Structure and biomechanics of trapping flower trichomes and their role in the pollination biology of Aristolochia plants (Aristolochiaceae)


Author for correspondence:
Birgit Oelschlägel
Tel: +49 (0)351 46331497


  • Catching insects to ensure pollination is one of the most elaborate and specialized mechanisms of insect–plant interactions. Phylogenetically, Aristolochiaceae represent the first angiosperm lineage that developed trap flowers. Here we report the structure and function of specific trichomes contributing to the highly specialized trapping devices.
  • Investigations were carried out on six Mediterranean Aristolochia species. The morphology and arrangement of the trapping trichomes were investigated by scanning electron microscopy (SEM) and cryo-SEM. To demonstrate frictional anisotropy of the trapping trichome array, a microtribological approach was used.
  • The results of our experiments support a hypothesis long proposed, but never tested, regarding the trapping mechanism in proterogynous Aristolochia flowers: that an array of highly specialized trichomes arranged eccentrically to the underlying surface is responsible for the easy entrance of insects into flowers but impedes their escape. As they enter the male stage of anthesis, flowers significantly modify their inner surface characteristics, allowing insects to leave.
  • We have demonstrated the substantial contribution of trapping trichomes to the capture, retention and release of pollinators, an important prerequisite for making cross-pollination possible in most Aristolochia species. Finally, we compare trapping trichomes of Aristolochia with similar structures found in other trapping flowers as well as in pitchers of carnivorous plants not optimized for insect release.


Specialized, species-specific pollinator–plant interactions probably evolved late in the evolution of flowering plants, and among basal angiosperms polymorphic pollination syndromes or unspecific insect pollination often occurs (Hu et al., 2008). To our knowledge, the genus Aristolochia (Aristolochiaceae), a member of the basal angiosperms, is the first plant group in which extensively modified trapping flowers, possibly targeting specific pollinators, developed. Similar specialized flowers are also recognized in the holoparasitic Hydnoraceae (Bolin et al., 2009) that according to molecular data from the nuclear and mitochondrial genome were recently placed close to Aristolochiaceae (Nickrent et al., 2002).

The representatives of the genus Aristolochia are well known because of their peculiar flowers, which attract pollinators by odour (Cammerloher, 1923, 1933; Daumann, 1971; Vogel, 1978; Burgess et al., 2004; Bänziger & Disney, 2006; Trujillo & Sérsic, 2006). Most of the insects attracted to the flowers belong to various families of Diptera, among others Calliphoridae, Ceratopogonidae, Cypselidae, Drosophilidae, Heleomyzidae, Muscidae, Phoridae, Sepsidae and Ulidiinae (Lindner, 1928; Vogel, 1978; Razzak et al., 1992; Sakai, 2002; Burgess et al., 2004; Trujillo & Sérsic, 2006; Rulik et al., 2008).

The flowers use an elaborate mechanism that enables insect trapping, retention and release, to ensure cross-pollination. Aristolochia flowers possess a monosymmetric, extensively modified perianth, which is subdivided into three main parts (Correns, 1891; Cammerloher, 1923; González & Stevenson, 2000): the limb, tube and utricle (Fig. 1a,b). The utricle is the most basal part of the perianth. It is a bloated structure that contains the gynostemium, a structure formed by the fusion of the style and the stamens. The tube above the utricle is a narrow zone having its smallest diameter at its proximal end. The apical end of the tube is extended by the limb, the only part of the flower that is not tubular but laminar in the plant species investigated.

Figure 1.

 Morphology of Aristolochia flowers. (a) Schematic drawing of a flower. (b) Longitudinal section through the tube and utricle. (c–e) Trapping trichomes are composed of a foot, a joint and a single row of cells forming the trichome body (modified and redrawn from Correns, 1891). (c) The base of a trapping trichome composed of a unicellular foot and a unicellular joint. The foot cell possesses a thickened outer cell wall, fixing the trichome to the tube wall. Generally, the joint (cell) is more than two times longer than it is wide and is conical in shape. It joins the foot cell at its narrow end. The wide end is located eccentrically (at the part facing the utricle) at the hemispherical base cell of the trichome. In addition to turgor pressure, the joint cell is stabilized by a thickened upper cell wall. In some species, the thickening narrows gradually toward the trichome and ends in an abrupt manner shortly before the foot cell, forming a pore-like structure of unknown function. (d) A trichome bending down. (e) A trichome bending up. This is possible only until the thickened trichome base rests against the tube wall.

The flowers show a pronounced proterogyny and attract flies by odour only during the first (female) flowering stage (Cammerloher, 1923, 1933; Daumann, 1971; Burgess et al., 2004). Previous authors have observed that insects first arrive on the flower limb and either directly slide into the tube (e.g. Aristolochia clematitis; Daumann, 1971) or walk inside the tube, lose their foothold and slip off (e.g. Aristolochia grandiflora; Cammerloher, 1923; Aristolochia lindneri; Cammerloher, 1933). Once captured, insects are fed by nectar produced by glands located on the utricle wall (Daumann, 1959; Trujillo & Sérsic, 2006). According to Brantjes (1980) and Rulik et al. (2008) pollinators must fulfill the following size requirements: they have to be small enough to pass through the smallest part of the tube but also big enough to be able to interact with the gynostemium to unload and upload pollen grains. Proximally around the gynostemium (Fig. 3b,d) the utricle wall shows a pale translucent circle (‘light window’; McCann, 1943). As trapped insects show a strong positive phototaxis, they are attracted by light at the bottom of the utricle, indicating a false exit from the trap (Cammerloher, 1923). The same behavior is found in flies trapped in Arum (Knoll, 1926) and in Ceropegia (Vogel, 1961). Whilst walking around the gynostemium the flies presumably deposit pollen on the stigmatic lobes, while the pollen sacs of this individual flower are still closed. The male flowering stage is usually observed on the second day of anthesis. The stigmatic lobes wilt and fold together. The anthers ‘burst explosively’ and thus the whole utricle is dusted with pollen grains (Razzak et al., 1992). Insects usually carry most pollen grains on their thorax (Razzak et al., 1992; Burgess et al., 2004; Trujillo & Sérsic, 2006; Rulik et al., 2008). The release of insects takes place 24–48 h after the beginning of anthesis (Razzak et al., 1992).

