Morphological Change of Trapping Flower Trichomes and ... · A. Open flower, showing a corolla tube...

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Morphological Change of Trapping Flower Trichomes and Flowering Phenology Associated with Pollination of Aristolochia debilis (Aristolochiaceae) in Central Japan

Takashi Sugawaraa,*, Sena Hirokib, Takaya SHiraib, Masayoshi Nakajia, Emiko oguric, Masahiro SueyoSHid and Akira SHimizue

aMakino Herbarium, Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1, Minami-Ohsawa, Hachioji, Tokyo, 192-0397 JAPAN;bDepartment of Biology, Tokyo Metropolitan University,

1-1, Minami-Ohsawa, Hachioji, Tokyo, 192-0397 JAPAN;cGraduate School of Science, Hiroshima University,

1-3-1, Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8526 JAPAN;dKyushu Research Center, Forestry and Forest Products Research Institute,

4-11-16, Kurokami, Kumamoto, 860-0862 JAPAN;eDepartment of Biological Science, Graduate School of Science and Engineering, Tokyo Metropolitan University,

1-1, Minami-Ohsawa, Hachioji, Tokyo, 192-0397 JAPAN*Corresponding author: [email protected]

(Accepted on November 4, 2015)

Morphological change of trapping flower trichomes and flowering phenology associated with pollination were investigated in a natural population of Aristolochia debilis (Aristolochiaceae) at Shiojiri, Nagano Prefecture in central Japan. This native species has flowers that attract mainly small flies (Milichiidae and Chloropidae). Like other species of the genus, the trap-like perianth of this species consists of the limb, tube and utricle. The specialized trichomes covering the inner surface of the corolla tube (trapping trichomes) are positioned perpendicular to the tube wall curving downwards, and trap the flies within the utricle for nearly a first day after anthesis. When the anthers dehisce at the second day, the trichomes gradually wilted, allowing the flies to escape from the utricle. The change of trapping trichomes from turgid to wilting is associated with the dehiscence of anthers, and provides the chance for the trapped flies to escape and transport the pollen grains from one flower to others.

Key words: Aristolochia debilis, Aristolochiaceae, Diptera, Milichiidae, pollination, protogyny, trapping flower trichomes.

J. Jpn. Bot. 91: 88–96 (2016)

Aristolochia L. is a woody or herbaceous liana distributed widely in tropical to subtropical and temperate regions in the world, and comprises more than 400 species (Mabberley 2008). Although the species show great diversity in floral morphology, their flowers commonly

share a tubular perianth composed of three parts: limb, tube and utricle (Proctor and Yeo 1972, Proctor et al. 1996, Matsuka 2001, Murata 2006), and are specially modified to trap the insect pollinators (Vogel 1978, Proctor et al. 1996). Their perianth limb is connected to the funnel-

April 2016 TheJournal of Japanese Botany Vol. 91 No. 2 89

like tube and is diversified in size, shape and color among the species. The funnel-like tube also differs among the species in size and shape, being straight, slightly bent, or remarkably bent (U-shaped). Most species have a particular tube, the inner surface of which is densely covered by downward directed trichomes (Proctor and Yeo 1972, Proctor et al. 1996, Oelschlägel et al. 2009), but some species lack such trichomes (Proctor et al. 1996). The utricle is usually like a balloon and contains the gynostemium formed by adnation of the styles and stamens. Flowers with such common features have been supposed to attract flies (Insecta: Diptera) and to be pollinated by them (Daumann 1959, 1971, Proctor and Yeo 1972, Vogel 1978, Faegri and van der Pijl 1979, Proctor et al. 1996, Willmer 2011).

