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RESEARCH PAPER
Spatio-temporal patterns in pollination of deceptive
Aristolochia rotunda L. (Aristolochiaceae)
B. Oelschl€
agel
1
, M. von Tschirnhaus
2
, M. Nuss
3
, T. Nikoli
c
4
, S. Wanke
1,†
,S.D
€
otterl
5,†
&
C. Neinhuis
1,†
1 Institut f€
ur Botanik, Technische Universit€
at Dresden, Dresden, Germany
2 Fakult€
at Biologie, Universit€
at Bielefeld, Bielefeld, Germany
3 Senckenberg Naturhistorische Sammlungen Dresden & Museum f€
ur Tierkunde, Dresden, Germany
4 Department of Botany, Faculty of Science, University of Zagreb, Zagreb, Croatia
5 Department of Ecology & Evolution, University of Salzburg, Salzburg, Austria
Keywords
Aristolochia rotunda; autogamy; Chloropidae;
deceptive pollination; floral life span; flower
biology; pollinator variability.
Correspondence
B. Oelschl€
agel, Institut f€
ur Botanik, Technische
Universit€
at Dresden, Zellescher Weg 20b,
01062 Dresden, Germany.
E-mail: birgit.oelschlaegel@tu-dresden.de
†
Authors contributed equally to this study.
Editor
A. Dafni
Received: 16 March 2016; Accepted: 22
August 2016
doi:10.1111/plb.12503
ABSTRACT
•Pollination success of highly specialised flowers is susceptible to fluctuations of the
pollinator fauna. Mediterranean Aristolochia rotunda has deceptive trap flowers
exhibiting a highly specialised pollination system. The sole pollinators are kleptopara-
sitic flies in search of food. This study investigates these pollinators on a spatio-tem-
poral scale and the impact of weather conditions on their availability. Two potential
strategies of the plants to cope with pollinator limitation, i.e. autonomous selfing and
an increased floral life span, were tested.
•A total of 6156 flowers were investigated for entrapped pollinators in 10 Croatian
populations. Availability of the main pollinator was correlated to meteorological data.
Artificial pollination experiments were conducted and the floral life span was recorded
in two populations according to pollinator availability.
•Trachysiphonella ruficeps (Chloropidae) was identified as dominant pollinator, along
with less abundant species of Chloropidae, Ceratopogonidae and Milichiidae. Pollina-
tor compositions varied among populations. Weather conditions 15–30 days before
pollination had a significant effect on availability of the main pollinator. Flowers were
not autonomously selfing, and the floral life span exhibited considerable plasticity
depending on pollinator availability.
•A. rotunda flowers rely on insect pollen vectors. Plants are specialised on a guild of
kleptoparasitic flies, rather than on a single species. Pollinator variability may result in
differing selection pressures among populations. The availability/abundance of polli-
nators depends on weather conditions during their larval development. Flowers show
a prolonged trapping flower stage that likely increases outcrossing success during peri-
ods of pollinator limitation.
INTRODUCTION
Deceptive pollination has evolved several times independently
in flowering plants (Proctor et al. 1996; Renner 2006). Decep-
tive flowers advertise a reward to their pollinators that they do
not provide in the end, and exploit the instinctive behaviour of
often non-typical flower visitors (e.g. Brodmann et al. 2008;
St€
okl et al. 2010). This results in highly specialised pollination
that is usually mediated by only one to a few pollinator species
(Dafni 1984; Proctor et al. 1996). Highly specialised plants (in-
dependent of whether deceptive or not) benefit from highly
specific pollen delivery and high outcrossing rates compared to
less specialised rewarding species (Scopece et al. 2010). How-
ever, they are counter-balanced by a high dependence on spa-
tiotemporal fluctuation of the pollinator fauna (Waser et al.
1996; Armbruster et al. 2000) and overall lower reproductive
success (Scopece et al. 2010). The Mediterranean climate is
characterised by strong seasonality and high variability of
short-term weather conditions (D€
unkeloh & Jacobeit 2003;
Martin-Vide & Lopez-Bustins 2006). It has been reported that
weather conditions during flowering influence the frequency of
pollinator visits and therefore pollination success (Baker et al.
2000; Jacquemyn et al. 2009). Such strong variability of polli-
nator abundance has earlier been shown for Mediterranean
Aristolochia species (Berjano et al. 2009).
The genus Aristolochia (Aristolochiaceae) comprises over
450 species and is distributed in the tropics and in temperate
regions (Wagner et al. 2014). In the Mediterranean about 60
species are known, representing a diversity hotspot for the
genus (Wanke 2007). Virtually all Aristolochia species exhibit
deceptive pollination strategies and it was assumed that flowers
mimic the brood sites of their fly pollinators (Vogel 1978;
Proctor et al. 1996). However, detailed knowledge on the
mechanism of pollinator attraction and deception is presently
only available for A. rotunda.A. rotunda flowers are klepto-
myiophilous, a pollination strategy recently described
Plant Biology 18 (2016) 928–937 ©2016 German Botanical Society and The Royal Botanical Society of the Netherlands928
Plant Biology ISSN 1435-8603
(Oelschl€
agel et al. 2015). Representatives of Chloropidae and
Ceratopogonidae were found pollinating this species
(Oelschl€
agel et al. 2015), and members of these families are
known kleptoparasites, stealing food of invertebrate predators
(e.g. Sivinski et al. 1999). The flowers of A. rotunda employ an
olfactory mimicry of the heteropteran prey to attract and
deceive their kleptoparasitic pollinators (Oelschl€
agel et al.
2015). Due to this particular attraction mechanism, A. rotunda
is a highly specialised plant species that could make the plant’s
reproductive success susceptible to fluctuations in pollinator
fauna.
Known strategies of plants to cope with pollinator limitation
and increase reproductive fitness are autonomous selfing and
increased floral longevity (e.g. Ashmann & Schoen 1994, 1996;
Fenster & Mart
en-Rodr
ıguez 2007; Castro et al. 2008).
Although flowers of Aristolochia show strong adaptations for
cross-pollination, the ability for delayed autonomous selfing
has been proven for several of its species (Razzak et al. 1992;
Hall & Brown 1993; Sakai 2002; Berjano et al. 2006; Bliss et al.