Previous authors have assumed that specialized trapping trichomes (Fig. 1c–e), located at the inner perianth surface, are responsible for the anisotropic frictional properties of the inner surface of the flower tube as a consequence of their peculiar suspension over the underlying surface. Representatives of the subgenera Pararistolochia and Aristolochia (González & Stevenson, 2000; Wanke et al., 2006) possess these prominent, conical, white or colored trichomes (González & Stevenson, 2000) with an enlarged base and a thin joint cell (Correns, 1891) within the flower tube. It was observed that insects can pass through the tube without any difficulty in the direction of the utricle, but are unable to leave the utricle and so are trapped. Both the eccentric location of the joint cell at the hemispheric trichome base and the orientation of the trichomes in the tube result in the trichomes bending inward relatively easily but bending outward much less readily (Correns, 1891; Cammerloher, 1923). This construction presumably creates an impenetrable barrier, which can imprison insects within the utricle during the female flowering stage. However, a rigorous biomechanical approach demonstrating frictional anisotropy of trapping trichomes is still lacking. This study was undertaken to measure friction forces within the tube in different directions and to combine these data with structural information derived from scanning electron microscopy (SEM). Flowers of six Mediterranean Aristolochia species, belonging to the subgenus Aristolochia, were subjected to comparative study, in order to obtain new insights into the trapping mechanism of the flowers.

This study combines both structural and experimental approaches, demonstrating the function of trapping trichomes in the flowers of Aristolochia specialized for the trapping, retention and release of insect pollinators. Since Correns’ investigation of the trapping devices of Aristolochia flowers using simple light microscopy in 1891, no further study has been undertaken using newer approaches. The morphological part of this study aimed to investigate the distribution and morphology of trapping trichomes, in particular by SEM, to obtain new structural information and further insights into the functionality of these highly specialized trapping devices. The experimental approach addresses the following questions: firstly, is more force required for an insect to escape than to enter the tube of an Aristolochia flower at the female flowering stage? Secondly, does the successful escape of an insect damage the trap in such a way that further insects may escape more easily, and is the flower able to trap further insects after such an event?

Materials and Methods

Species studied and their floral morphology

This study was performed on the six species Aristolochia baetica L., Aristolochia clematitis L., Aristolochia clusii Lojacono, Aristolochia navicularis Nardi, Aristolochia rotunda L. and Aristolochia sempervirens L. Plants used for this study were collected in their natural habitat and cultivated in the Botanical Garden of the Technische Universität Dresden, Germany (Supporting Information Table S1). The inner perianth surface of fresh flowers of the female and male flowering stages was investigated by light microscopy stereomicroscope (TSO Pulsnitz, Germany); magnification from ×6.4 to ×64 in nine distinct steps). Flower images were obtained with a digital SLR camera (Canon EOS 300D, Canon Inc., Tokyo, Japan) and a macro lens (Canon Macro EF 100 mm 1:2.8 II). Flower dimensions were measured either using a stereomicroscope with an ocular micrometer (inner tube diameter at its proximal end and the distance between the utricle wall and the gynostemium at the female flowering stage) or from digital pictures obtained by photography or SEM using Adobe Photoshop software (e.g. length of trapping trichomes at the proximal tube end).

Scanning electron microscopy

Samples of 5 × 5 mm were cut out of the limb and the tube of fresh flowers, dehydrated in an ascending series of ethanol and critical-point-dried in carbon dioxide in Emitech K850 (Emitech Ltd., Ashford Kent, UK) or CPD 030 (BAL-TEC AG, Balzers, Liechtenstein) critical-point driers. Then samples were fixed to aluminum sample holders (Plano GmbH, Wetzlar, Germany) using a carbon adhesive tape (Leit-Tabs; Plano GmbH), sputter-coated with a 20-nm-thick gold layer under an argon atmosphere using the sputter-coater Emitech K550 (Emitech Ltd.), and viewed in SEM LEO 420 (Leo Electron Microscopy Ltd., Cambridge, UK) at an acceleration voltage of 15 kV.

In order to obtain information about surface structures at a high resolution in freshly frozen material, a Cryo-SEM Hitachi S 4800 (Hitachi High-Technologies Corp., Tokyo, Japan) equipped with a Gatan ALTO-2500 cryo preparation system (Gatan Inc., Abingdon, UK) was used. Fresh flowers were cut in half with a sharp razor blade, fixed to a metal holder with TissueTek® mounting fluid (Sakura Finetek USA, Inc., Torrance, CA, USA), shock-frozen in liquid nitrogen, transferred to the cryostage of the preparation chamber (−140°C), and then sublimated at a temperature of −90°C for 3 min, to remove contamination by condensed ice crystals. The frozen sample was coated with gold-palladium (3 nm thickness) and investigated at −120°C and an acceleration voltage of 3 kV.

