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ORIGINAL PAPER
Mobility and oviposition site-selection in Zerynthia cassandra
(Lepidoptera, Papilionidae): implications for its conservation
Alessio Vovlas •Emilio Balletto •Enrico Altini •
Daniela Clemente •Simona Bonelli
Received: 7 February 2014 / Accepted: 22 June 2014
ÓSpringer International Publishing Switzerland 2014
Abstract The adults’ mobility and oviposition prefer-
ences of Zerynthia cassandra have been studied for the first
time, with the aim of integrating auto-ecological informa-
tion into recommendations for the habitat’s management of
this species. Results of our mark-release-recapture study
have highlighted that Z. cassandra is a strictly sedentary
species, since detected movements only occurred over very
short distances (B200 m) and mainly within the species’
reproductive habitat (i.e. around Aristolochia rotunda
stands), with males moving further than females. Our study
shows that the main oviposition habitat of Z. cassandra is
found where A. rotunda plants are growing in large stands;
sites where plants growing in half to full sun and mostly
oriented to the south are preferred. The distance of
deposited eggs from the plants’ roots was narrowly corre-
lated with the plants’ length. Eggs were deposited singly,
mainly on the underside of leaflets. Management strategies
necessary for improving the most important habitat fea-
tures for the conservation of this species are suggested.
Keywords Butterfly conservation Zerynthia cassandra
Oviposition Aristolochia rotunda
Introduction
Causes for the widespread decline of many European
butterfly species are primarily recognised in habitat deg-
radation and loss (New 1997; Fox 2012; Maes and Van
Dyck 2001; van Swaay et al. 2010). At least in principle,
however, any perturbation of the environment can nega-
tively affect species’ survival and may be at the core of
many extinction processes. As Samways (2007) suggests,
strategies for insect conservation must be planned at
regional scale, to reduce locally negative impacts. Steno-
topic and univoltine butterfly species are particularly
threatened worldwide by habitat destruction and climate
change, most particularly at the edges of their range (Hoyle
and James 2005; Bonelli et al. 2011). Habitat changes have
even stronger negative effects on species with low dis-
persal ability, including many terrestrial invertebrates
(Thomas et al. 2004) such as the Papilionid species of the
genus Zerynthia.
Zerynthia polyxena and Zerynthia cassandra are among
the potentially most vulnerable butterflies in the Mediter-
ranean area. Z. polyxena is a strictly European species,
ranging from southern France to the Urals, Italy and the
Balkans (Kudrna et al. 2011). In Italy, however, two sep-
arate species have been demonstrated to exist. Their spe-
cies-level separation was initially proposed by Dapporto
(2010) on the basis of genitalic characters and more
recently confirmed by genetic data (Zinetti et al. 2013). In
Italy, Z. polyxena occurs in the North of the Country,
mainly in the northern plains of the Po river valley and the
surrounding foothills of the Alps. The other species, known
A. Vovlas E. Balletto S. Bonelli (&)
Zoology Unit, Department of Life Sciences and Systems
Biology, University of Turin, Via Accademia Albertina 13,
10123 Turin, Italy
e-mail: simona.bonelli@unito.it
A. Vovlas
e-mail: alessio.vovlas@unito.it
E. Balletto
e-mail: emilio.balletto@unito.it
A. Vovlas E. Altini D. Clemente
A.P.S. Polyxena, Via Donizetti 12, 70014 Conversano, Bari,
Italy
e-mail: enricoaltini@yahoo.it
D. Clemente
e-mail: dadaclemente@yahoo.it; info@polyxena.eu
123
J Insect Conserv
DOI 10.1007/s10841-014-9662-4
as Z. cassandra, occupies most of the Peninsula, starting
from the northern Tyrrhenian divide and as far south as
Calabria, as well as in Sicily. Since they are almost
indistinguishable in external morphology, the two species
remained lumped under Z. polyxena for a long time. Since
Z. cassandra was separated by Z. polyxena (a EU Habitats
Directive species) responsibility for the conservation of
this species has become a matter of particular importance
for Italy, although not yet for the European Union (Maes
et al. 2013).
However, characteristics of the life history and ecology
of these species are not well understood and they can
probably differ in such characters as habitat selection,
oviposition behaviour and dispersal ability. As concerns
ecological characteristics, adults of Z. polyxena require a
sub-nemoral habitat and only spend a relatively short part
of the day in open herbaceous areas. Nothing is known, at
the moment, as concerns Z. cassandra.