2013). However, it is yet unknown if A. rotunda is outcrossing
or selfing. Aristolochia flowers possess an elaborated perianth
morphology evolved for trapping, retention and release of pol-
linators (Correns 1891; Oelschl€
agel et al. 2009; Fig. 1A). The
perianth consists of three main parts: the limb, the tube and
the utricle that contains the gynostemium. The flowers show
pronounced protogyny, with functional adaptations in the dif-
ferent sexual stages. Erect and receptive stigmas, closed pollen
sacks (Fig. 1B) and functional trapping devices characterise the
female flower stage (i.e. bendable trichomes inside the flower
tube; Fig. 1C). Pollinators are attracted and trapped during this
stage. During the interphase stigmas wilt and bend together
while pollen sacs open (Fig. 1D). The trapping devices remain
turgescent, enabling the flower to cover the trapped flies with
its pollen. During the male flower stage, the trapping devices
wilt (Fig. 1E) and enable the flies, loaded with pollen, to leave
the flower (Correns 1891; Daumann 1971; Gonz
alez & Steven-
son 2000; Oelschl€
agel et al. 2009).
Stotz & Gianoli (2013) hypothesised an adaptation of the
floral longevity within the genus to pollinator limitation fac-
tors. Species in habitats with low net primary productivity and
low precipitation that are more affected by pollinator limita-
tion evolved longer-living flowers compared to species in habi-
tats of high net primary productivity and high precipitation
(Stotz & Gianoli 2013). Longer floral lifespan increases floral
fitness as longer-lived flowers have a higher likelihood for polli-
nator visitation than shorter-lived flowers. In contrast to tropi-
cal Aristolochia species with a typical 1 day female flower stage
(Cammerloher 1923; Hall & Brown 1993; Sakai 2002; Burgess
et al. 2004; Trujillo & S
ersic 2006), a prolonged duration of the
female flower stage has been observed in A. baetica (3.5–
4.2 days), A. chilensis (2–3 days) and A. paucinervis (5.3 days),
all of which occur in Mediterranean and/or arid climates (Ber-
jano et al. 2009; Stotz & Gianoli 2013). A prolonged female
flower stage may be constrained by an increase in insect mor-
tality within the flowers, especially in hot and dry climates
(Stotz & Gianoli 2013). Besides the matter of whether the
flower is pollinated or not, the maximum duration of the trap-
ping flower stage is therefore also likely limited by the surviva-
bility of the pollinators (Stotz & Gianoli 2013).
The present study focuses on the pollinators of A. rotunda
(Fig. 1F) and their spatio-temporal variability among multiple
populations and years. The impact of weather conditions on
A. rotunda’s highly specialised pollination system is discussed
and potential mechanisms promoting reproductive success
during pollinator limitation are evaluated. With respect to the
highly specialised pollinator attraction we test the following
hypothesis: (i) A. rotunda flowers are pollinated by the same
pollinator species in different populations; (ii) pollinator avail-
ability is strongly influenced by weather conditions during
flowering of A. rotunda, and unfavourable weather conditions,
such as high precipitation or low temperatures, result in peri-
ods with pollinator absence; (iii) autonomous selfing is a strat-
egy of A. rotunda to ensure reproductive success in periods of
pollinator limitation; and (iv) flowers of A. rotunda show a
prolonged trapping flower stage (female stage and interphase)
as compared to tropical Aristolochia species.
MATERIAL AND METHODS
Sampling sites
Aristolochia rotunda is endemic to the northwestern Mediter-
ranean region and occurs from western Turkey to eastern
Spain. It colonises damp grassy areas in woodlands, on river-
banks and boulders (Nardi 1984, 1991). The study was
performed at ten localities along the Croatian coast (Fig. 1G):
Dragonja (45°27051.7″N, 013°37020.3″E, 0 m a.s.l.), Most Ra
sa
(45°03046.5″N, 014°02044.4″E, 2 m a.s.l.) both Istria;
Caska (44°32059.7″N, 014°55016.3″E, 2 m a.s.l.), Dinji
sko
polje (44°23.5940N, 015°07.9120E, 73 m a.s.l.), Mt. Sveti
Vid (44°30045.6″N, 014°57025.6″E, 40 m a.s.l.), Stari
grad (44°25048″N, 015°03052″), Vla
si
ci’ (44°19021.5″N,
015°12031.7″E, 20 m a.s.l.) all Isle of Pag; Omisalij lake
(45°10022.3″N, 14°34027.3″E 6 m a.s.l., Isle of Krk), Veli
Lo
sinj (44°31017.1″N, 014°30059.8″E, 5 m a.s.l., Isle of Los-
inje) and Dra
cevac Ninski (44°10054.9″N, 015°18036.6″E,
49 m a.s.l., Ravni Kotari, Croatia mainland coast). Climate of
the study region corresponds to the typical seasonal climate of
the Mediterranean, with dry summer and wet winter seasons.
Collection of flower visitors and pollinators, pollinator
identification
The different stages of anthesis were determined in all investi-
gated flowers based on the current state of the gynostemium
and the turgescence of trapping trichomes. Insects that carried
pollen and were trapped in a female stage flower must have pre-
viously been trapped in another flower. Only these insects have
proven to repeatedly visit Aristolochia flowers and thus are
potential pollinators (Rulik et al. 2008). Therefore, only arthro-
pods found in the utricle of female stage flowers were subject to
subsequent analysis. Potential pollinators, henceforth for sim-
plicity of reading called ‘pollinators’, were discriminated from
accidental flower visitors through the presence of a pollen load.
Flowers were collected during annual field trips in May
2009–2012, the main flowering period of A. rotunda. In 2009
to 2011 collections were conducted once per population and
year; in 2012 populations were sampled twice or three times to
observe changes in pollinator abundance during the flowering
period. Flowers were either frozen and dissected during the
field trip or preserved in 100% ethanol for later laboratory
investigation. All pollinators were identified to species level,
Plant Biology 18 (2016) 928–937 ©2016 German Botanical Society and The Royal Botanical Society of the Netherlands 929
Oelschl€
agel, Tschirnhaus, Nuss, Nikoli
c, Wanke, D€
otterl & Neinhuis Spatio-temporal patterns in pollination
and in the case of Ceratopogonidae to family level (Collin
1946; Dely-Draskovits 1981; Beshovski 1985; Narchuk et al.
