J. Jpn. Bot. 93(3): 147–154 (2018)

Comparative Anatomy of the Seeds of humile and uniflora (, )

Chiharu Ugajin and Yasuhiko Endo*

Graduate School of Science and Engineering, Ibaraki University, 2-1-1, Bunkyo, Mito, 310-8512 JAPAN *Corresponding author: [email protected]

(Accepted on November 27, 2017)

A comparative anatomical study on the endozoochorous seeds of Monotropastrum humile (D. Don) H. Hara in comparison to the wind-dispersed seeds of L. was conducted. The endozoochorous seeds were ovoid and had lignin-rich cell walls. These cell walls were six times thicker than those of wind-dispersed seeds. The ovoid seed was hypothesized to be an ancestral characteristic of the subfamily Monotropoideae (family Ericaceae). The evolution from the ancestral character state to the derived state (seeds having wing-like seed coats) was presumed to have happened four times independently in the subfamily.

Key words: Endozoochory, lignin, Monotropastrum humile, Monotropa uniflora, seed coat, wind dispersal.

The subfamily Monotropoideae (Ericaceae) crickets eat the and excrete the undigested is composed of 13 species, which are seeds as part of their feces, which then germinate achlorophyllous epiparasitic , and are (Suetsugu 2014, 2017). This type of seed classified into 10 genera (Wallace 1987, Qin and dispersal is called endozoochory (Cochrane et al. Wallace 2005). These plants have fine seeds, and 2005). their are berries in five genera and capsules In recent moleculer phylogenetic studies in five genera (Wallace 1975). (Bidartondo and Bruns 2001, Tsukaya et al. Three species of Monotropoideae are 2008), Monotropa unifloraand Monotropastrum distributed in Japan, namely, Monotropa humile were united into a monophyletic clade h y p o p i t y s L . , M. uniflora L . , a n d as sister species, and this clade basally branched Monotropastrum humile (D. Don) H. Hara from the clade of Monotropa . (Takahashi 1993). The former two species This means that the change of character have capsules that deposit numerous fine seeds states between the wind-dispersed seeds and (Olson 1980). These seeds have a wing (Olson endozoochorous seeds has happened among 1980), and are assumed to be dispersed by wind. closely related species in Monotropoideae. In contrast, the third species, Monotropastrum The present study examined the structural humile, has berries that contain numerous differences between wind-dispersed seeds and fine seeds that are dispersed by camel crickets endozoochorous seeds in detail. Specifically, in Japan (Suetsugu 2014, 2017). The camel we conducted a comparative anatomical study

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Table 1. Voucher specimens of the examined species Species Locality Voucher specimen1) Monotropa uniflora Japan, Ibaraki Pref., Mito-shi, Sakato-cho Ugajin 49, 99, 100 Monotropastrum humile Japan, Ibaraki Pref., Hitachiomiya-shi, Mt. Gozen-yama Ugajin 25, 85, 72 Monotropastrum humile Japan, Ibaraki Pref., Kuji-gun, Daigo-machi, Mt. Yamizo-san Ugajin 29 1)Voucher specimens are deposited in the herbarium of Ibaraki Nature Museum (INM).

Table 2. Seeds of Monotropastrum humile and Monotropa uniflora Size (mm) Number of seed-coat cells Seed-coat cell wall 1) Width Length Along polar axis Circumference thickness (μm) Monotropastrum humile av.2) 0.18 0.34 5.69 9.71 34.86 sd.3) 0.02 0.03 0.99 0.88 9.29 no.4) 21 11 13 7 22 Monotropa uniflora av. 0.11 0.51 10.17 9.86 5.39 sd. 0.01 0.17 1.34 1.12 1.20 no. 9 5 6 7 13 1)The thickness of seed-coat cell wall, glowing under epifluorescence microscope. 2)av.: Average. 3)sd.: Standard deviation. 4)no.: Number of seeds examined. of the wind-dispersed seeds of Monotropa Chemical Industries, Ltd., Osaka, Japan). uniflora against the endozoochorous seeds of Then, the sections were observed with a light Monotropastrum humile. Then, we infored microscope and an epifluorescence microscope the ancestral character state of the seeds of with a fluorescence filter cube G-2A C-FL Monotropoideae, and presume the diversification attached (Nikon instruments, Tokyo, Japan). process of seed characters in the subfamily. The filter cube employs an excitation passband from 510 to 560 nm (green excitation), and was Materials and Methods combined with a 590-nm cut-on wavelength Materials examined (longer wavelengths than those in the yellow The fruits, including the seeds, of Monotropa region), i.e., ≥ 590 nm, emission filter. The sizes uniflora and Monotropastrum humile were of seeds (width, length, number of seed-coat collected in the field, and fixed in FAA cells, and seed-coat cell wall thickness) were (Formaldehyde: acetic acid: 50% aqueous measured under a (epifluorescence-) microscope. ethanol = 0.5:0.5:9). The collection sites and The numbers of the seeds measured were shown voucher specimens are listed in Table 1. in Table 2.