Force measurements

Force measurements were performed on fresh female-stage flowers (n = 10–28 from one to 14 populations per species; see Table S1 for the origin of the plants). Flowers were prepared by cutting off the utricle and the limb (Fig. 2a). To avoid artificially increased traction forces in U-shaped tubes, flowers of A. sempervirens and A. baetica were cut off at the middle of the curvature (Fig. 2b) and only the proximal tube part was used, so that the tube measured was nearly straight. As the pollinators of the investigated species are not yet known, a small glass sphere, covered with a mixture of beeswax and colophony (weight ratio 1 : 1), simulated the body of an insect moving through the tube. Attached to a thin nylon thread (diameter 0.2 mm) by a droplet of beeswax/colophony, the sphere was pulled through the flower tube by a Zwick/Roell testing machine (Z2.5/TS1S, Zwick GmbH & Co. KG, Ulm, Germany) at a constant speed of 0.5 mm s−1. Forces applied to the sphere by the flower were measured at a sampling frequency of 50 Hz by the force transducer FORT 25 (World Precision Instruments Inc., Sarasota, FL, USA) (Fig. 2d). Each flower was tested four times in both directions to investigate the functionality of trapping trichomes at the female flowering stage. An insect entering the trap was simulated by the first traction of the sphere from the mouth toward the utricle in an inward direction (in1). The second traction of the sphere was performed in an outward direction from the utricle to the mouth (out1), simulating the successful escape of an insect. To study possible effects of the escape of an insect on the trapping efficiency of the flower, a third traction in an outward direction (out2) and a fourth traction inwardly orientated (in2) were performed. The sphere diameter for traction experiments had to be adjusted to floral size, indicating the possible pollinator size. During the first measurements in A. baetica, A. clusii and A. navicularis the sphere diameter was adapted to the flower size as follows: sphere diameter should be smaller than the average diameter of the proximal end of the flower tube; this represents the smallest tube diameter. During further measurements in A. clematitis, A. rotunda and A. sempervirens, sphere size was additionally adapted to the minimal pollinator size as follows: mean tube diameter (at its proximal end) > sphere diameter > mean distance between utricle wall and gynostemium (Fig. 2c). Selection of flowers for force measurements was performed randomly. In subsequent measurements, only the maximum measured force per traction was recorded, as this presents the amount of force an insect must generate to overbear the plant trap. Forces measured in different traction directions were compared for each tested flower as follows: traction in1 vs out1, out1 vs out2 and in1 vs in2; using the Wilcoxon signed-rank test for related samples.

Figure 2.

 Set-up for force measurements. (a) Section planes through the utricles and limbs of Aristolochia clematitis, Aristolochia clusii, Aristolochia navicularis and Aristolochia rotunda. (b) Section planes through the flowers of Aristolochia baetica and Aristolochia sempervirens. (c) Measurements on flowers: diameter of the proximal tube end and the distance between the gynostemium and the utricle wall. (d) Experimental set-up for force measurements. Outward-directed traction is shown; for inward traction the flower tube was turned upside down (180°). Hence, each flower was flipped twice for measurements. The arrow indicates the direction of the movement of the force transducer.


Morphology of flowers and trapping trichomes

Flower shape is rather similar within the investigated Aristolochia species, with the exception of tube shape and dispersion of trapping trichomes. Flowers of A. clematitis, A. clusii, A. navicularis and A. rotunda have a straight (Fig. 3a,b) to slightly curved tube whereas A. baetica and A. sempervirens have a U-shaped tube (Fig. 3c,d). However, the proximal end of the tube always opens in the middle of the distal utricle region.

Figure 3.

Aristolochia flowers. (a–b) Aristolochia rotunda. (c–d) Aristolochia sempervirens. (a, c) Frontal-lateral view. (b, d) Longitudinal section.

All six species have trapping trichomes within the flower tube (Fig. 3b,d) and, with the exception of A. clematitis, also have morphologically similar trichomes at least on parts of the limb. In A. baetica, the whole surface of the flower limb is covered by these trichomes, whereas in A. clusii, A. navicularis and A. sempervirens large regions are hairy. In A. rotunda only the border area of the lower limb has trichomes. Within the tube, trichomes are arranged irregularly (Fig. 4a). They are either erect or pointing toward the utricle. Thus, in flowers of A. clematitis, A. clusii, A. navicularis and A. rotunda, trichomes are generally orientated downward. In contrast, because of their U-shaped tubes, the trichomes within the proximal tube part of A. baetica and A. sempervirens are pointing upward in the direction of the utricle.

Figure 4.

 Scanning electron microscopy (SEM) micrographs of trapping trichomes within the flower tube. (a–f) Aristolochia rotunda. (a) Longitudinal section through the flower tube at the female flowering stage. Note that the trichome length increases in the proximal direction of the flower. (b) Junction of the tube and the utricle at the female flowering stage. In comparison with the distal parts of the tube, trichomes in this region stand closer together and are longer. (c) Junction of the tube and the utricle at the male flowering stage. Shriveled trichomes clear the tube diameter and provide many anchor points for insect claws. (d) Turgid trapping trichome at the female flowering stage within the tube. (e) A joint cell located eccentrically at the thickened trichome base. (f) A shrunken trapping trichome at the male flowering stage. (g) Trapping trichomes of Aristolochia baetica. (h) A trapping trichome of Aristolochia clusii. (i) A trapping trichome of Aristolochia sempervirens. (k) A trapping trichome of Aristolochia navicularis. (l) A trapping trichome of Aristolochia clematitis. (a–d and f–l: SEM + critical point dried; e: cryo-SEM). d, distal direction; j, joint; p, proximal direction; jtu, junction tube to utricle; t, tube; tr, trapping trichome; u, utricle.