The present paper was designed to gain information on
some population traits of Z. cassandra deemed particularly
useful for planning the conservation of this endemic spe-
cies. More in detail, our objectives were: (1) to obtain data
on adult mobility, by investigating whether Z. cassandra
adults moved all through the landscape matrix, (2) to
investigate the habitat factors and larval food-plant char-
acteristics positively influencing the choice of Z. cassandra
females in oviposition-site selection and (3) to consider
implications of results from this analysis for the conser-
vation of the species. Gaining this information will be a
first step for developing a specific action plan for the
conservation of Z. cassandra and will provide useful
guidelines for the management of its habitat.
Materials and methods
The study species
Similarly to Z. polyxena,Z. cassandra is single brooded
and the flight period of adults spans from late February, in
Sicily, to the beginning of June, depending on altitude and
latitude (Verity 1947; our data), with hibernation diapause
in the pupal stage. For an adult female, total fecundity is
about 50/60 eggs (personal observations). During the flight
period, which lasts around 15 days, females lay on the
Aristolochia leaves. In Italy, larvae generally feed on A.
rotunda or A. pallida, which always grow in small scat-
tered stands within semi-natural ecotonal grasslands,
between (0) 300 and 900 m.
At least 15 populations of Z. cassandra are known for
having become extinct in Italy during the past 50 years
(Bonelli et al. 2011), generally as a consequence of habitat
loss and/or demographic stochasticity, since this species
typically occurs only at low-densities. Populations of all
Zerynthia species are restricted to micro-habitats where
their larval food plants (Aristolochia spp.) grow, and their
restricted, spots-like distribution, is probably related to host
plants’ distribution, even though Zerynthia populations are
generally much rarer than those of their food-plants. Adults
of Z. polyxena do not move over great distances and sel-
dom fly far from their reproductive areas in search of
suitable host plants. In a population from Slovenia, the
maximum recorded flight distance was 400 m (C¸ elik
2012).
The study sites
Two disjunct areas were used in Italy as study sites for the
work presented here (Fig. 1). The ‘‘Capanne di Marcarolo’’
Regional Park (SCI IT 1180026) and the ‘‘Laghi di Con-
versano e Gravina di Monsignore’’ Regional Park (SCI IT
9120006). The two sites were chosen for their position at
the extremity of the distribution range of the species, as
well as because of logistic reasons. These two areas contain
large and persistent populations of Z. cassandra.We
investigated egg-laying behaviour at both sites, but we
carried out also mark-release-recapture only at ‘‘Laghi di
Conversano’’.
The ‘‘Laghi di Conversano e Gravina di Monsignore’’
Regional Park is in Apulia, in the southeast of the Italian
peninsula. The Natural Park area consists of a set of ten
karst ponds (dolines) located in a fragmented agricultural
matrix (Altini et al. 2007). Populations of Z. cassandra are
here under threat from habitat loss by anthropogenic dis-
turbance and spread of vineyards. We analysed the egg-
laying behaviour and the adults’ movement of Z. cassandra
in one of these dolines, i.e. the ‘‘Chienna lake’’
(48°5803700N, 17°0402100E), which is surrounded by orch-
ards and vineyards. For the mobility study, we divided the
site into 7 arbitrary plots from 0.68 to 3.32 ha in size.
Distances between occupied plots ranged from 110 to
477 m.
The oviposition study was conducted at 3 sites (a) Site 1:
a grassland area of 96 m
2
located in plot C, near a Medi-
terranean pond and with a 25 % canopy cover (40°
5801700N, 17°0402400E). (b) Site 2: a 170 m
2
area within plot
D (40°6801700N, 17°0403000E) with less than 10 % canopy
cover. (c) Site 3: an area of 78 m
2
in plot G, with 10 %
canopy cover (40°5804100N, 17°0404700E).
The Regional Park of ‘‘Capanne di Marcarolo’’ is
located in the North-west of Italy. Our field study was
conducted at three sites, where the butterfly apparently
reached its locally highest densities (a) Site 1: Woodland
edge. (44°3602200N, 8°4600100E): consisting of a 128 m
2
mesophilous meadow bordered by a Black locust (Robinia
pseudoacacia) grove and with 10 % canopy cover. (b) Site
J Insect Conserv
123
2: Riparian grassland (44°3602100N, 8°4505800E): having a
50 m
2
grassland with sparse alder (Alnus glutinosa) shrub
and hygrophilous vegetation Canopy cover was 25 %;
(c) Site 3: Dry grassland (44°380N, 8°510E): a 170 m
2
semi-
natural grassland with 5 % canopy cover.
