J. Jpn. Bot. 93(3): 147–154 (2018) Comparative Anatomy of the Seeds of Monotropastrum humile and Monotropa uniflora (Monotropoideae, Ericaceae) 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 Monotropa uniflora 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 berry and excrete the undigested is composed of 13 species, which are seeds as part of their feces, which then germinate achlorophyllous epiparasitic plants, 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 fruits 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 hypopitys. (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 —147— 148 植物研究雑誌 第 93 巻 第 3 号 2018 年 6 月 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 fruit 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 tree 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 genus Cheilotheca, Fig. 1F, H). for which mature fruits have not been reported (Wallace 1975). Discussion Pterospora 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) Allotropa virgata spindle B thin capsule Copeland (1938, 1941), Wallace (1975) shaped (–3)) Hemitomes congestum ovoid A – berry Copeland (1941), Wallace (1975) Monotropa hypopitys 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.