The density of trapping trichomes increases gradually from the mouth to the utricle. Trichomes are most densely packed at the junction of tube and utricle (Fig. 4b). Both trichome length and trichome cell number vary among regions of the flower tube and among the investigated species. Aristolochia rotunda and A. navicularis have very short, almost drop-shaped trapping trichomes within the distal parts of the tube. Trichome length increases rapidly in the proximal direction, whereas in other species trapping trichomes are already relatively long in the distal parts of the tube and thus their length increases less markedly in the direction of the utricle. Trichome length at the proximal end of the tube (Table 1) varies, but in all species studied, trapping trichomes either reach about half the length of the tube diameter in this area or are not much shorter. Cell number per trichome increases with trichome length in all investigated species. It ranges, for example, in A. rotunda from eight-celled trichomes in the mouth region to 38-celled trichomes at the proximal part of the tube (n = 10).

Table 1.   Measurements of Aristolochia flower parts
SpeciesFlower dimensions
n1Mean ± SDMinMax
  1. Tube diameter and trapping trichome length were measured at the proximal tube end.

  2. 1Sample size; overall number of flowers measured (in all populations).

  3. NA, not applicable.

Aristolochia baetica
Tube diameter (mm)303.5 ±
Distance gynostemium/utricle wall (mm) NA  
Trapping trichome length (μm)41498 ± 19813211672
Aristolochia clematitis
Tube diameter (mm)271.5 ±
Distance gynostemium/utricle wall (mm)270.7 ±
Trapping trichome length (μm)10804 ± 35723850
Aristolochia clusii
Tube diameter (mm)322.4 ±
Distance gynostemium/utricle wall (mm) NA  
Trapping trichome length (μm)10907 ± 847731053
Aristolochia navicularis
Tube diameter (mm)132.2 ±
Distance gynostemium/utricle wall (mm) NA  
Trapping trichome length (μm) NA  
Aristolochia rotunda
Tube diameter (mm)541.6 ±
Distance gynostemium/utricle wall (mm)540.6 ±
Trapping trichome length (μm)24766 ± 1465321070
Aristolochia sempervirens
Tube diameter (mm)212.2 ±
Distance gynostemium/utricle wall (mm)211.3 ±
Trapping trichome length (μm)10877 ± 1036971025

Trapping trichomes are in general multicellular and have a thickened base beneath the conical trichome shaft (Fig. 4d,g,h,k,l), except for A. sempervirens, whose trapping trichomes have a cylindrical shaft above a round trichome base and a short thin apex (Fig. 4i). Cells of the trichome shaft are, in general, wider than they are long (barrel-shaped). Unfortunately, it proved to be difficult to observe the joint cell because of its position below the thickened trichome base. Nevertheless, observations in A. rotunda revealed that the joint is in general one-celled (Fig. 4e) but it can rarely also be two-celled. Trichomes are densely covered with small wax rods.

The morphology of trapping trichomes changes considerably during the male flowering stage, when the trichomes tend to wilt and shrink. The lateral cell walls of each cell crumple (Fig. 4f), and thus the whole trichome shrinks in the longitudinal direction. Because all trapping trichomes within the flower tube shorten in the same way, a free space in the center of the tube will be created (Fig. 4c).

Anisotropic properties of the trichome array

Aristolochia flowers show a degree of diversity in their smallest tube diameter and in the distance between the gynostemium and the utricle wall (Table 1). Nevertheless, the smallest tube diameter is generally larger than the distance between the gynostemium and the utricle wall. Because sphere size was adapted to mean floral dimensions, in some flowers the tube diameter at the junction of the tube and the utricle proved to be too small for the sphere size used. Hence, in some cases the sphere became stuck inside the flower tube (Table 2). In many flowers, some loss of trapping trichomes could be observed after the first outwardly directed traction of the sphere. Some detached trichomes dropped down, and others jammed and stayed in their new upward orientated position within the flower tube.

Table 2.   Sphere sizes used for traction experiments and number of Aristolochia flowers (n) investigated
SpeciesSphere diameter (mm)nNumber of cases where the sphere stuck in the flower tube during tractionForce (mN)
Aristolochia baetica2.001100.10.940.390.36103.0334.920.7164.2232.70.134.940.34
Aristolochia clematitis0.80700.
Aristolochia clusii2.001814.32150.944.022.2193.272.63.37193.539.60.8999.117.7
Aristolochia navicularis1.05400.220.530.306.7322.310.17.1116.912.80.320.450.43
Aristolochia rotunda0.801700.050.810.140.1712.
Aristolochia sempervirens1.00400.1210.81.085.0725.610.31.6619.93.760.1223.20.52

Time–force curves show multiple force maxima during the pulling of the sphere in different directions relative to the trap. Maximum force was measured in both directions when the sphere passed across the junction of tube and utricle, which represents the narrowest part of the flower tube. Force measurements when the sphere was pulled toward the utricle (Fig. 5a) revealed that the force increased within the flower tube until the sphere had passed through this narrowest area. In the opposite direction several additional force peaks were registered after the sphere had entered the tube (Fig. 5b).