Egg-laying studies
We thoroughly investigated our sites to locate egg-laying
areas and to record details of food-plant’s characteristics,
eggs’ location and surrounding vegetation/habitat. In April/
May 2010, we exhaustively assessed the distribution of
Aristolochia plants and Zerynthia eggs at ‘‘Capanne di
Marcarolo’’ and in April 2013 at ‘‘Laghi di Conversano’’.
Z. cassandra occurred in small and often isolated popula-
tions, so that individually following females and observing
their egg-laying behaviour was not the appropriate field
method, in this case. To avoid any bias towards expected
habitat characteristics, we used intensive search method.
Some adults were still flying during the survey period, but
the season was nearing its end, so that we conducted egg
census shortly after the butterflies had completed
oviposition.
Observations were aimed at determining eggs’ distri-
bution on each host-plant. Z. cassandra eggs are not
morphologically different from those of Z. polyxena, but
since they are characteristic in shape, size and colour they
can be unequivocally identified even when hatched, since
egg shells remain on the leaves for at least 2 weeks. To
avoid recounting the same plant twice during the survey,
we marked each plant with a flag-bearing stick.
The characteristics of Z. cassandra oviposition habitat
were recorded at the landscape, patch and plant level. At
landscape level, the numbers of eggs and of A. rotunda
plants were assessed at all study sites. At patch level, the
geographical orientation (the direction the slope faces with
respect to the sun, or ‘‘aspect’’), number of plants (1 plant,
small stand with 2–5 plants, large stand with[5 plants) and
sun exposure (exposure to sunlight reaching the spot during
a sunny day) were collected.
Fig. 1 Location of our study
sites. Different habitat types are
represented with dots: white for
woodland edge, black for
riparian grassland, grey for dry
grassland
J Insect Conserv
123
At plant level, habitat parameters were recorded in
191 m sample quadrats having the plant in the centre.
Measurements of plant height, height of the surrounding
vegetation, vegetation coverage (estimated in 5 % units)
and distance from the nearest tree were recorded in each
quadrat. Finally, the number of eggs observed on each
plant, the height of each egg above the ground and its
position on the plant were registered. For data analysis we
calculated the difference between the egg’s position in
height and the average vegetation height, as a proxy for the
‘‘prominence’’ of the host plant. Positive values show
positive prominence of the eggs-bearing plants. These
parameters were then compared between occupied and
unoccupied host plants.
Butterfly mobility
From 10 to 20 April 2013, a mark-release-recapture study
(MRR) was conducted at the ‘‘Laghi di Conversano’’
Regional Park (40°580N, 17°040E, see Fig. 2) within the
peak flight periods of Z. cassandra adults. Since the pop-
ulation occurring at this general area is patchily distributed
and our target species occurred at a number of patches, we
conducted a preliminary study (Altini and Tarasco 2011),
to assess which patch contained the highest population
density. All the areas were walked during weather condi-
tions suitable for adults’ flight, and three to six people
participated in marking and capturing, summing up to 50
person-days in total. Occasional visits were also made to
apparently unsuitable areas, to check for the occurrence of
adults that might be moving outside their usual habitats. An
attempt was made to capture every observed individual and
since adults of Z. cassandra are not difficult to net, most
sightings resulted in captures. On each day we carried out
three capture sessions, each 30 min long, between 10:00
and 14:00.
On each survey event, butterflies were captured, indi-
vidually marked and immediately released. Larval food
plants density was estimated by counting the number of A.
rotunda plants per plot. Correlation between butterfly
abundance and food plant density was determined at each
plot. For each capture or recapture, the location (using a
hand-held GPS device, min. accuracy 3 m), date, time,
individual’s number and sex were recorded.
Statistical analysis
Adults’ mobility was estimated separately for the two
sexes, as the straight- line measurement of the distance
between consecutive captures.
