Hikobia 18: 1–6. 2019

A note on morphology of the heterogamous capitula rarely found in male inflorescences of Petasites japonicus (Siebold et Zucc.) Maxim. ()

Tamaki Hashimoto, Yuya Inoue and Masaki Shimamura*

Hashimoto, T., Inoue, Y. & Shimamura, M. 2019. A note on morph logy of the heterogamous capitula rarely found in male inflorescences of Petasi es japonicus (Siebold et Zucc.) Maxim. (Asteraceae). Hikobia 18: 1–6.

Petasites japonicus are typically dioecious, with functionally female and male florets on the capitula of separate . Each c pitulum of typ al female consists of two types of florets: numerous tubular female florets with ut stamens, and a few of bell-shaped florets morphologically he maphroditic but functionally sterile. On the other hand, each capitulum of typical male plant consists of only hermaphrodite florets but functionally male. Recently we found ome abnormal male plants bearing heterogamous capitula with a few ligulate female florets (functionally female) arranged at the periphery to surround the numerous hermaphrodite florets (functionally male) in the wild population. The finding provides circumstantial evidence for a plasticity of sex expression in P. japonicus The peripheral arrangement of ligulate female florets is a common feature in the capit la of the Asteraceae including the closely related genera such as Homogyne, and in other species of Petasites belonging to subgenus Nardosmia The occurrence of abnormal heterogamous capitula bearing marginal ligulate florets in the male inflorescence of P. japonicus could represent an evolutionary reversion.

Tamak Hashimoto, Laboratory of Plant and Ecology, Graduate School o Science, Hiroshima University, 1–3–1 Kagamiyama, Higashihiroshima-shi, Hiroshima, 739–8526, Japan. Yuya Inoue & Masaki Shimamura, Laboratory of Plant Taxonomy and Ecology, Graduate School of Integrated Sciences for Life, Hiroshima University, 1–3–1 Kagamiyama, Higashihiroshima-shi, Hiroshima, 739–8526, Japan. *mshima @hiroshima-u.ac.jp (author for correspondence)

do to Kyushu (Imazu & Fujishita 1962; Takagi Introduction 1994). The species has long been cultivated in The genus Petasites in the family Asteraceae Japan due to its unique flavor and pharmacolog- has ca. 20 species which distribute Asia, ical efficacy (Iwamoto 2009). After the flowering nd North America. They are typical perennial season, they produce vegetative leaves with a long plant with stems creeping underground and pro- petiole. The vegetative leaves are heart-shaped,15 uncorrectedduce umbrella-like leaves during the growing sea- to 30 cm long and petioles proofare 60 cm long or more. son. (Toman 1972; Cherniawsky & Bayer 1999; The plants with long petioles (sometimes reach to Steffen et al. 2016; Teofilovski 2018). Among 3 m in height) which distributed from Hokkaido them, Petasites japonicus (Siebold & Zucc.) Max- to northern Tohoku are sometimes distinguished im. is native to Japan, China, Korea and Sakhalin taxonomically at the subspecies level as P. japon- and typically found on moist disturbed areas, such icas subsp. giganteus Kitam. (Kitamura 1942; as slopes, forest edges and near streams. It grows Hind & Kay 2006). over a wide area of Japan, reaching from Hokkai- In early spring, they produce inflorescence 2 Hikobia Vol. 18, No. 1, 2019