Figure 5.

 Time–force curves obtained for a flower of Aristolochia clematitis. (a) Traction of the sphere into the flower (in1). (b) Traction of the sphere out of the flower (out1).

Comparison of maximum forces measured in different directions showed significant differences. In all investigated species, except for A. clusii, a significantly greater force had to be applied in the direction out of the flower (out1) than in the direction into the flower (in1) (Table 3a). Once a sphere had been pulled out (out1), a second attempt (out2) required significantly lower force in all investigated species (Table 3b). In addition, after one sphere had been pulled out of the flower (out1), subsequent experiments in pulling spheres into the same flower (in2) showed that a significantly greater force than before (in1) was required in A. clematitis and A. rotunda, whereas a significantly lower force was required in A. clusii (Table 3c).

Table 3.   Comparison of traction force measurements through the Aristolochia flowers in different directions by Wilcoxon signed-rank test for related samples
Speciesn1Median force2
Aristolochia baetica160.6750.11≤0.001136.0
Aristolochia clematitis171.2610.08≤0.001131.0
Aristolochia clusii2258.3679.790.080109.0
Aristolochia navicularis1013.7365.950.03741.0
Aristolochia rotunda240.172.03≤0.001298.0
Aristolochia sempervirens76.0415.340.01628.0
Speciesn1Median force2
Aristolochia baetica1650.1140.540.044−78.0
Aristolochia clematitis1710.081.06≤0.001−143.0
Aristolochia clusii2279.7944.440.001−197.0
Aristolochia navicularis1065.957.030.002−55.0
Aristolochia rotunda242.030.15≤0.001−296.0
Aristolochia sempervirens715.340.560.016−28.0
Speciesn1Median force2
  1. Medians for the whole sample for each species and traction type are given. Note that the median values given in this analysis cannot be compared among species, as the number of measurements of smaller and larger spheres varied among species (see Table 2).

  2. 1Sample size.

  3. 2Force values for in1, in2, out1 and out2 are given in (mN).

  4. 3Probability of error; P-values < 0.05 are considered significant.

  5. 4Wilcoxon signed-rank statistic.

Aristolochia baetica160.670.7150.782−12.0
Aristolochia clematitis171.268.80.005115.0
Aristolochia clusii2258.3617.68≤0.001−235.0
Aristolochia navicularis1013.7319.441.0001.0
Aristolochia rotunda240.170.760.004202.0
Aristolochia sempervirens76.044.290.5788.0

Depending on the ratio of the sphere diameter to the tube diameter, measured maximum forces differed greatly among individual flowers. A trend toward greater forces was found for higher ratios (Fig. 6). The difference between maximum forces for pulling the sphere in (in1) and out (out1) of the flower also varied considerably among flowers. In 9% of all measured flowers of the investigated species, force in1 was higher than force out1 (ratio out1 to in1 <1) and in another 9% of the flowers the sphere became stuck inside the flower tube. All other flowers (82%) showed a ratio of out1 to in1 >1. The highest recorded ratio of 294 (in1 = 0.35 mN; out1 = 103.0 mN) was found in a single flower of A. baetica.

Figure 6.

 The force measured when the sphere was pulled into the flower (in1) and out of the flower (out1) in different species of Aristolochia. Tube diameter was measured at the narrowest part, at the junction between the tube and the utricle.


The pollination mechanism in Aristolochia can be subdivided into four stages: insect attraction, trapping, retention and release. We have demonstrated that the last three stages are influenced by specialized structures or mechanisms, or a combination of these, facilitating a plant-insect interaction that was most likely to ensure specific pollination.

Force anisotropy

The experimental set-up was based on a model system and is only partly comparable to the natural insect–plant system. A small sphere was used as an insect substitute, and obviously did not replicate the complex shape of an insect with all its specialized surfaces and structures such as wings and legs, which would produce high resistance to locomotion inside the tube. Thus, measured forces correspond only to friction forces passively resisting the movement of the sphere inside the flower tube, in contrast to the traction force actively produced by an insect. In particular, the waxy surface of the trichomes reduces friction forces acting on the sphere, but will result in additional expenditure of energy for an insect negotiating the slippery trichomes (Harley, 1991).

When the sphere was moving into the tube, the maximum force was measured in the narrowest region, the junction of the tube and the utricle. After the sphere had passed through this region, an abrupt decrease in the force was recorded. Hence, in species with a straight flower tube, there is a high probability that an insect will drop down into the utricle, because of a lack of structures allowing the insect to cling to the wall. In contrast, in species with U-shaped tubes, insects have to overcome the proximal, upward-orientated part of the tube before they can enter the utricle. This is supported by the orientation of trapping trichomes, and a gradually paler coloration of the flower tube and utricle. Furthermore, the light window (see Fig. 3) around the gynostemium simulates a false exit at the proximal end of the flower (Knoll, 1926; Vogel, 1961; Masinde, 2004). As trapping trichomes can be bent only into an erect position, insects may use them as a ladder to climb into the utricle.

Force measurements in the outward direction are related to the hypothetical situation of an insect escaping from the flower during the female flowering stage. The trap morphology exhibits strong resistance to an outward-directed movement. This is not only a consequence of the structural organization and waxy surface of the trichomes, but also of the disadvantageous lever for an insect to apply force to the trichome array.