The summation of all single distances was taken to
represent the minimum cumulative distance travelled by
each individual. The maximum distance between any two
observations of each individual was also recorded. The
operational sex ratio was defined as the ratio between the
number of estimated males and the total estimated number
of females. To test if the total number of males and females
fitted the expected 1:1 ratio, a Chi squared test was per-
formed. At each plot, correlations between the following
parameters were computed: number of marked males,
number of marked females, total marked specimens,
recaptured butterflies, plot area and A. rotunda densities. A
a
b
c
d
e
f g
0 500 m
Fig. 2 Schematic map of the
population of Z. cassandra
investigated at Conversano S–E
Italy. Areas in grey represent
the investigated (a–g) habitat
patches
J Insect Conserv
123
non-parametric Mann–Whitney U test was used to analyse
intersexual differences in mobility, to test association
between sexes and moved distances. All statistical analyses
were performed on SPSS 21.
Data collected during MRR surveys were analysed by
Cormack-Jolly-Seber type constrained models (Schwarz &
Arnason 1996; Schwarz and Seber 1999) using MARK 5.1
program (White and Burnham 1999).
For the egg-laying study, statistical analyses were per-
formed on R-2.9.0 (R Development Core Team 2006).
Since it was impossible to determinate if eggs on a plant
belonged to one or more females, each eggs-bearing host
plant was treated as a single sample in our data set,
regardless of the number of batches it carried.
In those cases when data were normally distributed
(Komogorov-Smirnov test) and variances were homogenous
(Levene test), parameters for occupied and unoccupied
plants were compared using t-tests. Otherwise, the Mann–
Whitney U test was used. Data from all sites were merged for
evaluation of oviposition preferences at landscape levels. To
define the oviposition preferences at the landscape and patch
levels, the comparison of absolute frequencies for categor-
ical variables, between occupied and unoccupied plants was
assessed using Likelihood ratio statistic, to establish if
environmental variables differed between eggs-bearing and
unoccupied Aristolochia plants, at both the landscape and
the patch level. Standardized residuals were used to define
significant contributors to the overall Chi square value.
We used a generalized linear mixed-effects model to
recognise those parameters possessing the highest explan-
atory power for oviposition sites selectivity. The variable
‘‘egg presence’’ was set as a random factor to examine the
relationship between the occurrence of oviposition and
habitat variables. The best model was assessed using the
Akaike information criterion (AIC; cf. Zuur et al. 2009).
Using a multi-model inference, we examined the AICc
values for all possible models with all different combina-
tion of the explanatory variables mentioned above. Owing
to the large number of candidate models, we restricted
model averaging to models for which D AICc \4 com-
pared with model with the lowest AICc.
Statistical analyses were performed on SPSS 21 and R-
2.9.0 (R Development Core Team 2006). Multimodel
inference analyses were performed using ‘MuMIn’ pack-
age (Barton 2011) for R.
Results
Egg laying habitat
To evaluate how eggs’ occurrence and abundance were
affected by environmental variables, we surveyed 275 A.
rotunda plants potentially available for Z. cassandra ovi-
position. At landscape level, eggs were found at all sur-
veyed sites, but host-plant quantity differed between
patches (site 1: N =95, site 2: N =141, site 3: N =39;
Table 1b, c, d). Of 275 potential host plants for Z. cas-
sandra, 120 were selected for oviposition and 153 eggs
were found (site 1: 47, site 2: 40, site 3: 66) (Fig. 3). At
patch level, the occupied and unoccupied plants differed
significantly in all measured landscape and patch parame-
ters (Table 1a) except for the ‘‘Aspect’’ parameter: eggs
were predominantly laid on plants growing in large stands
(68.5 %), and in half or full sun (83.1 %), whereas single
plants or plants growing in small stands (2–5 plants)
(31 %), or in full shadow (16 %) were generally avoided
and played minor roles in eggs deposition. (The great
majority ([70 %) of occupied plants were found on south
or south-east facing slopes. More exactly, females prefer-
entially oviposited in south (44.9 %) or south-east facing
sites (29.1 %), while they avoided northern orientation
(Fig. 4) respect south and south-east facing sites
(v2=29.930; d.f. =1; p\0.001)). At site 2, eggs were
laid in full shadow (45 %), but females completely avoided
plants in full shadow at sites 1 and 3, except in one case at
site 1.
At plant level, the distribution of occupied and unoc-
cupied plants was best explained by the combination of
prominence (difference in height with respect to the sur-
rounding vegetation), number of plants per stand and
exposure to sun. The occupied and the unoccupied plants
also differed in height (Mann–Whitney U-test: N =275;
Z=3.468; p=0.001) and the more prominent A. rotunda
plants were significantly preferred p\0.05 by ttest
(Fig. 5).
