant on which they hatch
(BERNAYS&CHAPMAN 1994). So egg-laying females can make errors and larvae
can correct it to some degree, but if they are to survive, i.e. to maximise their fitness,
the females’ preference and the larvae’s performance should overlap as much
as possible. In the current study, the egg distribution of Z. polyxena was affected on
two levels, basically by habitat type and also by characteristics at food-plant level,
but not by factors at the microenvironment level.
The habitat differences found in the present study, i.e. the black locust plantation
and hummocks were more preferred than clearings, may well be interpreted as
the butterflies’ preference against areas that were more exposed to direct sunshine
or cold. FARTMANN (2006) reported that H. lucina preferred those sites for oviposition
in calcareous grasslands, where the food-plant (Primula veris) potentially
received direct insolation between 09:00 and 17:00. MCKAY (1991) found that
most eggs of G. rhamni were laid on isolated juvenile trees (Frangula alnus) growing
at sunny sites. These studies show that oviposition site preferences may ensure
an optimal microclimate for the development of the larvae.
In our case, patch size did not affect the number of eggs laid, while in the case
of Z. cretica DENNIS (1996) showed that larger food-plant patches had more eggs.
However, we have to call the attention to the fact that in the latter study the patches
were mapped on a much finer scale (food-plant patch size range: 25 to 2400 cm2)
than in our case (food-plant patch size range: 661 to 11954 m2, mean: 3384 m2).
At the microenvironment level, neither food-plant shoot density, nor foodplant
apparency seemed to act as a limiting factor on egg density. However, we
have to mention that food-plant shoots occurred in a very large number in each
habitat. Moreover, food-plant shoot density was highest in the clearings, generally
two times higher than in the black locust plantation and about 15% higher than in
the hummocks (Fig. 1). This also suggests that the habitat type effect is not connected
with the food-plant availability. In contrast to our results, DENNIS (1996) found in his fine scale study that food-plant shoot number was one of the most important
factors affecting the egg density of Z. cretica. Regarding food-plant apparency,
the height of egg-bearing shoots were significantly higher than those in
the microenviroment (paired t-test, t = 8.956, P < 0.001), however, it probably influences
only the food-plant selection of butterflies, but not the egg load.
The number of food-plant shoot leaves showed a positive effect on egg numbers
and more eggs were found on the lower leaves of food-plant shoots. In our earlier
study, we observed that at the beginning of flight period the food-plants were
quite scarce and just started to develop, therefore, the first females had limited choices
(ÖRVÖSSY et al. 2005). This probably means that early sprouting food-plants have
some advantage compared to the late sprouting ones, therefore, they could be more
apparent. Since food-plant shoots receive eggs at a younger stage, the eggs are situated
on lower leaves. Moreover, at this period the females have large egg load,
which could also cause that they lay eggs in clusters rather than single eggs. This
result suggests that temporal aspects are also important in oviposition. DENNIS
(1996) also emphasized the importance of the duration of food-plant patches; he
suspected that food-plant patches available for longer periods are more likely to
have more larvae. Investigating a congeneric species, Z. rumina, JORDANO &
GOMARIZ (1994) found that the freshly hatched larvae consumed the younger and
softer leaves of the food-plants. This could stand behind that the observed Z.
polyxena females also laid the eggs on young leaves, however, with egg searching
it was not possible to investigate the temporal effects. Another potential explanation
for the young leaf selection could be that females select these because of lower
concentrations of some defence chemicals. However, ROTHSCHILD et al. (1972)
described that Z. polyxena contained and stored efficiently two aristolochic acids
which were presumably present in its food-plant, in A. clematitis.
In this short study we showed that the egg distribution of Z. polyxena was affected
by several components acting at different spatial levels. We have to
emphasise that such a complex process, like oviposition, should also be investigated
by the direct tracking of females, which could illuminate further aspects of
egg distribution. As a conclusion, we also underline that spatially correlated data in
egg distribution studies need to be analysed considering an intrinsically hierarchical
structure of environmental factors (RABASA et al. 2005).
*
Acknowledgements – Two anonymous referees provided constructive remarks that considerably
improved the manuscript.We are indebted to LAJOS RÓZSA for valuable comments and linguistic
revisions on the manuscript. The study was supported by the National R&D Programme, entitled
“The origin and genezis of the fauna of the Carpathian Basin: diversity, biogeographical hotspots and
nature conservation significance” (contract no. 3B023–04).

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Revised version received December 14, 2007, accepted March 3, 2008, published November 21, 2008

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