Morphology, Taxonomy, Culm Internode and Leaf Anatomy, and Palynology of the Giant Reed (Arundo donax L.), Poaceae, Growing in Thailand

In this paper, we present the morphology, taxonomy, anatomy, and palynology of Arundo donax. A detailed morphological description and illustrations of the species are provided, along with information about the identification, distribution, the specimens examined, habitat and ecology, the International Union for Conservation of Nature (IUCN) conservation assessment, phenology, etymology, vernacular name, and uses. The species can be distinguished by its large, tall rhizomatous perennial reed; cauline leaves; an open, large, plumose panicle inflorescence; subequal glumes as long as the spikelets; glabrous rachilla; all bisexual florets; and a lemma with a straight awn and with long white hairs outside below the middle part. In this study, two names were lectotypified: Arundo bifaria and A. bengalensis, which are synonyms of A. donax. The culm internodes in the transverse section have numerous vascular bundles scattered in the ground tissue, and the parenchyma cells have significantly lignified cell walls. Vascular bundles are composed of phloem and xylem and are enclosed in a continuous sclerenchymatous bundle sheath. The chloroplasts in the transverse section of the leaf blades are found only in the mesophyll cells but are absent in the bundle sheath cells, which indicates that it is a C3 grass. The leaves have stomata on both surfaces and are confined to the intercostal zones. The stomata are typically paracytic, with two lateral subsidiary cells placed parallel to the guard cells. The stomatal density is higher on the abaxial surface [450–839/mm2 (606.83 ± 72.71)] relative to the adaxial surface [286–587/mm2 (441.27 ± 50.72)]. The pollen grains are spheroidal or subspheroidal [polar axis length/equatorial axis length ratio (P/E ratio) = 0.89–1.16 (1.02 ± 0.07)] with a single pore surrounded by a faint annulus, and the exine sculpturing is granular.

Arundo bengalensis was named by Retzius based on the specimen from Bengal, India (originally "Habitat in Bengala" on the first publication) but did not mention the collector number or the herbaria in which it was present [23]. We traced one sheet of the specimen Wallich Cat. 5018D (EICH 5018D) at K-W [K001104517], collected from Bengal, India (originally "Bengala inferior 1820" on the label), and following Art. 9.6 of the ICN [22], it constitutes a syntype. It is selected here as the lectotype, following Art. 9.3 and 9.12 of the ICN [22] (Figure 6).
Recognition. Arundo donax is characterised as a large, tall rhizomatous perennial reed, loosely tufted, 3-8.2 m tall (including inflorescence), bamboo-like; culms with numerous nodes and internodes, woody when mature; hollow culm internodes; cauline leaves, distichous (in two vertical ranks or rows on opposite sides of the culm); linear or linearlanceolate leaf blades, 24-73.2 × 2-11.7 cm; open large, plumose, panicle inflorescences, purplish, turning pale brown with age; spikelets solitary, laterally compressed, with 2-3(-4) florets, all bisexual; similar, subequal persistent glumes; lemma two toothed at apex, with a straight awn arising from sinus and with dense long white hairs (silky hairs) outside below the middle part. Distribution. This species is distributed in West and Central Asia to temperate East Asia: Transcaucasus (South Caucasus), Türkiye (Turkey), Cyprus, Egypt (Sinai), Syria, Lebanon, Palestine, Israel, Gulf States (Arab States of the Persian Gulf), Yemen, Kazakhstan, Uzbekistan, Turkmenistan, Tajikistan, Afghanistan, Pakistan, India, Nepal, Bhutan, Bangladesh, Myanmar, Tibet, China, Taiwan, Japan, Vietnam, Laos, Cambodia, and Thailand. This species was very widely introduced into many parts of the world: North and South America, Africa, Europe, Asia, and Australasia (Australia, New Zealand, and some neighbouring islands in the Pacific Ocean). It is an invasive plant species that invades some countries [24], but it is narrowly distributed in its native country or in its range of distribution, especially in Thailand. a straight awn arising from sinus and with dense long white hairs (silky hairs) outside below the middle part.         (Figure 8).
Distribution. This species is distributed in West and Central Asia to temperate East Asia: Transcaucasus (South Caucasus), Türkiye (Turkey), Cyprus, Egypt (Sinai), Syria, Lebanon, Palestine, Israel, Gulf States (Arab States of the Persian Gulf), Yemen, Kazakhstan, Uzbekistan, Turkmenistan, Tajikistan, Afghanistan, Pakistan, India, Nepal, Bhutan, Bangladesh, Myanmar, Tibet, China, Taiwan, Japan, Vietnam, Laos, Cambodia, and Thailand. This species was very widely introduced into many parts of the world: North and South America, Africa, Europe, Asia, and Australasia (Australia, New Zealand, and some neighbouring islands in the Pacific Ocean). It is an invasive plant species that invades some countries [24], but it is narrowly distributed in its native country or in its range of distribution, especially in Thailand.   Habitat and ecology. In Thailand, it is mostly found in wet habitats such as open areas along the riverbanks, streams, seasonally flooded areas, and irrigation canals, but also in drier habitats along the roadsides at elevations of 20-620 m above mean sea level. (Figure 9). In the lower Himalaya, from Kashmir to Nepal, it occurs up to c. 1070 m alt., and from Punjab to Sylhet, in the Naga Hills, it occurs at 1520-2440 m alt. [10]. Bor stated that it will grow in dry habitats when established, but it prefers plenty of moisture [11]. In Southeast Asia, it occurs chiefly in low, wet sites such as riverbanks and seasonally flooded places, but also in drier habitats at altitudes less than 1800 m [16]. In Japan and its neighbouring regions, it is occasionally found on sandy dunes along the coast [18].