Actually, the flies attracted to some flowers of Aristolochia belong to various families of Diptera: Calliphoridae, Ceratogpnidae, Chloropidae, Cypselidae, Drosophilidae, Helemyzidae, Milichiidae, Muscidae, Phoridae, Sepsidae and Ulidiinae (Vogel 1978, Wolda and Sabrosky 1986, Razzak et al. 1992, Disney and Sakai 2001, Sakai 2002, Trujiro and Sérsic 2006, Rulik et al. 2008). Regarding the mechanism for pollination, it has generally been reported that the flowers are protogynous and attract flies by odor during the female flowering stage (Daumann 1971, Murugan et al. 2006, Oelschlägel et al. 2009). After being trapped within the utricle, the flies are prevented from escaping by the downward directed trichomes on the inner surface of tube (Razzak et al. 1992, Trujillo and Sérsic 2006, Rulik et al. 2008, Oelschlägel et al. 2009), and release of the flies probably takes place 24–48 h after anthesis accompanied with wilting of the trichomes and dehiscence of the anthers (Razzak et al. 1992, Murgan et al. 2006, Trujillo and Sérsic 2006). Therefore, the trichomes (known as the trapping flower trichomes) on the inner surface of the corolla tube have been regarded as important structures for pollination of Aristolochia flowers

(Razzak et al. 1992, Trujillo and Sérsic 2006, Oelschlägel et al. 2009). However, it is less well known how the exact timing of male and female maturation might influence the morphological change of the trapping flower trichomes. In addition, the duration of the male and female phases is supposed to differ among the species.

In Japan, there are six native species of Aristolochia (Murata 2006), but little is known on their pollination biology, including flowering phenology. Among the Japanese species, the flower of A. debilis Siebold et Zucc. is characterized by having a straight or slightly curved funnel-like tube, inner surface of which is densely covered with the trapping flower trichomes (Yamazaki and Kadota 1998). In this study we aim to describe the morphological change of the trichomes and the flowering phenology associated with pollination of Aristolochia debilis occurring in central Japan. We also examine flower visitors effective for pollination of this species.

Materials and MethodsStudy species and site

Aristolochia debilis Siebold & Zucc. is a herbaceous liana distributed widely in the Japanese archipelago (Murata 2006) and China (Hwang et al. 2003). This study was performed mainly in a natural population of A. debilis occurring at Seba (36°7ʹ31ʺN, 137°55ʹ42ʺE), Shiojiri City, Nagano Prefecture in central Japan. In this site, A. debilis occurs on the bank along a river and grows together with Pueraria lobata (Willd.) Ohwi, Achyranthes bidentata Blume var. tomentosa (Honda) Hara, and Sicyos angulatus L. Its flowers usually open from July to August, and possess an extensively modified perianth composed of three floral parts: limb, tube and utricle (Fig. 1).

Observation on morphological changes of the trapping trichomes and the gynostemium

To examine maturation of the stigma and anthers and change of the trapping flower

90 植物研究雑誌　第 91巻　第 2号 2016年 4月

Fig. 1. Flowers of Aristolochia debilis. A. Open flower, showing a corolla tube entrance (mouth part). B. Outer surface of utricule, showing two round thickenings. C. Longitudinal section of the flower, showing the gynostemium and the trichomes on the inner surface of corolla tube. D. Two nectariferous patches found in the inner surface of utricle. E. Turgescent trichomes on the inner surface of corolla tube. F. Wilted trichomes on the inner surface of corolla tube.

trichomes during a flowering period, we made two small holes on the utricle at the level of the gynostemium and on the corolla tube (see Fig. 2). Three flowers were monitored from

the flower bud stage to the wilting stage of the trapping trichomes, using a waterproof digital camera (Pentax Optio W90, Rico Co.) and photographs were taken at intervals of 10


Fig. 2. Temporal change of gynostemium and trichomes on the second day after anthesis in Aristolochia debilis. A. 03:06 h, anthers remain undehiscent and trichomes are turgescent. B. 05:26 h, anthers begin to be dehiscent, but trichomes still remain turgescent. C. 07:46 h, anthers are completely dehiscent and trichomes begin to wilt. D. 09:16 h, anthers are dehiscent and trichomes completely wilt.