1989; Oosterbroek 2006; Nartshuk & Andersson 2013). To con-
firm that pollen load was indeed Aristolochia pollen, 49% of all
pollen samples were subject to scanning electron microscopy
using a Supra 40 VP SEM (Carl Zeiss, Oberkochen, Germany).
Meteorological data
The Meteorological and Hydrological Service Croatia (DHMZ)
provided meteorological data for April and May 2009–2012.
Daily data of mean, minimum and maximum temperature,
precipitation, sunshine hours and wind velocity were collected
from two meteorological stations close to the study localities:
station Zadar (44°080N, 15°130E; 5 m) and station Pula air-
port (44°540N, 13°550E; 63 m). As data were not normally dis-
tributed Kruskal–Wallis test (software SigmaPlot 12; Systat
Software, San Jose, CA, USA) was employed to test for differ-
ences of the single parameters among years and stations. In
case of statistical significance, the Student–Newman–Keuls
method (SigmaPlot 12) was used for pair-wise comparisons.
Monthly precipitation was compared to a reference period
(1961–2000) using the online facility of DHMZ (http://kli-
ma.hr/klima_e.php?id=SPI; DHMZ starting page –Climate –
Drought monitoring, accessed 10/23/2014).
Coupling meteorological data with pollinator availability
During our fieldwork we observed high variability in pollina-
tor availability and abundance during the flowering period in
May as well as among years. The observed variation could be
potentially explained by fluctuations in weather conditions.
To test for a possible correlation, meteorological data were
analysed in dependence to temporary availability of the main
pollinator, Trachysiphonella ruficeps (Macquart, 1835)
(Oelschl€
agel et al. 2015), in all investigated populations where
T. ruficeps occurred (Tables 1 and S1). Other pollinators were
not included because of the limited number of collected speci-
mens. The irresistible nature of pollinator attraction and
deception of A. rotunda flowers has been shown in a previous
study (Oelschl€
agel et al. 2015). We therefore assumed that the
pollinators start visiting A. rotunda flowers as soon as they are
(A) (B) (C)
(D)
(F) (G)
(E)
Fig. 1. Aristolochia flower morphology and study sites. A: Floral morphology and characteristics of the female and male flower stage. B: Female stage gynos-
temium showing receptive stigmata and closed pollen sacs. C: Trapping trichomes at the junction between tube and utricle during the female flower stage. D:
Male stage gynostemium showing dried stigmata and open pollen sacs. E: Wilted trapping trichomes at the junction of tube and utricle in the male flower
stage. F: Flower of A. rotunda. G: Study sites in Croatia (dots) and position of meteorological stations used (asterisks).a–anther, g –gynostemium, l –limb,
p–peduncle, s –stigma, t –tube, tr –trapping trichome, u –utricle. Images (C) and (E) are from Oelschl€
agel et al. (2009).
Plant Biology 18 (2016) 928–937 ©2016 German Botanical Society and The Royal Botanical Society of the Netherlands930
Spatio-temporal patterns in pollination Oelschl€
agel, Tschirnhaus, Nuss, Nikoli
c, Wanke, D€
otterl & Neinhuis
available and conditions allow it. As for the majority of
dipterans, the biology and ecology of T. ruficeps,i.e. duration
of larval stages, the occurrence of imagines during the year,
preconditions to complete the life cycle, the substrate of larval
development, etc., remain unknown. We therefore linked
meteorological data to periods of trapping events within Aris-
tolochia flowers (see Collection of flowers visitors and pollina-
tors, pollinator identification) and up to 1 month before
pollinator collection. The following time spans were analysed:
(a) the day of collection in a flower, (b) the day before the
collection date, (c) days 1–5 before collection, (d) days 5–15
before collection, and (e) days 15–30 before the collection
date. Time spans (a) and (b) represent the period of attraction
to and trapping in the investigated flower. Time span (c)
likely fits the period when the pollinators were attracted,
trapped, covered with pollen and released by (an)other flower
(s). Hence the chosen time spans are based on the variable
floral trapping stage, as outlined in the Results section.
Finally, time spans (d) and (e) represent most likely weather
conditions during periods of pollinator development (i.e. lar-
val and pupal s stages).
For all collection dates of the populations
Caska and Vlasici
(Isle of Pag) meteorological data were obtained from station
Zadar. Meteorological data for the Istrian populations Dra-
gonja and Most Ra
sa were obtained from the station Pula air-
port. Data on daily precipitation, daily sunshine hours, average
daily temperature, maximum daily temperature and minimum
daily temperature were recorded for the time spans (a) to (e),
and data on wind velocity for the time spans (a) to (c). For the
time spans (a) and (b) absolute daily values were used. For the
time spans (c) to (e) cumulative values for precipitation
and sunshine hours and average daily values for temperature
and wind velocity were calculated for subsequent analyses,
respectively.
Scatter plots were drawn on the individual meteorological
parameters using R 3.1.1 (R Core Team 2014) and RStudio
0.98.978 (RStudio 2014). Normalised values of meteorological
parameters underwent principal components analysis (PCA)
as implemented in the software Primer 6 (version 6.1.15) and
Permanova+(version 1.0.5; Clarke 1993; Clarke & Gorley
2006) to visualise similarities and differences in meteorologi-
cal parameters among the collected samples and according to
pollinator availability. The contribution of single meteorologi-
cal parameters to the ordination of the samples is plotted.
Permutation test (PERMANOVA) as implemented in Primer 6
and the Permanova+add on was conducted on Euclidean dis-
tances of normalised meteorological parameters of individual
time spans using the factor pollinator availability (presence/
absence) and controlling for year. Additional two-way Simi-
larity percentages (SIMPER) analyses (Primer 6) using the fac-
tor pollinator availability, and again controlling for year, was
performed to determine the contribution of individual meteo-
rological parameters to the differences among pollinator avail-
ability groups.
Breeding system
Aristolochia rotunda’s ability for autonomous selfing, geitonoga-
mous and xenogamous pollination was tested on 24 plants col-
lected from 15 natural populations in France, Italy and Croatia.