stems before the vegetative leaves develops. The buffer (pH 7.2) and evacuated by a water aspirator inflorescence of Petasites is composed by flower until the specimens sank to the bottom of the vi- heads (capitulum) with numerous florets and sur- als. After the fixation for 3 h. at room temperature rounding involucral bracts. All species of Peta- in the fixative solution, the samples were rinsed sites show a sexual dimorphism, with individuals in 0.05 M phosphate buffer and Post-fixed in 1 % being either male or female functionally (Bremer buffered osmium tetroxide at room temperature 1994; Cherniawsky & Bayer 1998a, b). Howev- for 2 h. Then the samples were dehydrated in grad er, each capitulum is basically gynomonoecious. ed ethanol series and immersed in t-butyl alcohol. Each male capitulum consists of many morpho- The samples were immersed n t-butyl alcohol logically hermaphroditic florets (functionally then freeze-dried in an evacu or (VFD-21; Vac- male) and a few pistillate female florets. The ster- uum Device, Ibaraki, Japan). The samples were ile pistil of the hermaphroditic florets in male ca- mounted on an aluminum stag with double-sided pitula is modified to form the pollen presenter and carbon tape and sputt r-coated with gold at 200 during its elongation, the large stigmas sweeps Å using a JFC-1500 (JEOL, Tokyo). The images out the pollen from the anthers fused into a tube were then cap ured using a JCM-6000 scanning (Haratym & Weryszko-Chmielewska 2012). Each electron microsc pe (JEOL, Tokyo). female capitulum consists of many pistillate fe- male florets and a few morphologically hermaph- Results roditic florets functionally sterile (Toman 1972; Haratym & Weryszko-Chmielewska 2012; Stef- I the survey area, the typical female (Fig. 1) fen et al. 2016). Therefore, it is interpreted that the and male plants (Fig. 2) and some male plants status of dioecy of the genus Petasites is “cryp b ar ng abnormal heterogamous capitula (Fig. 3) tic” with nonfunctional sex organs are substan- were found. tially developed (Yu et al. 2011). In the genu P. The capitulum of female plants consists of japonicus is considered as the only exception with many pistillate female florets and a few hermaph- no female florets on male capitula (Toman 1 72; roditic florets functionally sterile (Fig. 1a, b). The Sakai et al. 2008). Recently, w found an bnor- female florets are filiform tubular with a reduced mal male plants of P. japonicus earing some het- whitish or pinkish corolla which is shallowly five erogamous capitula comp sed of morphological- lobed or obliquely truncate, but not ligulate (Fig. ly hermaphroditic florets (functionally male) and 1c). The female florets have no stamens, and the pistillate florets (functionally female) in the wild pistil has a long style terminated in a two parted population in Fukushima p efecture, northeastern stigma (Fig. 1b, e). Some hermaphroditic florets Honshu, Japan In his paper, we discuss the di- with a huge sterile pistil and shriveled anthers rection of evolution of the capitulum of the genus were found in the center of a capitulum. However, Petasites through the description of the shape of the matured pistils have no ovule and anthers also the abnormal male capitulum. never produce pollen (Fig. 1d, f). The capitulum of typical male plants consists only of many morphologically hermaphroditic Materials and Methods florets (Fig. 2a, b). Although the outer morpholo- The male and female inflorescences of Peta- gy of the hermaphroditic florets of male capitula ites japonicus (Siebold et Zucc.) Maxim. were are similar to the sterile florets of female capit- collected in March to May 2019, in Tsuchiyu-on- ula, the anthers produce many pollen grains and uncorrectedsen Town, Fukushima City, Fukushina Pref., Ja- the modification of the upperproof portion of the style pan. The voucher specimen of the plants bearing functions as a pollen presenter (Fig. 2c). As far as abnormal heterogamous capitula were deposited we investigated, in the hermaphroditic florets of in Herbarium of Hiroshima University (hiro-my male capitula, ovules did not develop. 141869). In addition to the typical male and female, For scanning electron microscopy, the samples some inflorescences producing the heterogamous were placed in vials containing the fixative solu- capitula with many hermaphroditic (functionally tion with 3 % glutaraldehyde in 0.05 M phosphate male) florets and a few female florets were discov- Has himoto, T., Inoue, Y. and M. Sh imamura 3

ered (Fig. 3a, b). The number of female florets per Discussion a capitulum varied between 0 to 11 in individual inflorescence. In the absence of female florets, it In the many species of genus Petasites, the was not possible to distinguish them from the ca- establishment dioecism is incomplete and the pitulum of typical male plants. The female florets occurrence of the female florets at the margin of the were always arranged in periphery to surround male capitula is known in many species (Yu et al. the numerous hermaphrodite florets (Fig. 3c). Co- 2011; Haratym & Weryszko-Chmielewsk 2012) rolla of most of the female florets were distinctly The male capitula of P. japonicus bearing only ligulate. The anthers of the hermaphroditic flo- male florets (morphologically hermaphr ditic, rets produce many pollen grains and a brush-like functionally male) seem to be n exceptional case pollen presenter same as to those of typical male of the genus. However, Imazu and Fujisita (1961) plants (Fig. 3e). Although the hermaphroditic flo- reported that abnormal male capitula bearing a rets never produced seeds, some of the ligulate few female florets sometimes occur in Japanese female florets were fertilized and produced seeds population of P. japonic . The abnormal male (Fig. 3f). capitula reported in Imazu and Fujisita (1961)

uncor ected proof

Fig. 1. Female capitula and florets. a: Cross section of a female capitulum. Asterisk and female signs indicate the hermaphroditic florets (functionally sterile) and female florets, respectively. b: A female floret and hermaphroditic florets (functionally sterile). c: Magnified image of the top of the corolla of female florets. d: Cutting open of ovaries of female florets and hermaphroditic florets (functionally sterile). The arrow indicates an ovule. e: Cutting open of corolla of a female floret. There is no trace of stamens around the pistil (arrow). f: Cutting open of anther cylinder (arrows) surrounding a thick pistil in a hermaphroditic floret. No pollen grains are formed in anthers. 4 Hikobia Vol. 18, No. 1, 2019