Fig. 3. Morphological change of trichomes at the female and male stages in a flower of Aristolochia debilis. A. Trichomes are turgescent at the female stage. B. Trichomes that begin to wilt from the female to the male phases. C. Trichomes completely wilt at the male stage.

minutes at the study site on August 3–5 in 2012. Flower images were obtained using a

digital camera (Olympus E-520, Olympus Imaging Corp., Tokyo, Japan) with a macro lens (Olympus 50 mm 1:2 Macro). The change of

the flower trichomes on the inner surface of the corolla tube was also investigated with a light microscope (Olympus BH2, Olympus Co.).

Observation on flower visitors and their


behaviorFlower visitors were observed in the Seba

population of Aristolochia debilis at the daytime on August 2–3, 2010 and on August 3–5, 2012. In addition, we labeled flower buds with vinyl tape and monitored its flowers, focusing on the flowering stage. Following this treatment, we collected flowers at random in three different stages after anthesis: a) 15–16 h of the first day (half a day after anthesis), b) 8–9 h of the second day (one day after anthesis), and c) 15–16 h of second day (one and a half days after anthesis), and put them in a small sampling bag. Subsequently, we moistened these flowers with 100 % ethanol and counted the number of flowers catching flies to estimate the percentage of flowers with flies in each flowering time. Furthermore, we counted the number of flies per flower for the flower frequency of catching flies. The flies collected from the flowers were morphologically determined.

Statistical analysesStatistical test for the flower frequency

with insect visitors was performed using Excel Statistics for Macintosh ver. 5.0 software (Esumi Co. Ltd.).

ResultsFloral morphology of Aristolochia debilis

The flowers of Aristolochia debilis, like those of other congeneric species possess an extensively modified perianth composed of three floral parts: the limb, tube and utricle (Fig. 1). The limb is well developed only on one side, ovate-lanceolate, 2–3 cm long, and pubescent (Fig. 1A, C). The tube is straight or slightly curved, 2–2.5 cm long, 3–4 mm wide at base. Its inner surface is densely pubescent with multicellular trichomes (Fig. 1C, E, F). The utricle is subglobose, 8–9 mm in diameter and possesses two round thickenings (Fig. 1B). Its inner surface is densely pubescent and possesses two patches composed of particular trichomes distinguishable from the surrounding ones (Fig.

1D). These patches are always wet and therefore probably function as secretary glands. The stamens and styles form a gynostemium within the utricle (Fig. 1C).

Morphological changes of trapping flower trichomes and gynostemium

There were no major differences among the three examined flowers in flowering phenology. When the flower opened at early morning, the trapping trichomes were turgescent and oriented downwards (Figs. 1A, 3A). At this stage, the anthers did not dehisce, whereas the stigmatic lobe with turgescent papillae was functional and allowed the acceptance of pollen grains, which indicates that the flower was protogynous and was in the female phase (stage 1). This phase lasted up to the early morning of the second day after anthesis (Fig. 2A). At the early morning of the second day, the trapping trichomes gradually wilted from the base (Figs. 2C, 3B), and the anthers started to dehisce (Fig. 2B), while the margins of the stigmas were gradually folded upward and covered the stigmatic surface (Fig. 2C). In the morning of the second day, the trapping trichomes completely wilted (Figs. 2D, 3C) and all anthers were completely dehiscent, which means the flower was in the male phase (stage 2). This phase lasted up to the evening of the second day after anthesis. Subsequently, the perianth was retained for four to five days and finally abscised (stage 3), although the trapping trichomes had completely wilted.