To avoid resource limitation that has been reported for natural
Aristolochia populations in the Mediterranean (e.g. Berjano et al.
2006, 2010) pollination experiments were conducted under
greenhouse conditions. Flower buds were enclosed with small
gauze bags to exclude visitors. Test for autonomous autogamy
was performed on 15 plants employing up to nine replicates
(flowers) per plant. For this experiment, flowers were left
untreated within the gauze bags. Artificial geitonogamous polli-
nation was performed on 15 plants with up to 13 replicates, and
artificial xenogamous pollination on 17 plants with up to 13
replicates. At the beginning of anthesis the perianth was cut in
the middle of the utricle and pollen from the same or another
plant was transferred to the stigmatic lobes using a toothpick.
Subsequently, flowers were re-bagged till abortion to exclude fur-
ther pollination. Fruit and seed set were recorded for each flower.
Average fruit and seed set per plant were calculated. Kruskal–
Wallis rank sum test was employed for global and Wilcoxon rank
Table 1. Number of pollinators entrapped in Aristolochia rotunda flowers at female flower stage in seven Croatian populations on the Isle of Pag and in Istria.
total (%)
Pag Istria
Caska Dinji
sko polje Mt. Sveti Vid Stari Grad Vla
si
ci Dragonja Most Ra
sa
no. flowers
a
4424 375 74 895 173 1777 377 753
no. pollinators 303 12 6 6 2 20 96 161
no. pollinators/100 flower 6.8 3.2 8.1 0.7 1.2 1.1 25.5 21.4
Chloropidae
Aphanotrigonum femorellum
b
2(<1%) 1 ––––1–
Oscinimorpha koeleriae 2(<1%) –– 2––––
Oscinimorpha minutissima
b
14 (5%) 2 1 4 1 –24
Trachysiphonella ruficeps
b
242 (80%) 9 –––16 61 156
Tricimba humeralis 6 (2%) –5––1––
Ceratopogonidae
b
35 (12%) –– – 1 1 32 1
Milichiidae
Leptometopa niveipennis 1(<1%) –– – – 1––
Neophyllomyza acyglossa 1(<1%) –– – – 1––
a
Only pollinator-bearing collections considered (see Table S1).
b
Taxa revealed as pollinators by Oelschl€
agel et al. (2015).
Plant Biology 18 (2016) 928–937 ©2016 German Botanical Society and The Royal Botanical Society of the Netherlands 931
Oelschl€
agel, Tschirnhaus, Nuss, Nikoli
c, Wanke, D€
otterl & Neinhuis Spatio-temporal patterns in pollination
sum test for pair-wise comparisons of fruit set among treatments
(both executed in R 3.1.1 and RStudio 0.98.978).
Duration of trapping stage
The trapping flower stage is characterised as the ability to trap
and retain flower visitors by means of fully turgescent trapping
trichomes within the floral tube (Oelschl€
agel et al. 2009). This
stage includes the female flower stage and the interphase. As
flower damage might shorten the floral lifespan (Berjano et al.
2009), the turgescence of the trapping trichomes, which were
visible from the entrance of the floral tube, was used as indica-
tor of the trapping stage in order to avoid flower damage dur-
ing the investigation. Investigations were performed in Vla
si
ci
(2010/05/09–2010/05/18) and Most Ra
sa (2012/05/27–2012/
06/01). A total of 64 and 27 flower buds, respectively, were
labelled and, following flower opening, the flower stage was
recorded daily in the morning. Flowers that did not enter the
male stage during the observation period (30 in Vla
si
ci and
one in Most Ra
sa) were excluded from statistical analysis. Wil-
coxon rank sum test (R 3.1.1) was employed to compare trap-
ping flower stages among sites and years.
RESULTS
Flower visitors, pollinators and pollinator variability among
populations
Altogether 6156 female-stage flowers were investigated on 31
different collection dates and sites. Arthropods were found
entrapped in about 9% of the flowers, but only 3% contained
arthropods carrying pollen. A total of 1081 arthropods was
found, 84% of which were Diptera. Among the arthropods,
303 individuals (28%) carried Aristolochia pollen and thus
proved to have repeatedly visited A. rotunda flowers. All polli-
nators were Diptera belonging to the families Chloropidae
(88%), Ceratopogonidae (12%) and Milichiidae (<1%;
Table 1). Chloropid pollinators were: Aphanotrigonum femorel-
lum Collin, 1946 (2♀), Oscinimorpha koeleriae Narchuk, 1970
(2♀), Oscinimorpha minutissima (Strobl, 1900) (14♀), Trachysi-
phonella ruficeps (221♀20♂, 1 unknown sex) and Tricimba
humeralis (Loew, 1858) (6♀). Milichiid pollinators were identi-
fied as Leptometopa niveipennis (Strobl, 1898) (1♀) and Neo-
phyllomyza acyglossa (Villeneuve, 1920) (1♀). Among the
Ceratopogonid pollinators were 26 females, and nine individu-
als of unknown sex.
In seven out of 10 sampled populations, pollinators were
found (Fig. 2, Table S1). Chloropid pollinators were found in
all pollinator-bearing populations, but ceratopogonid and mili-
chiid pollinators in only four and one of the populations,
respectively (Table 1). Throughout the study period pollinators
were found more consistently in Istrian than in the Pag popu-
lations (Fig. 2). The pollinator with overall highest numbers of
individuals trapped in flowers, T. ruficeps, was found in only
four populations (Table 1).
Despite the well-defined pollinator guild, the spectrum of
flower visitors is much broader (Table S1). Dipterans of alto-
gether ten families were identified: besides Chloropidae, Cer-
atopogonidae and Milichiidae, non-pollinating representatives
of Cecidomyidae, Chironomidae, Hybotidae, Phoridae, Psy-
chodidae, Scatopsidae as well as Sciaridae. However, with an
abundance of 68%, Chloropidae represented both the main
flower visitors and pollinators of A. rotunda. Additionally, a
diverse set of other arthropods (e.g. Acari, Aphididae, Collem-
bola, Colleoptera, Formicidae, Lepidoptera), mostly larvae, was
also found trapped within the flowers (Table S1).