Anatomical study Presumption of an ancestral character state We made transverse and longitudinal The reconstruction of the evolution of the sections of FAA-fixed seeds that were embedded and seed characteristics (termed character in paraffin, using a rotary microtome (RV-240, states) was done based on the most recent and Yamato Kohki Industrial, Co., Ltd., Saitama, comprehensive molecular phylogenetic of Japan). The sections were stained with a Monotropoideae (Bidartondo and Bruns 2001), combination of Heidenhain’s hematoxylin, using MacClade 4.06 software (Maddison and safranin, and fast green FCF (Wako Pure Maddison 2003). The character states observed June 2018 The Journal of Japanese Botany Vol. 93 No. 3 149 in the present study were treated as unordered green FCF and then excited by a light at 510 to and multistate. 560 nm wavelength (near the blue excitation) under an epifluorescence microscope (Fig. 1B, Results D, F, H). As these conditions are considered Monotropastrum humile (Table 2, Fig. 1A–D) to be almost equivalent, this part of the cell The seeds are ovoid, approximately 0.18 mm wall is likely lignin-rich. The lignin-rich cell wide (Fig. 1A, Table 2) and approximately 0.34 wall of Monotropastrum humile seeds was mm long (Table 2, Fig. 1C). The seed coats have approximately six times thicker than that of the a single-cell layer, and contain approximately six wind-dispersed seeds of Monotropa uniflora. cells along the polar axis (Table 2, Fig. 1C). The Lignin has a strengthening function, as well as circumference contains approximately 10 cells providing protection against consumption by (Table 2, Fig. 1C). The embryo is composed insects (Swain 1979). Therefore, the thicker of two cells (Fig. 1C) and is surrounded by lignin-rich seed coat of Monotropastrum endosperm cells. The seed coat glows orange humile probably protects its embryo from being under an epifluorescence microscope (Fig. 1B, damaged by the digestive tract of camel crickets. D). The thickest glowing part is approximately Actually, the seeds in the feces of camel crickets 34.86 μm thick (Fig. 1B, D). were anatomized by Suetsugu (2017), and their lignified seed coats (about 40μm thick) were Monotropa uniflora(Table 2, Fig. 1E–H) found to be maintained. The seeds are approximately 0.11 mm wide (Table 2, Fig. 1E) and approximately Anatomical characteristics of the seeds of other 0.51 mm long (Table 2, Fig. 1G). The seed Monotropoideae species coats have a single-cell layer (Fig. 1E), and This study distinguished two types of seeds contain approximately 10 cells along the polar in Monotropoideae: A) ovoid seeds having axis (Table 2, Fig. 1G). The circumference seed coats fitting to the endosperm (e.g., contains approximately 10 cells (Fig. 1E, F). Monotropastrum humile), and B) long spindle- The embryo is composed of two cells and is shaped seeds having seed coats much larger than surrounded by endosperm cells (Fig. 1G). The the endosperm (e.g., Monotropa uniflora). These seed coats, especially their adaxial walls and two seed types have been reported in the other tangential walls, glow under an epifluorescence species of Monotropoideae. The distribution of microscope (Fig. 1F, H). The thickest glowing the two seed types in the subfamily is shown part is approximately 5.39 μm thick (Table 2, in Table 3, except for the Cheilotheca, Fig. 1F, H). for which mature fruits have not been reported (Wallace 1975). Discussion andromedea has rounded seeds, Glowing cell wall section under the which have a thin, rounded and membranous epifluorescence microscope seed coat, attached at one end of the seed Lignin-rich cell walls stained with safranin (Wallace 1975). The seed coat is much larger and excited under a light at 488 nm wavelength than the endosperm. Therefore, we classified the (blue excitation) exhibit red or orange seed into type B. fluorescence under a confocal fluorescence In the examined species of Monotropoideae, microscope (Bond et al. 2008). In the present six species have A-type seeds and four species study, we observed orange glowing parts have B-type seeds. that were stained with a combination of Heidenhain’s hematoxylin, safranin, and fast 150 植物研究雑誌 第 93 巻 第 3 号 2018 年 6 月