Fig. 2. Typical male capitula and florets. a: A side view of a typical male capitu m consis ing only of male florets. b: All florets contained in a capitulum are morphologically identical. c Cutting open of anther cylinder of a hermaphroditic floret (functionally male). Many pollen grains and a brush-like pistil that functions as a pollen presenter (asterisk).

uncorrected proof

Fig. 3. Abnormal heterogamous capitula and florets. a: A young male inflorescence bearing abnormal capitula with some ligulate female florets. b: A side view of a capitulum. Male and female signs indicate the hermaphroditic (functionally male) and female florets, respectively. c: Overhead image of a capitula. d: A simple pistil (arrowhead) and distinctly ligulate corolla (arrow) of a female floret. e: Cutting open of anther cylinder of a hermaphroditic floret (functionally male). Many pollen grains and a brush-like pistil that functions as a pollen presenter (asterisk). f: A fruited female floret and seedless hermaphroditic floret (functionally male). Has himoto, T., Inoue, Y. and M. Sh imamura 5

were different from the plants observed here in tive success under mate-limited conditions (e.g that the central hermaphroditic florets were sterile Korpelainen 1998). Cossard and Pannell (2019) which producing no pollen grains. demonstrated that leakiness in dioecious plants In the genus Petasites, the occurrence of some might be plastic and sensitive to mating oppor hermaphroditic central florets (functionally ster- tunities. Although P. japonicus sometimes form ile) with showy corolla on female capitulum isolated unisexual populations at landscap lev- seems to increase the pollinator attraction and el due to vegetative propagation with rhizom enhance female reproductive success (Sakai et al. extending underground, the chance of sexu l re- 2008). On the other hand, the ecological signif- production might not be completely lost even in icance of the occurrence of a few female florets isolated male population. Th occasional female within male capitula is unknown (Yu et al. 2011). florets in the male capitulum m y facilitate evo- In the many species of Asteraceae, the large ligule lutionary transitions from dioecy to monoecy un- of peripheral female florets serves to attract pol- der mate-limited cond tion as uggested in other linators. However, the ligule of peripheral female angiosperms (Ehlers & B aillon 2007; Crossman florets of P. japonicus seem to be too small to & Charleswor h 2014; Käfer et al. 2017). Further attract pollinators (Fig. 3c). investigations ar required to examine the ger- The abnormal male inflorescence which we mination bility o seeds of female florets in the newly discovered here seems to be the monoe- male c pitul and sex expression pattern of their cious plants producing both male and female offsprings functional florets (Fig. 3). Although further in- The three types of the floret arrangements vestigations are required to confirm whether found in the wild population of present survey such abnormal male inflorescence is common in ar ummarized in Fig. 4. The peripheral ar- P. japonicus, our finding provides circumstan- rangement of ligulate female florets (Fig. 4c) is a tial evidence for a plasticity of sex expression in common feature in the capitula of the Asteraceae P. japonicus at least in the area surveyed in this including some species of Petasites belonging to study. In angiosperms, the males and f males of subgenus Nardosmia and closely related genera dioecious plants commonly display inc nstant or such as Homogyne and Tussilago. The character leaky sex expression, with the o casional produc- seems to be an ancestral form and the incidental tion of a few flowers of the opposit sex (Cossard ligulate florets in the male inflorescence of P. ja- & Pannell 2019). Such labil ty in sex expression is ponicus could represent an accidental evolution- considered to be adaptiv by ssuring reproduc- ary reversion. uncorrected proof

Fig. 4. Schematic illustrations of capitula of P. japonicus in cross sections observed in present study. a: Typical male capitulum. b: Female capitulum. c: Heterogamous capitulum rarely found in male plants. Asterisk, male and female signs indicate the sterile florets, functionally male and female florets, respectively. 6 Hikobia Vol. 18, No. 1, 2019

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