Weather conditions during the study years
Main weather trends were similar between meteorological sta-
tions Pula airport and Zadar, although minor differences were
found in temperature (slightly lower monthly mean values in
Pula airport; Table S2), wind velocity (variation in daily values)
and precipitation (stations show similar precipitation pattern
over time, but considerable differences in daily precipitation
rates; Fig. 2). In contrast, considerable variation in weather
conditions was detected among years (Table S2). Total
monthly precipitation values varied strongly among the study
years (Table S2). In comparison to the reference period 1961–
2000 (data not shown) extremely wet periods were observed in
May 2010 for both stations and April 2012 in station Zadar
only (outside 90% percentile), and very dry periods in May
2009 and April 2011 for both stations (outside 10% percentile).
Both 2009 and 2011 were characterised by a significantly war-
mer May than the other years (Table S2). May 2012 showed
alternating sunny weather and rainfall events, in contrast to the
more steady weather conditions of the remaining years that
were mostly sunny (2009 and 2011) or very wet (2010; Fig. 2).
Temporal pollinator variability and dependence on climate
factors
The period of pollinator availability and the pollinator abun-
dance varied strongly among years, populations and in the course
of May (Fig. 2). In general, pollinator collections were most suc-
cessfulfrommidandthroughtotheendofMay.Fieldtripsin
2009 and 2011 were characterised by predominantly warm and
sunny weather conditions and abundant T. ruficeps pollinators in
early May, although in considerably different amounts between
years. In contrast, frequent rainfall and relatively low tempera-
tures characterised the field period in 2010, during which over
1250 investigated flowers revealed no T. ruficeps pollinators. Year
2012 showed alternating sunny weather and rainfall with abun-
dant T. ruficeps pollinators in the second half of May.
Overall, a significant effect of weather conditions on the
availability of the main pollinator was found in a period 15–
30 days before pollinator collection (Pseudo-F
1,22
=3.65,
P=0.02; Fig. 3B). This effect was dependent on the year stud-
ied (pollinator 9year: Pseudo-F
2,22
=4.29, P=0.002). Struc-
ture of data allowed only for 2012 to test for a weather effect
on pollinator availability (>100 permutations possible), and
this test revealed a highly significant effect (Pseudo-
F
1,9
=15.73, P=0.005). SIMPER analysis of meteorological
parameters during this time span, controlling for factor year,
revealed a contribution of nearly 80% by maximum tempera-
ture, sunshine hours and average temperature to the differences
in samples with pollinators present versus absent.
In all other time spans weather conditions did not explain
the availability of pollinators (PERMANOVA,P>0.10), although
scatter plots point towards a negative correlation between pre-
cipitation and the occurrence of the main pollinator on the col-
lection day (Fig. 3).
Plant Biology 18 (2016) 928–937 ©2016 German Botanical Society and The Royal Botanical Society of the Netherlands932
Spatio-temporal patterns in pollination Oelschl€
agel, Tschirnhaus, Nuss, Nikoli
c, Wanke, D€
otterl & Neinhuis
Breeding system
Pollination experiments revealed significant differences in fruit
set among autogamic, geitonogamic and xenogamic experi-
ments (Kruskal–Wallis v
2
=7.97, df =2, P=0.02). All flowers
that were not hand-pollinated yielded no fruits. Hand-pollina-
tion experiments yielded fruit sets of 18.4% by geitonogamic
and 12.7% by xenogamic pollination. No significant difference
was found between geitonogamic and xenogamic hand-pollina-
tion treatments (W=130, P=0.93), and both treatments
yielded an average of 11 seeds per fruit.
Variability of the trapping flower stage
In Vla
si
ci in 2010 the trapping flower stage averaged 6.1
1.6 days (mean SD; min =2 days, max =8 days, n =34).
However, 12 flowers were observed as being in the trapping
stage for at least 9 days. Their total lifespan could not be
recorded as the observation period ended at day 9. A signifi-
cantly shorter trapping stage of 2.3 0.6 days on average
(min =1, max =3, n =26) was observed in Most Ra
sa 2012
(W=17, P<0.001). One flower was recorded in the trapping
stage for 3 days but did not enter the male stage during the
observation period. Pollination conditions differed consider-
ably among sites and years. In Vla
si
ci in 2010 pollinators were
absent (534 investigated flowers), whereas in Most Ra
sa in
2012 a high pollinator frequency was observed (74 pollinators
per 100 flowers, 86 investigated flowers; Fig. 2, Table S1).
DISCUSSION
Pollinators of Aristolochia rotunda
Employing a broad sampling spanning 10 populations
throughout Croatia, this study reveals kleptoparasitic T. rufi-
ceps being the most important pollinator of A. rotunda in the
study region, thus confirming findings of Oelschl€
agel et al.
(2015). The data presented here however complement the
previously identified pollinator guild by adding the species
Oscinimorpha koeleriae,Tricimba humeralis (Chloropidae),
Leptometopa niveipennis and Neophyllomyza acyglossa (Milichi-
idae) (Table 1). Nevertheless, T. ruficeps was the only species
that at least temporarily occurred in high abundance. Addi-
tionally and in contrast to the other chloropid pollinators that
are geographically more restricted (Nartshuk 2013), T. ruficeps
has a wide western Mediterranean distribution range, including
Corsica, Sardinia and Sicily (Nartshuk 2013), where A. rotunda
also occurs (Nardi 1984). Therefore, we assume T. ruficeps is
the most important pollinator throughout the whole distribu-
tion area of the plant, even though this species was not found
in all investigated populations.