The trapping mechanism

Trapping trichomes of Aristolochia flowers play an important role in the trapping, retention and release of insects during anthesis and therefore in the specialization of Aristolochia pollination biology, through the size and force restrictions they impose on pollinators. The force measurements support the trichome functionality previously described by Correns in 1891. Trichomes can easily be bent downward because of the eccentric location of the joint cell at the trichome base (Fig. 1d). If the trichome is bent in the opposite direction, it locks the tube (see Fig. 1e; Correns, 1891). In our experiments, the sphere pulled through the flower tube experienced considerably higher resistance during outward-orientated traction than when directed inward. Only measurements in A. clusii showed no significant difference between forces measured during inward- and outward-orientated movements of the sphere, although the data indicate a tendency similar to that observed in the other species investigated.

In a few flowers the measured inward force (in1) was greater than the outward force (out1). A similar peculiar finding was obtained in a few cases where the hypothetical insect became stuck in the flower tube. Also Rulik et al. (2008), investigating flowers collected and preserved in the wild and the insects trapped at the time of preservation, found that in nature it seldom happens that insects block the flower tube. The consequence for the flower of an insect blocking the tube is that no more insects are able to enter and any trapped insects are unable to escape. Hence, any such flower arising through natural morphological variation is unable to distribute its own pollen, but can probably be pollinated by already trapped insects.

The retention mechanism

What happened within the flower tube during outward traction that resulted in a significant increase in resistance force? The broad barrel-shaped cells of the trichome shaft and high turgor pressure cause stiffness of the trapping trichomes (Correns, 1891). In order to bend such a stiff trichome, large forces are required. In our experiments, some trichomes were even ripped off the tube wall by the movement of the glass sphere. Because of the specific anatomy of the trichome foot (Correns, 1891), ripping of a trapping trichome from the tube wall requires large forces. Furthermore, the dense arrangement of trichomes, as well as the small tube diameter, prevents single trichomes from being tilted sidewards by moving insects. Otherwise, trichomes in general not only constitute a mechanical barrier for trapped insects but may also provide anchor points for their claws and attachment pads (Gorb & Gorb, 2002) for climbing out of the trap. To ensure the functioning of the trapping device, Aristolochia flowers have to avoid this undesirable side effect. It is known that insects use their claws to cling readily to thin trichomes but rely on their attachment pads to adhere to thicker ones (Voigt et al., 2007). The trapping trichomes of Aristolochia flowers are relatively thick and have a smooth surface without any cuticular texture. Hence, according to Dai et al. (2002) and Gorb & Gorb (2002) they are assumed to provide almost no anchor points for insect claws. Additionally, the functioning of attachment pads is thought to be impaired by the dense wax covering of the entire trichome surface (according to investigations by Gaume et al., 2002, 2004; Gorb & Gorb, 2002, 2006; Gorb et al., 2005). Consequently, the morphological and biomechanical properties of trapping trichomes result in a highly effective device for the trapping and retention of insects.

However, in the event that trapping trichomes could not prevent the escape of a hypothetically powerful insect, the trap would be likely to be at least partially destroyed by the moving insect. Force measurements showed that, after the first pulling of the sphere outwards, another attempt required a significantly lower force to allow the sphere to pass through the trapping trichomes in all investigated plant species. Partial destruction of trichomes was also seen during further insect trapping attempts. In the case of such damage to the trichomes, the access of insects may be hindered by outwardly orientated interlocked trichomes as well as detached trichomes which are likely to block the flower tube. In A. clematitis and A. rotunda this kind of damage means that a greater force is required for a second entrance of the sphere. Aristolochia baetica, A. navicularis and A. sempervirens did not show differences between the tractions of in1 and in2, whereas in A. clusii a significantly lower force was required for traction of in2 than for that of in1. The results obtained for A. clusii are probably attributable to the fact that the trichome length at the most narrow diameter of the tube is much smaller (1.3 times) than half of the tube diameter. This might also be the reason for the nonsignificant difference between the forces measured in in1 and out1. Aristolochia clematitis and A. rotunda have trichomes as long as or even longer than half the tube diameter, which causes jamming of the trichomes and therefore higher resistance to the second traction trial into the flower (in2). In A. baetica and A. sempervirens, trichomes are only a little smaller than half the tube diameter.

The release mechanism

As cross-pollination is based on the transfer of pollen from one individual to another, flowers have to disengage their retention mechanism and allow captured insects to escape and perhaps become trapped in another flower. Disengagement of the retention mechanism is ensured by the specific way in which trapping trichomes wilt after opening of the anthers. Nearly the entire tube diameter is cleared by the shrinking of the trichomes, providing enough space for insects to move through the former trap. On the basis of the findings of Betz (2002) and Dai et al. (2002) on beetle attachment performance on smooth and rough surfaces, we conclude that the heavily corrugated surface of the folded trichomes provides for insects ample opportunity to interlock within the flower tube by means of their claws. Insects may use the trichomes like a ladder to climb out of the trap. The shrinking of trichomes may be a result of water loss and their barrel-shaped cell morphology which promotes crumpling only of the lateral cell walls. In addition, programmed cytoskeleton activity may also play a role in the shrinking process of trapping trichomes providing the particular geometry of trichomes after shrinkage.