Fig. 1. Seed anatomy of Monotropastrum humile (A–D) and Monotropa uniflora (E–H). Transverse (A, B, E, F) and longitudinal (C, D, G, H) sections of seeds. Photographs under a light microscope (A, C, E, G) and under an epifluorescence microscope (B, D, F, H). s. seed coat. en. endosperm. em. embryo. Scale bars = 0.05 mm. June 2018 The Journal of Japanese Botany Vol. 93 No. 3 151

Table 3. Distribution of seed and fruit characteristics in Monotropoideae Seed Type of Seed coat cell Species Fruits Literature shape seeds1) wall thickenss2) virgata spindle B thin Copeland (1938, 1941), Wallace (1975) shaped (–3)) congestum ovoid A – berry Copeland (1941), Wallace (1975) spindle B thin capsule Koch (1882), Copeland (1941), Pyykkö (1968), shaped (–) Wallace (1975) Monotropa uniflora spindle B thin capsule Campbell (1889), Copeland (1941), Wallace shaped (ca. 5μm) (1975), Olson (1980), present study Monotropastrum humile ovoid A thick berry Wallace (1975), present study (ca. 35μm) odorata ovoid A thick berry Copeland (1939, 1941), Wallace (1975) (ca. 24μm) californicus ovoid A thick berry Copeland (1941), Wallace (1975), Massicotte et (ca. 26μm) al. (2007) fimbriolata ovoid A thick berry Copeland (1937,1941), Wallace (1975) (ca. 18μm) Pterospora andromedea rounded4) B thin capsule Copeland (1941), Bakshi (1959), Wallace (1975) (–) sanguinea subovoid5) A thick capsule6) Oliver (1890), Copeland (1932, 1941), Wallace (ca. 20μm) (1975), Merckx et al. (2012) 1)A. Seeds have seed coats fitting to endosperm. B. Seeds have seed coats much larger than endosperm. 2)thick: With thickening of cell walls. thin: without thickening of cell walls. The thickness of cell walls, measured from photos or sketches appeared in the literatures, shown in parenthesis. 3)–: Unknown. 4)rounded: Wallace (1975) described the seed as having a thin, rounded, membranous wing attached at one end of the seed. 5)subovoid: Oliver (1890) described the seeds as lacking any wings, and Wallace (1975) described the seeds as brown to reddish, sticky, often adherent to one another, and falling from capsules in clumps. 6)capsule: Wallace (1975) described the capsule as unusual almost indehiscent capsule, and Merckx et al. (2012) described the capsule as irregulary dehiscent capsule.

Correlation between the seed coat size and seed (1932, 1937), Pterospora andromedea seed by coat cell wall thickness Bakshi (1959), and Sarcodes sanguinea seed by The seed coats of Monotropastrum humile Copeland (1932); and photos of the section of were thickened by lignin-rich materials (Fig. Pityopsis californicus seed by Massicotte et al. 1B, C). However, such thickening could (2007). The types of the seed coat cell wall and not be observed in the seed coat cells of the thicknesses of the cell walls, measured from Monotropa uniflora (Fig. 1F, H). We call the the sketches and photos of Monotropoideae, are former type of seed coat cells, thick cell wall shown in Table 3. The thick cell walls are more type, and the latter type, thin cell wall type, than three times thicker than the thin cell walls in the present study. These two types of cell (Table 3). wall could be recognized in the other species Within the species of which seed coat cell of Monotropoideae based on the sketches or wall types have been known, type A seed has photos of the seed coat sections by previous thick seed coat cell walls and type B seed has researchers as follows; sketches of the section thin seed coat cell walls. This may mean that of Allotropa virgata seed by Copeland (1938), type A seeds have more hard seed coats suitable Monotropa hypopitys seed by Copeland (1941), to endozoochory and type B seeds have lighter Monotropsis odorata seed by Copeland (1939), and larger seed coats functioning as wings Pleuricospora fimbriolata seed by Copeland suitable to wind dispersion. 152 植物研究雑誌 第 93 巻 第 3 号 2018 年 6 月

Fig. 2. Optimization of the seed character states on a molecular phylogenetic tree (from Bidarutondo and Bruns 2001, modified in the present study) inMonotropoideae . CI = 0.25.