It is most likely that the newly found pollinator species are
also deceived by the flower’s kleptomyiophilous pollination
strategy. Representatives of both milichiid genera and the
chloropid genus Oscinimorpha sp. are known to be attracted by
hexyl butyrate and other aliphatic esters (Sugawara & Muto
1974; Zhang & Aldrich 2004; Heiduk et al. 2010), the key
temperature (°C)
Precipitation (mm)
Date
Zadar
5101520253035
temperature (°C)
Pula
5 101520253035
0 5 10 15 20 25
Precipitation (mm)
0 5 10 15 20 25
April 20
April 25
April 30
May 05
May 10
May 15
May 20
May 25
May 30
April 20
April 25
April 30
May 05
May 10
May 15
May 20
May 25
May 30
April 20
April 25
April 30
May 05
May 10
May 15
May 20
May 25
May 30
April 20
April 25
April 30
May 05
May 10
May 15
May 20
May 25
May 30
April 20
April 25
April 30
May 05
May 10
May 15
May 20
May 25
May 30
April 20
April 25
April 30
May 05
May 10
May 15
May 20
May 25
May 30
April 20
April 25
April 30
May 05
May 10
May 15
May 20
May 25
May 30
April 20
April 25
April 30
May 05
May 10
May 15
May 20
May 25
May 30
MR
50
O
99
Va
55
DP
46 SV
65
Ca
103
O
391
Va
534
SV
22
Ca
49
VL
68
MR
18
St
173
MR
345
Dr
67 MR
DN
107
Ca
92
Va
1040
MR
150
MR
86
Dr
26
Dr
199
MR
122
Ca
259
SV
101
Ca
185
Va
270
Va
467
SV
830
DP
28
Dr
85
Dr
Minimum temperature
Maximum temperature
Precipitation
Meteorological parameter
Study sites
DP - Dinjiško polje (Pag)
Va - Vlašići (Pag)
Ca - Časka (Pag)
SV - Mt. Sveti Vid (Pag)
St - Stari grad (Pag)
VL - Veli Lošinj (Losinje)
DN - Dračevak Ninski (Ravni Kotari)
MR - Most Raša (Istria)
Dr - Dragonja (Istria)
O - Omisalij lake (Krk)
<1
1–4
9–14
36
74–173
not determined
Present, but
Pollinators per 100 flowers
Study site
Chloropidae pollinators
Pollinators absent
No. female-stage flowers
Sample details
Dr
85
Ceratopogonidae pollinators
Milichiidae pollinators
Fieldwork period
Ca
24
(A)
(B)
2009 2010 2011 2012
Fig. 2. Pollinator collection, pollinator abundance and weather conditions during April and May 2009 to 2012. Daily minimum and maximum temperature
and daily amount of precipitation are shown for the periods 20 April to 31 May every year recorded by the weather stations (A) Pula airport in Istria and (B)
Zadar in Ravni Kotari. Aristolochia rotunda populations are assigned to the nearest weather station. For each collection event from top to bottom the amount
of pollinators per 100 flowers and dipteran family, the locality and the sample size (number of investigated female-stage flowers) are given. Samples of several
days’ duration (Table S1) are assigned to the latest collection date. Fieldwork periods are highlighted with pale green bars. For ease of comparison among
years. the periods 1 May to 15 May are highlighted in grey.
Plant Biology 18 (2016) 928–937 ©2016 German Botanical Society and The Royal Botanical Society of the Netherlands 933
Oelschl€
agel, Tschirnhaus, Nuss, Nikoli
c, Wanke, D€
otterl & Neinhuis Spatio-temporal patterns in pollination
0
5
10
15
0 50 100 150
Pollinators
Precipitation (mm)
0
5
10
15
0 50 100 150
Pollinators
Precipitation (mm)
0
10
20
0 50 100 150
Pollinators
Cum. prec. (mm)
0
20
40
0 50 100 150
Pollinators
Cum. prec. (mm)
0
25
50
75
0 50 100 150
Pollinators
Cum. prec. (mm)
0
5
10
15
0 50 100 150
Pollinators
Sunshine hours (h)
5
10
0 50 100 150
Pollinators
Sunshine hours (h)
20
30
40
50
60
0 50 100 150
Pollinators
Cum. sun. hours (h)
50
75
100
125
0 50 100 150
Pollinators
Cum. sun. hours (h)
60
80
100
120
140
160
0 50 100 150
Pollinators
Cum. sun. hours (h)
12.5
15.0
17.5
20.0
22.5
25.0
0 50 100 150
Pollinators
Tmean (°C)
15
18
21
24
0 50 100 150
Pollinators
Tmean (°C)
15.0
17.5
20.0
0 50 100 150
Pollinators
Av. Tmean (°C)
14
15
16
17
0 50 100 150
Pollinators
Av. Tmean (°C)
11
12
13
14
15
16
17
0 50 100 150
Pollinators
Av. Tmean (°C)
18
21
24
27
0 50 100 150
Pollinators
Tmax (°C)
20
25
0 50 100 150
Pollinators
Tmax (°C)
17.5
20.0
22.5
25.0
0 50 100 150
Pollinators
Av. Tmax (°C)
18
20
22
0 50 100 150
Pollinators
Av. Tmax (°C)
17
19
21
23
0 50 100 150
Pollinators
Av. Tmax (°C)
5.0
7.5
10.0
12.5
15.0
17.5
0 50 100 150
Pollinators
Tmin (°C)
7.5
10.0
12.5
15.0
17.5
0 50 100 150
Pollinators
Tmin (°C)
9
11
13
0 50 100 150
Pollinators
Av. Tmin (°C)
8
10
12
14
0 50 100 150
Pollinators
Av. Tmin (°C)
6
8
10
12
0 50 100 150
Pollinators
Av. Tmin (°C)
2.0
2.5
3.0
3.5
4.0
4.5
020406080
Pollinators
Wind velocity (m s–1)
2
3
4
5
0 50 100 150
Pollinators
Wind velocity (m s–1)
3
4
5
6
0 50 100 150
Pollinators
Av. wind vel. (m s–1)
Legend
Pollinators absent
Pollinators present
(A) Coll. day One day
before coll.
Days 1 to 5
before coll.
Days 5 to 15
before coll.
Days 15 to 30
before coll.
(B) Coll. day One day
before coll.
Days 1 to 5
before coll.
Days 5 to 15
before coll.
Days 15 to 30
before coll.