The distributions of host plants and egg-deposition
heights were more or less bell-shaped (Fig. 6) and eggs
were in significantly higher number (N: 65 =62 %) than
expected on plants 21–40 cm high. More than a half of the
egg-bearing plants were 21–60 cm high (min: 17 cm, max
75 cm) (Fig. 7).
The vast majority of eggs (N: 136 =88.88 %) were laid
on the underside (abaxial) surface of the leaves, while a
small fraction were laid on the upper surface (N:
15 =9.8 %), or even more rarely (N: 2 =1.3 %) on
flower buds (v
2
=199,991 df =2, p\0.001). Usually
one egg per plant was found. Eggs, in fact, were laid
mainly singly (N: 56 =63 %), sometimes in pairs (N:
16 =18 %), or rarely in small batches of 3 (N: 7 =8 %),
4 (N: 7 =8 %), 5 (N: 2 =2%)or6(N:1=1 %) eggs.
The vegetation cover was over 70 % in all plots and areas.
Most of the eggs (40 %) were observed in plots with grass
coverage between 80 and 90 %.
GLM analysis showed that the likelihood of an A.
rotunda plant being selected for oviposition was positively
J Insect Conserv
123
Table 1 Absolute (N) and relative (%) frequencies of landscape and patch parameters of foodplants (A.rotunda), either occupied, or unoccupied by Z. cassandra eggs in NW Italy
Parameters (a) All sites (b) Site 1 (c) Site 2 (d) Site 3
Unoccupied
(N =186)
Occupied
(N =89)
Unoccupied
(N =63)
Occupied
(N =32)
Unoccupied
(N =111)
Occupied
(N =30)
Unoccupied
(N =12)
Occupied
(N =27)
N % N% N% N% N % N% N% N%
Habitat type LR =30.767, df =2, p\0.001
Forest edge 63 33.33 32 35.96
Grassland 12 6.35 27 30.34
Woodland 111 58.73 30 33.71
Aspect LR =2.468, df =2, p=0.481 LR =5.779, df =2, p=0.052 LR =0.532, df =2, p=0.766 LR =0.572, df =1, p=0.449
N 20 10.75 5 5.62 9 14.29 2 6.25 11 9.91 3 10 0 0
S 86 46.24 40 44.94 27 42.86 22 68.75 59 53.15 18 60 0 0
SE 47 25.27 26 29.21 0 0.00 0 0 41 36.94 9 30 6 50.00 17 62.96
SW 33 17.74 18 20.22 27 42.86 8 25 0 0.00 0 0 6 50.00 10 37.04
Sun exposure LR =10.400, df =2, p=0.006 LR =2.670, df =2, p=0.263 LR =1.186, df =2, p=0.553 LR =0.572, df =1, p=0.449
Full sun 85 45.70 33 37.08 27 42.86 11 34.38 52 46.85 12 40.00 6 50.00 10 37.04
Medium 50 26.88 41 46.07 36 57.14 20 62.50 8 7.21 4 13.33 6 50.00 17 62.96
Full shadow 51 27.42 15 16.85 0 1 3.13 51 45.95 14 46.67 0 0
Size of A. rotunda stand LR =6.347. df =2, p=0.042 LR =0.323, df =2, p=0.851 LR =0.815, df =1, p=0.367 LR =1.913, df =1, p=0.167
Individual (1 plant) 2 1.08 2 2.25 1 1.59 1 3.13 1 0.90 1 3.33 0 0
Small (2–5 plants) 31 16.67 26 29.21 22 34.92 12 37.50 0 0.00 0 0.00 9 75.00 14 51.85
Large ([5 plants) 153 82.26 61 68.54 40 63.49 19 59.38 110 99.10 29 96.67 3 25.00 13 48.15
Likelihood ratio statistics (LR) are shown for comparisons of absolute frequencies between occupied and unoccupied plants (a–d)
J Insect Conserv
123
correlated with the distance from the nearest tree (Table 2).
The likelihood of a site being accepted as oviposition
habitat increased with host plant presence and with
southerly orientation. Most egg-bearing A. rotunda plants
were found in areas with no tree cover.