Flower visitors and their behavior The flower visitors collected within the

flowers belong to two families of the Diptera: Milichiidae and Chloropidae. Most of the flies we collected are Desmometopa sordida (Fallén, 1820) of Milichiidae (Fig. 4B). Rarely, we observed the visitation of Polydaspis sp. of Chloropidae (Fig. 4C). These flies were small, about 2 to 3 mm in length. In the field, they often attempted to intrude into the utricle through the tube (Fig. 4A). We could not find pollen grains


dehisce, which indicates that the flower is in the male stage. The male stage in A. debilis begins at about 24 h after anthesis, which coincides with that of A. bracteolata (Razzak et al. 1992) and A. argentiana (Trujillo and Sérsic 2006), but differs from that of A. tagala (Murugan et al. 2006). Dehiscence of the anthers provides the chance that the pollen grains adhere to the flies. Actually, we observed that the flies having escaped from the flower carried the pollen grains on the thorax dorsally. Therefore, the time matching of the trapping trichome wilting and the anther dehiscence may be significant for the performance of pollination in this species.

Like other species of Aristolochia, visitation of small flies belonging two families of Diptera (Milichiidae and Chloropidae) was often observed in A. debilis. Although the behavior of these flies within the utricle still remains unclear, the flies escaping from the flower occasionally possess pollen grains on their thorax dorsally. We therefore suggested that the effective pollinators of A. debilis were probably small flies belonging to Milichiidae and Chloropidae. In earlier studies, visits of such flies were also reported in A. pilosa distributed in Panama (Wolda and Sabrosky 1986).

In many species of Aristolochia examined, it has been reported that nectaries are present on the inner wall of the utricle (Proctor and Yeo 1972, Proctor et al. 1996, Sakai 2002, Trujillo and Sérsic 2006), or are situated below the anthers (Razzak et al. 1992). The nectar exuded from the nectaries has generally been considered only as a food source to ensure the fly’s survival during captivity rather than a reward for flies (Razzak et al. 1992, Sakai 2002, Trujillo and Sérsic 2006). In A. debilis, two patches with particular trichomes on the inner surface of the utricle probably exude nectar. These patches seem to function as a food source for the flies trapped within the utricle.

In earlier studies, it was reported that the fruit production of Aristolochia debilis was rare under natural conditions (Yamazaki and Kadota

on the bodies of most flies collected from the flowers. However, we occasionally observed that some flies, which had escaped from the flowers, actually possessed pollen grains on the thorax dorsally (Fig. 4C).

During the flowering period, flower sampling was performed at three different stages after anthesis: half a day (at 15–16 h of the first day of anthesis, assigned to stage 1), one day (at 8–9 h of the second day of anthesis, a transitional stage from stage 1 to stage 2), and one day and a half (at 15–16 h of the second day of anthesis, stage 2). Among them, the percentage of the flowers catching flies is the highest in half a day after anthesis and is significantly reduced at the other two stages (Fisher’s exact test, P < 0.05; Fig. 5). The number of flies captured within the utricle ranged from one to six, and is more at the stage of half a day than at the other stages (Fig. 6).

DiscussionThe present study clearly indicates that

the change of the trapping trichomes from the turgescent condition to the wilted condition synchronizes with anther dehiscence, as suggested by Yamazaki and Kadota (1998), and that the latter starts at about 24 h after anthesis in Aristolochia debilis. Like other fly-trapping species of Aristolochia (Proctor et al. 1996, Trujillo and Sérsic 2006, Rulik et al. 2008), in A. debilis the female stage precedes the male stage; the stigmatic lobes are ready to receive pollen grains, but the anthers still remain undehiscent after anthesis. In this stage, the trapping trichomes are turgescent and oriented downwards, and it is consequently difficult for the trapped flies to escape from the utricle, as suggested in earlier studies (Razzak et al. 1992, Trujillo and Sérsic 2006, Rulik et al. 2008, Oelschlägel et al. 2009). Indeed, frequency of the flowers trapping flies is significantly higher at this stage than at the other stages. At the early morning of the second day after anthesis, the trapping trichomes start to wilt and should allow the flies to escape easily from the utricle. At the same times, the anthers start to