PC1: 44.8 %
PC2: 26.1 %
PC1: 48.6 %
PC2: 22.2 %
PC1: 55.3 %
PC2: 28.8 %
PC1: 45.2 %
PC2: 31.5 %
PC1: 60.4 %
PC2: 24.8 %
–4 –2 0 2 4
PC1
–2
0
2
4
PC2
PC2
PC2
PC2
PC2
WP
Sh
Tmean
Tmax
Tmin
–4 –2 0 2 4
PC1
–4
–2
0
2
W
P
Sh
Tmean
Tmax
Tmin
–4 –2 0 2 4 6
PC1
–2
0
2
4
W
SumP
SumSh
Tmean
Tmax
Tmin
–4 –2 0 2 4
PC1
–4
–2
0
2
4
SumP
SumSh
Tmean
Tmax
Tmin
–4 –2 0 2 4
PC1
–4
–2
0
2
SumP
SumSh
Tmean
Tmax
Tmin
Fig. 3. Variability of meteorological parameters during and up to 30 days before pollinator collection from 2 009 to 2012. Absolute values of mete orological parameters
are drawn on the collection day and the day before the collection day, whereas for the remaining time spans cumulative values (sunshine hours and precipitation) and
averages (windvelocity andtemperature) of parameters wereused. Samplesare discriminatedby colour according to presence andabsence of Trachysiphonella ruficeps
pollinators. A: Scatter plots of meteorological parameters in relation to pollen-carrying T. ruficeps individuals per 100 investigated female stage flowers. B: PCA con-
ducted on normalised meteorological parameters of collection samples.Variation explained by first two principal components (PC) is given and thecontribution of indi-
vidual parameters to ordination is plotted in the diagrams. P - daily precipitation, Sh - daily sunshine hours, SumP - cumulative precipitation, SumSh-cumulative
sunshine hours, Tmax - maximum daily temperature, Tmean - average daily temperature, Tmin - minimum daily temperature, W - wind velocity.
Plant Biology 18 (2016) 928–937 ©2016 German Botanical Society and The Royal Botanical Society of the Netherlands934
Spatio-temporal patterns in pollination Oelschl€
agel, Tschirnhaus, Nuss, Nikoli
c, Wanke, D€
otterl & Neinhuis
compounds of the kleptomyiophilous pollination system of
A. rotunda (Oelschl€
agel et al. 2015). Additionally, kleptopara-
sitism is a common phenomenon in Milichiidae and was
reported for female Neophyllomyza sp. on heteropteran prey
(Coreidae and Pentatomidae; Eisner et al. 1991) as well as for
N. acyglossa on beetle prey (Brake 2015). Knowledge about the
habit of the chloropid genus Tricimba is currently unavailable.
Although A. rotunda flowers show low ecological specialisa-
tion (several pollinator families and species), these findings
substantiate the high functional specialisation of the flowers
attracting kleptoparasites through olfactory mimicry of their
heteropteran prey. In scent-based pollination systems, com-
plex composition of the floral scent may promote the attrac-
tion of several pollinator species, as different scent
compounds or differences in their quantity may attract differ-
ent insect taxa. The flower scent of A. rotunda is composed of
numerous aliphatic esters and hydrocarbons, many more
compounds than were shown to be crucial for attraction of
the main pollinator T. ruficeps (Oelschl€
agel et al. 2015). It
needs to be shown in future studies whether these compounds
are involved in attraction of pollinators other than T. ruficeps.
Complex blends of floral scent are also known from other
deceptive pollination systems (Brodmann et al. 2008; St€
okl
et al. 2010). It has been shown that different pollinator species
detect different compounds of complex scent blends (St€
okl
et al. 2010). Consequently, it is likely that different pollinator
taxa of the same plant species are attracted to different com-
pounds of the floral scent.
High pollinator variability among populations
In contrast to our hypothesis that A. rotunda flowers trap the
same pollinator species among different populations, we
detected considerable variability of its pollinator composition
on a spatial scale. The occurrence of individual pollinator spe-
cies and their relative abundance varied strongly among popu-
lations (Table 1). This might give rise to different selection
pressures on floral traits among populations (Fenster et al.
2004; G
omez et al. 2009). The reason for the differing pollina-
tor guilds remains unknown but might be caused by differences
in habitat-based biotic and/or abiotic conditions (i.e. humidity,
salinity, vegetation) or floral traits (i.e. scent).
The existence of geography-related, community-based dif-
ferences in the pollinators has also been shown in other decep-
tive plants, such as Arum maculatum (Esp
ındola et al. 2010).
A. rotunda plants tolerate highly anthropogenic environments
such as field margins and dykes. We assume that the original
habitat of the species might have been grassy border areas of
the Mediterranean sclerophyll forests, i.e. treefall gaps, clearings
derived from forest fires, open areas along rivers or the coast.
Such habitats typically underlie constant modifications that
might lead to varying pollinator climates. We hypothesise that
a pollination system that is able to attract a functional group of
pollinators, i.e. carnivorous kleptoparasites, instead of a single
pollinator species is more likely able to adapt to altering and
unpredictable environmental conditions.
Another reason for the different pollinator guilds could be a
hitherto not studied variability of floral scent among popula-
tions. A scent-driven pollinator variability among conspecific
populations has been shown for deceptive Ophrys spp. in
southern Italy (Mant et al. 2005). Especially in highly
specialised pollination systems even small changes in qualita-
tive and/or quantitative floral traits (i.e. scent chemistry) may
provoke pollinator-induced reproductive isolation (Schiestl &
Ayasse 2002; Peakall et al. 2010). This may be the case in Din-
ji
sko polje where A. rotunda is almost exclusively pollinated by
Tricimba humeralis (Table 1). However, we do not know,
whether the absence of a specific pollinator species in a popula-
tion is due to the general absence of this species in the respec-
tive locality or is due to different scent traits. This still needs to
be tested and is the subject to on-going studies.
Temporal pollinator variability and dependence on climatic
factors
During the study period considerable inter-annual variations
of meteorological conditions were found along with differences
in pollinator availability and abundance. Similar variations in
pollinator availability were reported in other Mediterranean
Aristolochia species (Berjano et al. 2009) and are likely also
common outside the Mediterranean region.