Comparison of trapping-release mechanisms among Aristolochia species

The force required for the sphere to pass through the flower tube tends to be affected by the size of the tube, the length and density of the trapping trichomes, and the sphere diameter used. Variability in both flower size and measured force among individual flowers of the same species is rather high. Because of the irregular arrangement of trichomes on the inner surface of the tube, insects have to push several trichomes simultaneously to move through the flower tube. A relatively small force is required to bend a single trichome, but the total force required to bend dense arrays of trichomes can be rather large and thus impossible to generate for small insects.

Small dipteran flies of the genus Megaselia (Phoridae) are recognized as pollinators of the closely related Mediterranean species Aristolochia pallida (Rulik et al., 2008), but have also been recorded as the second most frequent flower visitors in A. baetica and A. paucinervis in Spain (Berjano et al., 2009). This indicates not only that Phoridae should be regarded as prevalent pollinators but also that the diameter for our hypothetical insect, inferred from flower morphology, is appropriate. Flies of this genus are c. 0.8 mm high (Rulik et al., 2008) and have an average fresh weight of 0.5 mg for males and 0.9 mg for females (B. Rulik, pers. comm.). As A. pallida has similar floral morphology and flower size as the species investigated in this study (B. Oelschlägel, unpublished), it is likely that they also have pollinators of similar size. Assuming that in forward-orientated movements these insects are only able to generate pushing forces comparable to their weight, they will not be able to bend a trapping trichome array outward during the female flowering stage. This conclusion is supported by resistance measurements of the trichome array, under the assumption that the linear regression models presented by Gorb et al. (2001, 2002), describing the correlation between the friction force generated by an insect and its weight, can be applied to phorid fly species. According to that regression model, F = 0.601 + 0.019 × m (where F is the friction force in mN, and m is the insect mass in mg), individuals of the genus Megaselia should be able to generate forces of up to 0.62 mN. The forces measured during our experiments (for out1, from an average of 7.7 mN for A. rotunda to an average of 99 mN for A. baetica; and for in1, from an average of 3.3 mN for A. rotunda to an average of 69 mN in A. clusii) suggest that insects of this small size will probably not be able to bend trapping trichomes in either the inward or outward direction during the female flowering stage, as the forces applied must be at least six times higher than those the fly can potentially generate. This finding is inconsistent with the fact that insects enter the flower for pollination. The explanation for the inconsistency is probably that insect forces have been underestimated. Data by Gorb et al. (2001) show adhesion and adhesion-mediated friction of insects on a smooth surface. Traction force generated by interlocking on a slightly rough surface by means of the claws might be up to two times stronger than traction generated by adhesive pads (Gorb et al., 2005). The relationship of insect mass and generable force might deviate in very small insects like representatives of the genus Megaselia. Additionally, the sphere used in our experiment generally bent several trichomes at the same time whilst being pulled through the trap and thereby experienced a significantly higher traction force than a single trichome would induce. We assume that an insect slipping off the lip will initially be stopped somewhere in the middle of the tube by several trichomes. However, because of the slippery surface and a lack of anchor points it cannot climb out of the trap. Every time it tries to get a foothold within the flower tube, it will slide deeper by gravity and actively bend single trichomes downward (B. Oelschlägel, unpublished).

As only insects of a certain maximum size can enter the flower through its tube, it must be ensured that insects too large to enter do not congest the trap. In contrast to the situation in the larger flowering tropical Aristolochia species, insects cannot turn around after entering the flowers of Mediterranean Aristolochia, because of the small tube diameter. Smaller insects can go through the tube, but are only of accidental benefit to the plants with respect to pollination. Even if they carry pollen by chance and deposit some grains on the stigmatic lobes, they can probably escape by slipping through the trapping trichomes before the flower enters the male flowering stage, consequently carrying no new pollen. Rulik et al. (2008) revealed a high specificity in pollinator attraction, probably attributable to pheromone-like substances, because only male representatives of the genus Megaselia (Phoridae) were found in A. pallida flowers collected in their natural habitat. Hence, pollinator specificity in Mediterranean species seems more likely to be effected by flower scent than by tube diameter, thus ensuring that the flowers attract only insects of a specific size.

To fully understand the trapping mechanism in Aristolochia, the next step must be to rigorously investigate the attracting mechanisms, which are probably scent and pheromone driven. We believe that the Mediterranean Aristolochia species are a good model for such a study, as specific attraction has already been demonstrated and size restrictions defined by the flower might be more specific in smaller Aristolochia flowers than in the larger ones of the tropics.

Trapping mechanisms and their functions in other angiosperm pitfall traps

During plant evolution, pitfall traps have been developed independently in different plant lineages and for different purposes. In carnivorous plants, such as Nepenthes (Nepenthaceae) and Sarracenia (Sarraceniaceae), traps evolved for additional nutrition using a completely different trapping mechanism without a specialized release function (Barckhaus & Weinert, 1974; Gaume et al., 2002, 2004; Bohn & Federle, 2004; Gorb et al., 2004, 2005). For pollination purposes, pitfall traps have been developed in representatives of the angiosperm families Araceae, Aristolochiaceae, Apocynaceae s.l., Hydnoraceae and Orchidaceae. In contrast to pitcher plants, flowers must ensure successful release of caught insects after a certain time.