1998, Matsuka 2001). This species usually grows in wastelands and human-affected lands such as riverbanks and parks, and thus its rare fruit production was supposed to be caused by the deficiency of flower visitors for pollination. In the Seba population, however, small flies frequently visited the flowers and occasionally

carried pollen grains on their thorax dorsally. Therefore, deficiency of flower visitors may not be a main factor for the rare fruit production in A. debilis. It is more likely to be caused by the self-incompatibility system of the species. In Aristolochia, both self-compatible and self-incompatible species have been reported (Razzak et al. 1992, Trujillo et al. 2006, Nakonechnaya et al. 2008). Although we did not examine whether A. debilis is self-incompatible or not, the species appears to be self-incompatible because the hand self-pollinated flowers do not set fruits in the natural population examined (Sugawara personal observation). Furthermore, if A. debilis is self-compatible, it may be easy for the species to set fruits by geitonogamy since several flowers with different stages open on the same plant. However, no fruit was found in the plants examined in the population. Thus, the rare fruit production in A. debilis is probably caused by the self-incompatibility system. In our subsequent investigations it will be necessary to characterize the incompatibility system of this species by experiments of self- and cross-pollination.

We thank Drs. Satoshi Shinonaga, Kenkichi Kanmiya and Nozomi Utsugi for identification of small flies. We also appreciate Mr. K. Watanabe for comments the manuscript, and

Fig. 4. Flower visitors of Aristolochia debilis. A. A small fly (arrow) landed on limb of the perianth. B. Desmometopa sordida (Milichiidae), a small fly frequently observed within utricles. C. An unidentified species of Polydaspis (Chloropidae) escaped from the flower. It possesses pollen grains on the thorax, dorsally.

Fig. 5. Percentage of flowers catching flies on each of three flowering times (15–16 h of 1st day, 8–9 h of 2nd day, 15–16 h of 2nd day) after anthesis examined at the Seba population of Aristolochia debilis. Small letters (a, b, c) indicate significant differences at p < 0.05 determined by Fisher’s exact test following the Bonferroni correction for multiple test. N: Number of flowers examined.


Messrs. Yukihiko Tsuda and Kiyotaka Hori for providing us important information on the plants.

ReferencesDaumann E. 1959. Zur Kenntnis der Blütennektarien von

Aristolochia. Preslia 31: 359–372.Daumann E. 1971. Zur Bestäubungsökologie von

Aristolochia clematitis L. Preslia 43: 105–111. Disney R. H. and Sakai S. 2001. Scuttle flies (Diptera:

Phoridae) whose larvae develop in flowers of Aristolochia (Aristolochiaceae) in Panama. Eur. J. Entomol. 98: 367–373.

Faegri K. and van der Pijl. 1979. The Principles of Pollination Ecology, Third Revised Edition. Pergamon Press, Oxford.

Hwang H.-M., Kelly L. M. and Gilbert M. G. 2003. Aristolochiaceae. In: Wu Z.-G. and Raven P. H. (eds.), Flora of China 5: 246–269. Science Press, Beijing and Missouri Botanical Garden, St. Loius.

Mabberley D. J. 2008. Mabberley’s Plant-Book, Third Edition. Cambridge University Press, Cambridge.

Matsuka H. 2001. Natural History of Birdwing Butterflies. Matsuka Printing Co., Tokyo.

Murata J. 2006. Aristolochia L. In: Iwatsuki K., Boufford D. E. and Ohba H. (eds.), Flora of Japan IIa: 366–368. Kodansha, Tokyo.

Murugan R., Shivanna K. R. and Rao R. R. 2006. Pollination biology of Aristolochia tagala, a rare species of medicinal importance. Curr. Sci. 91: 795–798.

Nakonechnaya O. C., Sidorenko V. S., Koren O. G., Nesterova S. V. and Zhuravlev Y. N. 2008. Specific features of pollination in the Manchurian birthwort, Aristolochia manshuriensis. Biol. Bull. 35: 459–465.

Oelschlägel B., Gorb S., Wanke S. and Neinhuis C. 2009. Structure and biomehanics of trapping flower trichomes and their role in the pollination biology of Aristolochia plants (Aristolochiaceae). New Phytol. 184: 988–1002.

Proctor M. and Yeo P. 1972. The Pollination of Flowers. Taplinger Publishing Co., New York.