Distribution in
IUCN Conservation Status: Least Concern (LC) [25]. This species is widely distributed from West and Central Asia to temperate East Asia and has been widely introduced into many parts of the world. The Global Invasive Species Database stated that dense populations of Arundo donax affect riversides and stream channels, compete with and displace native plants, interfere with flood control, and are extremely flammable, increasing the likelihood and intensity of fires. It may establish an invasive plant-fire regime as it both causes fires and recovers from them 3-4 times faster than native plants. It is also known to displace and reduce habitat for native species. Long 'lag times' between introduction and development of negative impacts are documented in some invasive species; the development of A. donax as a serious problem in California may have taken more than 400 years. The opportunity to control this weed before it becomes a problem should be taken, as once established, it becomes difficult to control [24]. It is appropriate to consider its status as LC.
Phenology. Flowering from October to December; fruiting from November to December. Etymology. Arundo donax was named by Carl Linnaeus (1707-1778), a Swedish botanist, physician, and zoologist [26]. The generic name "Arundo" comes from the old Latin name for a reed. The specific epithet "donax" from the old Greek name for a reed [27,28]. Uses. In the northern region of Thailand, the woody culms of Arundo donax are locally used in light construction and are used in building rough fences, walls, and benches. When they are crushed and sun-dried, they can be woven into rough mats to be used as walls. They are also used to make giant reed tubes (giant reed cylinders) with sand, uncooked rice, or water contained inside the hollow culm internodes used in the traditional Lanna rites of "Suep Chata" (the prolongation rituals of human life) (based on the observations of C. Ngernsaengsaruay et al. and interviews conducted in Mae Hong Son, Chiang Mai, and Lamphun Provinces). A. donax is also grown in the garden of Suan Luang Rama IX Park (King Rama IX Park) as an ornamental grass to provide botanical education to the people (C. Ngernsaengsaruay et al. own observations and interviews). In the northern and central regions of Nakhon Sawan and Chai Nat Provinces, respectively, it is cultivated as a medicinal grass. The young culms and young leaves are cut and boiled in water for bathing and used as a treatment for an itching rash (called "Pa dong") by the local people. (C. Ngernsaengsaruay et al. own observations and interviews). In the Chaiyaphum province, located in the eastern part of Thailand, the whole plant of this species is boiled in water for bathing or drinking and is used in the treatment of eczema (dry, itchy skin) by the Kyah Kur (from the specimen K. Kertsawang 3241). A. donax has long been used in the European region; the dried culm internodes are used in musical instruments as reeds for clarinet, oboe, and bassoon [9]. Dr. Nattapon Banjatammanon, a lecturer and clarinet player from the Department of Music, Faculty of Humanities, Kasetsart University, has been purchasing dried culms of giant reeds from France for making clarinet reeds himself ( Figure 10).   Previous studies reported that the culms of Arundo donax are a useful source of cane for light construction and for making woodwind instrument reeds [6,7]. The culms are used for making clarinet reeds and organ pipes [24,29] and are extensively cut to make mats, trays, and baskets, with the Romans using the culms to make pens and sometimes paper [17]. The dried, crushed culms can be woven into rough mats to be used in making walls and roofs. The hollow culms are also used to make herdsmen's pipes. In Texas, this grass has been planted to protect against wind erosion [11] and has been extensively used by the highway department for erosion control and beautification along the culverts and bridges [30]. It has long been grown as a windbreak in horticultural plantings, but more recently it has been brought into cultivation for reed production [8]. The culms are also used to make sticks and fishing rods [15]. The large, thick, and fluffy flower plumes (inflorescences) are used in floral arrangements [24].
The names "Ban Sop O", "Huai Mae O", "Ban Mae O Nok", and "Ban Mae O Nai" are located in Mae Na Subdistrict., Chiang Dao District, Chiang Mai Province, the northern region of Thailand. They are geographical names (place names or toponyms) that are applied to topographical features and settled places in Arundo donax. The words "Ban" means the village, "Sop" means the junction of rivers, "Huai" refers to the stream, and "O" is the local name of A. donax.