In general, two different trapping mechanisms have evolved, with one or both being present in all pitfall traps: sliding areas and specialized trapping trichomes. In addition to the genus Aristolochia, trapping trichomes have been reported for pitfall flowers of some representatives of the genus Ceropegia (Apocynaceae s.l.; Müller, 1926; Vogel, 1961; Masinde, 2004) as well as for pitchers of the carnivorous plant Sarracenia purpurea (Barckhaus & Weinert, 1974). Large, horizontally orientated tentacles derived from modified male flowers, long known to be trapping trichomes, obstruct the entrance to the pitfall chamber in Arum nigrum (Araceae). According to Knoll (1926), insects captured by Arum nigrum try to escape not by flying but by walking, and he assumed that these tentacles do not block the passage of insects as do those of Aristolochia and Ceropegia but have slippery areas that prevent their escape. In an experiment with living insects, he showed that they lose foothold on the surface as long as the female flowers of the inflorescence are receptive, but on the second day of anthesis, when female flowers have wilted and male flowers are open, the surface properties of the tentacles have changed and insects can easily cling to them.

Flowers of the genus Ceropegia have developed two different types of long, unicellular trapping trichomes (Vogel, 1961). Ceropegia woodii has simple, conical trichomes that are anchored at an acute angle in the epidermal layer by a broad foot and point downward with their narrowed tip (Müller, 1926; Vogel, 1961). Insects that move inward can easily bend the trichome tip while the thicker trichome base remains straight. If the trichomes are bent in the opposite direction they become entangled. The second trichome type occurring, for example, in Ceropegia ampliata looks very similar to the trichomes of Aristolochia. Above a globose foot that fixes the trichome to the tube wall, the trichome has a short, narrow zone that functions as a joint. The remaining part of the trichome is conical. Similar to the trapping trichomes of Aristolochia, the joint is located eccentrically at the trichome base, resulting in a downward-pointing orientation of the trichome. The function of these trichomes is similar to that of the trichomes in Aristolochia trap flowers described above. Since Ceropegia trichomes are unicellular they maintain their stiffness through turgor pressure and a spiral structure of the cuticle (Vogel, 1961). As in the genus Aristolochia, the trapping trichomes of Ceropegia wilt in the male flowering stage (Müller, 1926; Vogel, 1961), probably by actively losing water. Trichomes collapse to a flat and corkscrew-like band (Vogel, 1961), providing many anchor points for insect claws. Representatives of Aristolochia and Ceropegia exhibit a similar degree of specialization in the plant–pollinator interaction (e.g. Rulik et al., 2008; Ollerton et al., 2009). Although the trapping trichomes of Aristolochia and Ceropegia have the same function (trapping, retention and release), there is at least in some species a basic difference in morphology, which is probably attributable to the specific way in which pollen is loaded onto insects. Pollinators of Aristolochia carry loose pollen grains on the back of the thorax, which are easily wiped off by stiff structures such as trichomes. Hence, flowers have to indirectly ensure pollen transport through the flower tube into the utricle by having trichomes that easily bend through narrow joints. However, Ceropegia does not necessarily need such a mechanism. In contrast to the situation in Aristolochia, Ceropegia pollen is transported as clusters (pollinia), which are attached securely to the insect by a clamp body.

Trichomes of the trapping zone of Sarracenia pitchers are anatomically distinct from Aristolochia trichomes. The needle-like trichomes of Sarracenia are one-celled and point downward. In comparison to the cell lumen, the cells have a very thick cell wall. The trichome foot has a secondary cell wall two to three times thicker than the surrounding epidermal cells, fixing it securely to the pitcher wall. The trichome foot and the remaining trichome form an acute angle at the proximal side that results in a downward orientation of the trichome. Trichome stiffness is achieved through an irregular thickening of the secondary cell wall, especially at the tip. Only a ring-like cuticle covers the trichome base, whereas the surface of the remaining trichome is smooth (Barckhaus & Weinert, 1974). In contrast to the trapping trichomes of Aristolochia and Ceropegia, the anatomical properties of Sarracenia trichomes do not allow any active change in morphology or even trichome shrinkage as a result of water loss. This is probably the most parsimonious adaptation to a pure trapping, nonreleasing function in Sarracenia pitchers.


The force measurements performed in this study confirm the important role of trichomes in the trapping mechanism of Aristolochia pitfall flowers. Insects do not need to exert large forces to enter the trap. In some species (with a straight tube), gravity and a lack of anchor points may allow insects to slide along the trapping trichome array into the trap. Any attempts to cling to the inner tube surface lead to the insect losing foothold (B. Oelschlägel, unpublished). To escape from the flower during the female flowering stage, a large effort is required which is probably not achievable by insects of this size. Even if an insect were theoretically able to bring sufficient force to bear on the trichome array in the outward direction to allow it to push through, the form and arrangement of the trichomes within the flower tube would not allow the insect to anchor itself sufficiently securely to enable it to apply that force. During the male flowering stage, traps allow insects to escape by clearing the flower tube and providing sufficient anchor points on the withered trapping trichomes.


We would like to thank Markus Günther, Ralf Helbig, Hafez Mahfoud, Andreas Metzker, Walter Amos Müller, Björn Rulik and Dagmar Voigt (in alphabetical order) for technical assistance, the contribution of plant material and helpful discussions. Vicky Kastner provided linguistic corrections which are gratefully acknowledged. This work was partly supported by the SPP 1420 priority program of the German Science Foundation ‘Biomimetic Materials Research: Functionality by Hierarchical Structuring of Materials’ (project GO 995/9-1) and by a grant from the Landesstiftung Baden-Württemberg (project ‘Plants as Source for Biomimetics of Anti-Adhesive Surfaces’) to SG. Last but not least, we gratefully acknowledge helpful suggestions for improving the manuscript from three anonymous reviewers.