Correlation between the seed types and fruit capsule by Wallace (1975), and described as an types of Monotropoideae irregularly dehiscent capsule by Merckx et al. The fruits of Monotropoideae are berries or (2012) (Table 3). We need further investigation capsules (Copeland 1939, 1941, Wallace 1975). about differences of the mechanisms of the How these two types of fruit are distributed in dehiscence of capsules between S. sanguinea the subfamily is shown in Table 3. Based on and other species of Monotropoideae. Table 3, it may be inferred that the distribution of the seed types is correlated to the distribution Diversification of seeds and fruits in of the fruit types in Monotropoideae, i.e., Monotropoideae type A seeds occur in berries, whereas type B A molecular phylogenetic tree for seeds occur in capsules. However, the seeds of Monotropoideae, on the basis of the analysis Sarcodes sanguinea Torrey are classified into of nrDNA (ITS + 28S) sequence data, was type A but occur in a capsule. This combination proposed by Bidartondo and Bruns (2001). The of seed type and fruit type is an exceptional case tree branches into two clades basally. The first of Monotropoideae. The capsule of S. sanguinea clade is composed of Pterospora andromedea was described as an unusual almost indehiscent and Sarcodes sanguinea, whereas the second June 2018 The Journal of Japanese Botany Vol. 93 No. 3 153

Fig. 3. Optimization of fruit character states on a molecular phylogenetic tree (from Bidarutondo and Bruns 2001, modified in the present study) inMonotropoideae . CI = 0.25. clade is composed of the other genera of the This work was partially supported by JSPS subfamily. Here, we attempted to optimize KAKENHI Grant number 26440205 (to Y.E.). the seed and fruit types on the tree (Figs. 2, 3). The optimization indicates that the ancestral References character state of the first clade is the capsule Bakshi T. S. 1959. Ecology and morphology of Pterospora andromedea. Bot. Gaz. 120: 203–217. containing ovoid seeds, whereas the ancestral Bidarutondo M. I. and Bruns T. D. 2001. Extreme state of the second clade is the berry containing specificity in epiparasiticMonotropoideae (Ericaceae): ovoid seeds (Figs. 2, 3). Notably, in the first widespread phylogenetic and geographical structure. clade, the winged seeds evolved once, and in the Mol. Ecol. 10(9): 2285–2295. second clade, the winged seeds evolved three Bond J., Donaldson L., Hill S. and Hitchcock K. 2008. Safranin fluorescent staining of wood cell walls. times independently (Fig. 2). This means that Biotech Histochem 83: 161–171. the wind dispersal seeds would be evolved four Campbell D. H. 1889. Monotropa uniflora as a subject for times in Monotropoideae. In the second clade, demonstrating the embryo sac. Bot. Gaz. 14: 83. the capsule evolved from the berry three times Cochrane J. A., Friend J. A. and Hill S. J. E. 2005. Endozoochory and the Australian bluebell: independently. consumption of Billardiera fusiformis (Labill.) Payer Further research is required to know the (Pittosporaceae) seeds by three species at adaptive significance of these changes. Two Peoples Bay Nature Reserve, Western Australia. J. 154 植物研究雑誌 第 93 巻 第 3 号 2018 年 6 月