Proctor M., Yeo P. and Lack A. 1996. Natural History of Pollination. Harper Collins, London.

Razzak M. A., Ali T. and Ali S. I. 1992. The pollination biology of Aristolochia bracteolata Lamk. (Aristolochiaceae). Pakistan J. Bot. 24: 79–87.

Rulik B., Wanke S., Nuss M. and Neinhuis C. 2008. Pollination of Aristolochia pallida Willd. (Aristolochiaceae) in the Mediterranean. Flora 203: 175–184.

Sakai S. 2002. Aristolochia spp. (Aristolochiaceae) pollinated by flies breeding on decomposing flowers in Panama. Am. J. Bot. 89: 527–534.

Trujillo C. G. and Sérsic A. N. 2006. Floral biology of Aristolochia argentina (Aristolochiaceae). Flora 201: 374–382.

Vogel S. 1978. Pilzmückenblumen als Pilzmimeten. Flora 167: 329–398.

Willmer P. 2011. Pollination and Floral Ecology. Princeton Univ. Press, Princeton.

Wolda H. and Sabrosky C. W. 1986. Insect visitors to two forms of Aristolochia pilosa in Las Cumbres, Panama.

Fig. 6. Frequency distribution of flowers with different number of flies in each of the three different flowering times after anthesis at the Seba population of Aristolochia debilis. A. 15–16 h of 1st day after anthesis. B. 8–9 h of 2nd day after anthesis. C. 15–16 h of 2nd day after anthesis. N. Number of flowers examined.


Biotropica 18: 295–299. Yamazaki Y. and Kadota Y. 1998. Fruits of Aristolochia

debilis Siebold & Zucc. fed by butterfly (Byasa

alcinous; Papilionidae) caterpillars. J. Jpn. Bot. 73: 333–334 (in Japanese).

菅原　敬 a，広木瀬名 b，白井貴也 b，中路真嘉 a，小栗恵美子 c，末吉昌宏 d，清水　晃 e：ウマノスズクサの送粉に関連する開花フェノロジーと花筒内壁の毛の変化　ウマノスズクサ属（ウマノスズクサ科）の花は一般に筒状に変化し，また花筒部に内向する毛を密生することで，送粉昆虫を花筒内に閉じ込めて送粉をおこなう，いわゆるトラップ状花とみなされている．日本に分布するウマノスズクサについては実際どうなのか，また開花に伴って花筒内壁の毛や葯の裂開がどのようなタイミングで変化するのか，送粉昆虫は何かなどについて，長野県塩尻市の集団においてカメラによるインターバル撮影を駆使して調査した．その結果，この集団では花にハエ目クロコバエ科 (Milichiidae) やキモグリバエ科 (Chloropidae) の小さなハエが頻繁に訪花していることが確認された．これらのハエの体には花粉の付着も確認されることから，有効な送粉者と考えられた．花は早朝に開き，花冠が開いた時には雌性期で，葯はほぼ 1日遅れて翌朝に裂開した．一方，花筒内壁の毛は，花冠が開いた時にはまっすぐ伸びた状態であるが，翌朝になる

と徐々に短く萎縮してしまった．毛の萎縮は葯裂開のタイミングとよく合致していた．これは，花筒内に侵入したハエが伸長する毛のために雌性期の間は花外へ逃げ出しにくいが，葯が裂開する雄性期には毛が萎縮することで花粉を付着したハエが花外へ逃げ出しやすい構造といえるであろう．実際野外では毛の伸長した雌性期においてハエを捕獲した花の割合が有意に高く，雄性期では低いことが確認された．従って，日本のウマノスズクサにおいても花筒の毛の変化が送粉に重要な役割を演じているように思われる．

（a首都大学東京大学院理工学研究科牧野標本館，b首都大学東京都市教養学部生命科学，

c広島大学大学院理学研究科，d森林総合研究所九州支所，

e首都大学東京大学院理工学研究科生命科学）

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