To evaluate eggs’ occurrence at ‘‘Laghi di Conversano’’,
we surveyed 82 A. rotunda plants potentially available for
Z. cassandra oviposition. Eggs were found at all surveyed
sites, but host-plant quantity differed between patches (site
1: N =21, site 2: N =21, site 3: N =40). Of 82 potential
host plants, 15 (18 %) were selected for oviposition (site 1: 7,
site 2: 7, site 3: 25). Eggs were predominantly laid on
plants growing in large stands (N =296; 82 %), whereas
single plants or plants growing in small stands were avoi-
ded, and in half (24.3 %) or full sun (75.6 %). The great
majority of occupied plants were found on dry stone wall
surfaces (58.5 %) and plants on dry stone wall bore more
eggs (N =28, 73.6 %). Females preferentially oviposited
in south- (66.6 %), or east facing sites, but the latter bore
more eggs (N =23; 59 %).
The great majority ([70 %) of occupied plants were
found on south facing slopes. Eggs were laid mainly singly
(N: 67 =43 %). The maximum number of eggs per plant
was 6 at Marcarolo and 14 at Conversano. Probably the
smaller number of plants on the site of Conversano induces
individuals to lay a larger number of eggs on a single plant.
Fig. 3 Number of available food plants and number of eggs observed
in the three habitat types
Fig. 4 Polarplot of the geographical orientation (in %) of A. rotunda
plants, either unoccupied (black line,N=189), or occupied (grey
line,N=89) by Z. cassandra eggs
unoccupied plants [N=186] occu
p
ied
p
lants [N=86]
Fig. 5 Prominence of unoccupied and occupied plants. Prominence
was calculated as the difference between the host plant’s and the
turf’s height. The dotted line indicates turf height
Host Plant height
Oviposition height above ground
Fig. 6 Distribution of plant and oviposition height in NW Italy
J Insect Conserv
123
At Marcarolo the chosen plants grew in very thick vege-
tation cover ([80 %), whereas in southern Italy plants
carrying more eggs were found on bare soil. Most sites
with egg-bearing A. rotunda plants were found in areas
with no tree cover.
Mobility
In total 34 individual were marked (23 males and 11
females) and 14 (41 %) of them were recaptured at least
once (Table 3). Based on these data, we estimated a total
population size of 116 (±19) individuals, with 79 (±11)
males and 37 females (±6). No individuals were captured
in nearby plots, showing no exchange between populations.
The sex ratio of captures was male biased (v
2
=4.235,
d.f. =1, p=0.039) and males were recaptured more often
than females (v
2
=4.083, d.f. =1, p=0.043). Grouped
for sexes, distances between captures did not markedly
vary and ranged from 8.8 to 96.7 m. The longest detected
movement between successive captures was 110.63 m.
Nevertheless, most (75 %) of the movements were within
60 m from the release spot. Residence time provides a
rough estimate of maximum adult life span. The maximum
recorded time between the first and the last capture was
6 days and was similar for males and females.
Discussion
Egg laying habitat
In this study we investigated the oviposition microhabitat
of Z. cassandra. Characteristics of the oviposition site play
an important role in determining habitat suitability since,
according to several studies (e.g. Rausher 1983; Janz 2002)
the choice of the deposition site is generally not random
and is structured according to various maternal behaviours.
Probably the most interesting result of our eggs-laying
study is that irrespectively of a lower food-plants density,
females lay in the more open areas (v
2
=7.098, d.f. =2,
p=0.028) at both study sites (Fig. 3).
The habitat requirements by Z. cassandra for egg laying
are best explained by a combination of presence in small
stands of Aristolochia plants growing in medium sun
conditions, with no other preference for any type of veg-
etation structure and/or feature of host plant quality.
Although Aristolochia plants preferentially tend to colonise
ecotonal areas and are generally less abundant in full
sunlight, Z. cassandra prefers the more open habitats. This
is probably a consequence of the fact that Zerynthia cat-
erpillars are chemically protected ectotherms in no need of
concealing in the shadow, while in the early Spring, when
adults are flying, weather is surely more unpredictable than
in other seasons. Aristolochia plants growing the in the
shadow potentially represent ecological traps for strictly
sedentary larvae. This is in contrast with data from the
Hungarian population of Z. polyxena recorded by Bata
´ry
et al. (2008). Considering that Z. cassandra occurs in
unoccu
p
ied
p
lants occu
p
ied
p
lants
plant height
Fig. 7 Differences in height between plants receiving or not receiv-
ing eggs, versus plant height
Table 2 GLMM statistic: relationship between probability of
occurrence (binomial response variable: presence [N =89 occupied
plants] or of absence [N =186 unoccupied plants] of A. rotunda, in
relation to environmental parameters (predictor variables: host plant
height, height of surrounding vegetation, distance from the nearest
tree and vegetation cover)
Estimate Std.Error t value Pr([|t|)
(Intercept) -0.0607251 0.2090534 -0.29 0.77168
Host plant height 0.0030851 0.0025819 1.195 0.23319
Height
surrounding
vegetation
0.0023708 0.0020736 1.143 0.25392
Distance from the
nearest tree
0.0007594 0.000245 3.158 0.00177**
Turf height 0.0357912 0.0572253 0.625 0.53221
Southern
orientation
0.0109205 0.0703549 0.155 0.87676
Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘’ 1
(Dispersion parameter for gaussian family taken to be 0.1914006).