Contrary to our expectations, no statistically significant cor-
relation between meteorological data and pollinator availability
was found during the flowering period. However, the collected
data are biased because observations were mainly performed
under dry weather conditions, and potentially differing micro-
climatic conditions of individual populations could not be
considered. However, a significant correlation was found
between the availability of the main pollinator T. ruficeps and
the weather conditions 15–30 days before pollinator collection
(Fig. 3B). Unfavourable weather conditions in this period, i.e.
low values for temperature and sunshine hours, lead to strong
pollinator limitation and failure of reproductive success of the
flowers later on. This period is assumed to include at least
some developmental stages of the flies, and most probably
occurs ahead of the flowering period of the plants. It is well
known that insects strongly depend on weather conditions dur-
ing development (e.g. Coulson et al. 1976; Tolley & Niemczyk
1988) and our results show that a high number of sunshine
hours, and high daily maximum and average temperatures
favour the appearance of T. ruficeps 2 or 3 weeks later.
Breeding system
We reject our hypothesis that autonomous selfing is a strategy
to ensure reproduction in A. rotunda flowers, as autonomous
selfing was not observed. A. rotunda flowers consequently rely
on insects as pollen vectors for successful sexual reproduction.
The inability for autonomous selfing, together with the
observed periods of pollinator absence during flowering, might
strongly decrease the reproductive success of the plants. Artifi-
cial pollination of the flowers yielded relatively low numbers of
ripe fruits (12–18%) independent of whether the pollen origin
was from a different flower of the same or another plant. We
assume that inefficient transfer of pollen to the stigmatic lobes
using a toothpick or damage to the stigmatic lobes might be
the reason for the low fruit set (Bliss et al. 2013), as well as pos-
sible resource limitation in the greenhouse. Quantitative data
under natural conditions are not available for A. rotunda.
However, in most studied Aristolochia species artificial pollina-
tion resulted in considerably higher fruit set than natural polli-
nation (Sakai 2002; Berjano et al. 2006; Murugan et al. 2006;
Plant Biology 18 (2016) 928–937 ©2016 German Botanical Society and The Royal Botanical Society of the Netherlands 935
Oelschl€
agel, Tschirnhaus, Nuss, Nikoli
c, Wanke, D€
otterl & Neinhuis Spatio-temporal patterns in pollination
Trujillo & S
ersic 2006). Fruit set of artificial-pollinated flowers
ranges from 7–19% in A. baetica (Berjano et al. 2010) up to
94% in A. tagala (Murugan et al. 2006). Fruit set of open-polli-
nated flowers, in contrast, ranges from 2% in A. maxima (Sakai
2002) to about 40% in A. chilensis and A. gigantea (Hip
olito
et al. 2012; Stotz & Gianoli 2013), but in most investigated spe-
cies a fruit set <20% was found (Sakai 2002; Berjano et al.
2006, 2010; Murugan et al. 2006; Trujillo & S
ersic 2006). Sev-
eral factors were suggested as promoting pollen limitation and
consequently low fruit set in the genus: scarcity of pollinators,
limited ability of small dipterans to cover larger distances,
insufficient amount of pollen grains transported by single polli-
nators, the need for several pollen grains to fertilise one ovule
(e.g.A. baetica –2.75 pollen grains per seed, A. paucinervis –
4.5 per seed), short pollen viability, and a rapid decline in
stigma receptivity after anthesis (Sakai 2002; Berjano et al.
2006, 2010; Murugan et al. 2006; Trujillo & S
ersic 2006).
Variability of the trapping flower stage
Flowers of A. rotunda show a significantly longer trapping
flower stage compared to short-lived tropical species (e.g. Bur-
gess et al. 2004; Trujillo & S
ersic 2006) and similar floral long-
evity as other Mediterranean species (Berjano et al. 2009).
These findings substantiate the hypothesis of Stotz & Gianoli
(2013). Additionally, high plasticity of the duration of the trap-
ping flower stage was observed. The trapping phase of flowers
in the Vla
si
ci population in a period with virtually no pollina-
tors lasted significantly longer compared to the trapping phase
of flowers in the Most Ra
sa population experiencing a period
with high pollinator frequency. A possible influence of weather
conditions on the duration of the flower trapping stage in
A. rotunda also cannot be excluded. However, we assume that
successful pollination, instead of weather, might be the major
influencing factor on the floral life span (Berjano et al. 2009).
A prolonged trapping flower stage enhances both, the female
and male fitness of a flower, as the chance to trap scarce polli-
nators and, hence, to receive foreign and distribute own pollen
increases. Bliss et al. (2013) showed, for two tropical
Aristolochia species, that fruit set is possible in flowers polli-
nated on day 2 or 3 of anthesis. In contrast, it has been shown
for at least one Aristolochia species that stigmata receptivity
decreases rapidly during the first day of anthesis (Murugan
et al. 2006). In this case, prolongation of the trapping stage of a
flower would increase its male rather than its female fitness.
Additionally, by contributing to a flower surplus in the popula-
tion, an expanded lifespan of a flower may increase the female
fitness of neighbouring, younger flowers, as flower surplus in a
population is assumed to attract pollinators more efficiently
(Podolsky 1992; Berjano et al. 2010).
ACKNOWLEDGEMENTS
We dedicate this publication to the late Professor Dr Stefan
Vogel. We thank Monika
Skegro (University Zagreb), Anna–
Magdalena Barniske (University Kassel), Markus G€
unther,
Cindy Thomas, Claudia P€
atzold and Sarah Wagner for their
valuable assistance during fieldwork, and Katharina Doll for
assistance in pollination experiments. Dirk Pavlik and Thomas
Pluntke (all TU Dresden) gave valuable advice for analysis of
meteorological data. The identification of flower visitors by
Frauke Nielsen and Andr
e Reimann (Senckenberg Museum f€
ur
Tierkunde, Dresden) and of milichiid pollinators by Irina
Brake (Burgdorf) is acknowledged. Aristolochia plants used for
pollination experiments are cultivated in the Botanical Garden
Dresden. Meteorological data were provided by the Meteoro-
logical and Hydrological Service Croatia. The Croatian author-
ities issued the collection permit (UP/I-612-07/12-33/0292,
517-12-02).
SUPPORTING INFORMATION
Additional Supporting Information may be found online in the
supporting information tab for this article:
Table S1. Arthropods entrapped in female stage A. rotunda
flowers in Croatia.
Table S2. Analysis of meteorological data for the months
April and May 2009 to 2012.
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