Roy. Soc. West Aust. 88: 191–196. Pyykkö M. 1968. Embryological and anatomical studies on Copeland H. F. 1932. The development of seeds in certain Finnish species of the Pyrolaceae. Ann. Bot. Fenn. 5: . Amer. J. Bot. 20: 513–517. 153–165. Copeland H. F. 1937. The reproductive structures of Qin H. and Wallace G. D. 2005. Monotropa, Pleuricospora. Madroño 4: 1–40. Monotropastrum. In: Wu Z. Y. and Raven P. H. (eds.), Copeland H. F. 1938. The structure of Allotropa. Madroño Flora of China (Apiaceae through Ericaceae). 14: 255– 4: 137–168. 257. Science Press, Beijing, and Missouri Botanical Copeland H. F. 1939. The structure of Monotropsis and Garden Press, St Louis. the classifying of the Monotropoideae. Madroño 5: Suetsugu K. 2014. Diverse interactions of heterotrophic 105–136. plants with their hosts, and seed dispersers Copeland H. F. 1941. Further studies on Monotropoideae. (Abstract). http://dx.doi.org/10.14989/doctor.k18605. Madroño 6: 97–144. Issue date: 24 Sept. 2014. Koch L. 1882. Die Entwicklung des Samens von Suetsugu K. 2017. Independent recruitment of novel seed Monotropa hypopitys L. Jahrb. Wiss. Bot. 13: 202–252, dispersal system by camel crickets in achlorophyllous with plates 9–11. plants. New Phytologist 217: 828–835. Maddison D. R. and Maddison W. P. 2003. MacClade Swain T. 1979. Tannins and lignins. In: Rosenthal G. A. and version 4.06: Analysis of Phylogeny and Character Janzen D. H. (eds.), Herbivores: Their Interaction with Evolution. Sinauer, Sunderland. Secondary Metabolites. pp. 657–682. Academic Press, Massicotte H. B., Melville L. H., Tackaberry L. E. and New York. Peterson R. L. 2007. Pityopus californicus: structural Takahashi H. 1993. Pyrolaceae. In: Iwatsuki K., Yamazaki characteristics of seed and seedling development in a T., Boufford D. E. and Ohba H. (eds.), Flora of Japan. mycoheterotropic species. 17(8): 647–653. IIa: 64–70. Kodansha, Tokyo. Merckx V. S. F. T., Freudenstein J. V., Kissling J., Tsukaya H., Yokoyama J., Imaichi R. and Ohba H. 2008. Christenhusz M. J. M., Stotler R. E., Crandall-Stotler Taxonomic status of Monotropastrum humile, with B., Wickett N., Rudall P. J., Maas-van de Kamer H. and special reference to M. humile var. glaberrimum Maas P. J. M. 2012. 2 and classification. (Ericaceae, Monotropoideae). J. Pl. Res. 121(3): 271– In: Merckx V. S. F. T. (ed.), Mychoheterotrophy: 278. the Biology of Plants Living on Fungi. pp.19–102. Wallace G. D. 1975. Studies of the Monotropoideae Springer Science+Business Media, New York. (Ericaceae): Taxonomy and distribution. The Wasmann Oliver F. W. 1890. On Sarcodes sanguinea Torr. Ann. Bot. Journal of Biology 33: 1–88. 4: 303–326, with plates 17–21. Wallace G. D. 1987. Transfer of Eremotropa schiaphila to Olson A. R. 1980. Seed morphology of Monotropa uniflora Monotropastrum (Ericaceae: Monotropoideae). Taxon L. (Ericaceae). Amer. J. Bot. 67: 968–974. 36: 128–130.

宇賀神智晴，遠藤泰彦：ギンリョウソウとアキノギンリ ョウソウの種子の比較解剖（シャクジョウソウ亜科，ツ ツジ科） ツツジ科シャクジョウソウ亜科のギンリョウソウ ギンリョウソウ種子に見られる「種皮が内乳を取り囲む Monotropastrum humile (D. Don) H. Hara（周食型動物散 以上に伸展し翼状になる」という状態は，ギンリョウソ 布種子）とアキノギンリョウソウ（ギンリョウソウモド ウに見られる「種皮が伸展せず内乳を取り囲むだけ」の キ）Monotropa uniflora L.（風散布種子）の種子の比較 状態と比較し，派生的であると推定された．そして，こ 解剖を行った．ギンリョウソウの種子は卵形で，種皮は の派生的な形質状態（風散布に適していると考えられる リグニンを多く含み，種皮細胞壁は，アキノギンリョウ 状態）への進化がシャクジョウソウ亜科の多様化過程の ソウのおよそ 6 倍の厚さに達した（アキノギンリョウ 中で独立に 4 回起きたことが推定された． ソウの種子は比較的長い紡錘体である）．シャクジョウ （茨城大学理工学研究科） ソウ亜科における種子の多様化の過程において，アキノ