Null deviance: 59.840 on 275 degrees of freedom. Residual deviance:
51.487 on 269 degrees of freedom. AIC: 333.67 Number of Fisher
Scoring iterations: 2
Table 3 Summary of the MRR data
No. of marked
individuals
No. of
recaptured
individuals
No. of
captures
No. of
recaptures
Males 23 8 31 9
Females 11 6 17 5
Total 34 14 48 14
J Insect Conserv
123
Mediterranean areas, we would expected the opposite
result. The majority of eggs, however, were placed on the
abaxial surface of the leaflets, which may be explained by
the fact that eggs need sufficient humidity to avoid desic-
cation (Anthes et al. 2008), as well as detection by pre-
dators or parasites. Eggs and young larvae, in fact, are not
as chemically protected as the older larvae and adults
(Albanese et al. 2008). Elevated ambient temperatures
appear to be important for many butterfly species because
they may increase rates of larval development, decrease
mortality (McKay 1991), improve females’ fecundity by
increasing the time available for egg-laying, and therefore
generally increase egg-laying rates (Davies et al. 2006).
The significant relationship between landscape, patch and
plant parameters of Z. cassandra oviposition habitat
showed that, as is common for butterflies in general
(Dennis 2010), selection of oviposition sites is determined
by characteristics operating at different hierarchical levels,
reflecting their importance in the process of egg-laying site
selection. A sufficient amount of food is essential for larval
survival, in particular for species having ‘‘sedentary’’ cat-
erpillars. Visual attraction is an important factor when
searching for a suitable host plant (Porter 1992). For ovi-
position, females frequently choose the most conspicuous
host plants (Porter 1992; Garcia Barros and Fartmann
2009). Compared to plants growing in shadier areas, shoots
that grow higher than their surrounding vegetation are also
less shaded and offer better microclimatic conditions for a
quick development of eggs and larvae (Ku
¨er and Fartmann
2005). Furthermore, Z. cassandra females laid their eggs
on the intermediate parts of plants probably also due to
better food quality for the larvae, and this may be a direct
consequence of female oviposition choice. Upper plant
parts generally have lower amounts of alcaloids, as well as
nitrogen contents (Agerbirk et al. 2010), compared to lower
parts. Between occupied and unoccupied host plants,
plants’ height was the most important discriminating var-
iable and larger plants bore more eggs, while Dennis
(1996) showed that in Allancastria (or Zerynthia)cretica,
larger food plants bore more eggs.
At least in some cases, laying eggs on the higher parts of
the plant may provide some advantage. In Z. rumina,
Jordano and Gomariz (1994) found that the freshly hatched
larvae consumed the younger and softer leaves of the food
plant (A. pistolochia). This could be related with the lower
concentration of defence chemicals (Bata
´ry et al. 2008).
Early instars of Lepidopteran larvae are known to be sen-
sitive to environmental and chemical changes (Zalucki
et al. 2002). Some larvae are known to shelter inside the
flower buds during their first instar, when they are less
mobile and more prone to suffer from environmental stress
(Pinto et al. 2011) and can thereby increase larval survival
and growth. Anthes et al. (2003) noted a similar behaviour
in Euphydryas aurinia. They suggested that this strategy
eliminated the risks of predation and of exposure to adverse
weather conditions, associated with moving along the host
plant or to another plant.
Mobility
No movement of adult individuals was detected away from
their breeding areas, which reveals that Z. cassandra is
strongly sedentary and has relatively closed population
structure. Similarly to many other butterfly species, Z.
cassandra is strongly dependent upon particular micro-
habitats, both at the larval and at the imaginal stages. Due
to strong human influence, suitable habitats have become
increasingly fragmented, thereby restricting gene flow
among populations as well as chances for a successful re-
colonization of the remaining but isolated patches. Fitness
benefits of intermediate-distance dispersal will therefore
become strongly reduced, which will finally impose strong
selection against dispersal (Bonelli et al. 2013). Regional
extinctions, in such a situation, represent a very likely
scenario, perhaps the most likely at least in many cases.
Conclusion and implications for conservation
Protecting Z. cassandra populations requires that areas
containing suitable nectar plants are also protected. In the
northern parts of the species’ range, the main problem is in
the abandonment of rural areas. Thus, it is vital to maintain
the few remaining meadows, by promoting cyclical grass
cutting or light cattle grazing, as well as to create new
grasslands, wherever possible. In the study area, which is
part of the EU NATURA 2000 network, appropriate agro-
environmental schemes have already been used to allocate
the necessary funding for the maintenance of traditional
land management, within the framework of the regional
Rural Development Program (RDP). Grazing or grass
cutting, whenever implemented in the end of June or in late
summer, does not affect the survival of Z. cassandra pupae,
which shelter at the base of their (unpalatable) host plant.
In Apulia, in contrast, the ‘‘tendone’’-type vineyards
have become largely dominant since their plastic covering
is particularly suitable to protect the (mainly table-con-
sumed) grapes from excessively high summer tempera-
tures, strong winds and frequent hail. This particular
cultivation type, however, is highly and negatively
impacting on Z. cassandra populations, since adults are
unable to fly across or over the vines. In our study area, as a
consequence, butterfly populations tend to become
increasingly fragmented by the still spreading vineyards.
Mobility data obtained in our study are alarming, and
urgent action is needed. To counteract the currently heavy
fragmentation, suitable habitats should be created within
J Insect Conserv
123
the framework of current agro environmental schemes, to
ensure host plant stands connectivity.
In south Italy Aristolochia plants that grow on dry stone
walls will act as stepping stones. Dry-stone walls are
important landscape elements in this area, but their
importance has only been recognised in their aesthetic and
cultural dimension. Current promotion policies officially
aimed at preserving dry stone walls need to be better
implemented to prevent any further loss of this irreplace-
able asset, at communitarian level.
In general, schemes based on the application of the
integrated organic production rules financed by RDPs will
be less impacting for butterfly populations occurring in
cultivated areas and particularly in vineyards. These
include the insertion of buffer stripes between the fields,
the (cyclical) abandonment of some mown areas and the
encouragement of spontaneous re-vegetation in the alley-
ways and areas around crops. In the current economical
crisis, reaching a trade-off compromise between agro-
industrial needs and biodiversity conservation may locally
generate important revenues, both by guaranteeing sus-
tainability and by preserving the touristic attractiveness of
landscapes (Lasanta et al. 2001).
We also agree with Thomas et al. (1992) and with Maes
et al. (2004), that installing a stepping stones system of
suitable habitat patches is the most efficient way to restore
a healthy meta-population structure, which surely works
much better than ‘generalistic’ corridors in enhancing the
conservation status of many invertebrates.
In the case of butterflies, each ontogenetic instar
requires its own specific resources. In Z. cassandra, how-
ever, resources are spatially overlapping, since the adults’
habitat closely matches that of the immature stages, at least
insofar as suitable nectar sources are maintained. The way
to a successful management is in keeping the sites open and
free from invading scrub. In both study sites, Z. cassandra
is distributed mainly in the meadows and along the sur-
rounding hedges. Managing marginal lands to preserve
their biodiversity values and traditional farming systems,
with mowing once or twice a year, could contribute to
species persistence in a fragmented landscape. An optimum
mowing heights for this species could range from 5 to
15 cm.
Conservation efforts are generally focused on main-
taining species in situ, with considerable debate about the
possible merits of reintroductions. Natural recolonisation
of suitable habitats will be a slow process, provided that
recolonisation rates will not match extinction rates. So we
suggest that at least in some case the colonisations of some
particularly suitable patches should be artificially encour-
aged. A well-connected network of suitable habitats ought,
however, to be established well before any reintroduction
scheme is implemented.
Acknowledgments Our work was conducted in collaboration with
the ‘‘Capanne di Marcarolo’’ Nature Park and the ‘‘Laghi di Con-
versano e Gravina di Monsignore’’ Regional Park. Permission to
collect species listed under the 92/43/EEC Annex IV was granted by
the Italian Ministry of the Environment (U. prot. PNM-2011-
0010400-13/05/2011).
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