Culm Internode Anatomy
The culm internodes of Arundo donax are circular with a wide hollow central pith (central pith cavity), leaving a cylindrical area of tissue (solid tissue). A monolayer of epidermis and several layers of cortex that are both small cells. There is a continuous sclerenchyma ring (also called a zone of sclerenchymatous cells or a fibre band) between the cortex and ground tissue close to the periphery. The outermost small vascular bundles are embedded in a continuous sclerenchyma ring. Numerous larger vascular bundles are scattered in the ground tissue, and the parenchyma cells have significantly lignified cell walls. Vascular bundles are composed of phloem and xylem (conducting tissue) and are surrounded in a continuous sclerenchymatous bundle sheath (also called a bundle sheath ring or fibre ring). The size of the vascular bundles (including sclerenchyma rings) increased gradually with the distance inward from the epidermis (Figure 11). Measurements of the vascular bundles, vessels, and parenchyma cells are shown in Table 3.   According to Kawasaki et al. [9], it turned out that the acoustic quality of a reed is mainly ascribed to the shape and configuration of vascular bundles and the size of parenchyma cells. A reed where vascular bundles with continuous bundle sheath rings are homogeneously distributed with a higher proportion among a softer network of small parenchyma cells enables musical performance, and Veselack [31] found that clarinet reed quality was related to the size of the parenchyma cells, the size of vascular bundles and the associated bundle sheath ring, and the number of twisted and broken bundle sheath rings. The results of Kawasaki et al. [9] are consistent with Kolesik et al. [8]. Furthermore, from our study, we found the vascular bundles are surrounded in a continuous sclerenchymatous bundle sheath, homogenously distributed, and enclosed by small parenchyma cells. These are good characteristics of culm internodes as the material for clarinet reeds made from Arundo donax, in agreement with Veselack [31], Kolesik et al. [8], and Kawasaki et al. [9].

Leaf Blade Anatomy
Transverse section of leaf blades: The outline of the leaf blades in the transverse section is flat. Bulliform cells are present on the adaxial surface in discrete, regular groups, each cell pyriform, and combine with colourless mesophyll cells to form narrow groups penetrating the mesophyll. All the veins appear parallel to each other. The vascular system consists of three orders of collateral vascular bundles (variously sized: large-sized, middle-sized, and small-sized); the median vascular bundle (first-order vascular bundle) is larger than the lateral vascular bundles (second-order vascular bundles and third-order vascular bundles). Vascular bundles are composed of phloem and xylem (vascular tissue) and are enclosed in two layers of bundle sheath cells: outer bundle sheath cells and inner bundle sheath cells. The outer bundle sheath cells have thin walls and are colourless (parenchyma cells), and the inner bundle sheath cells have thick walls (sclerenchyma cells). The chloroplasts in the transverse section of the leaf blades are found only in the mesophyll cells but are absent in the bundle sheath cells, which indicates that Arundo donax is a C3 grass, in agreement with Watson and Dallwitz [7] (Figure 12).
Leaf blade epidermis: The leaf blade epidermis of Arundo donax consists of long cells alternating with short cells arranged in the costal and intercostal zones on both surfaces. The long costal cells are elongated and rectangular. The intercostal long cells are irregularly elongated with sinuous anticlinal walls (arranged in vertical rows and having sinuous radial walls), and the intercostal short cells (silica cells) have silica bodies. The styloid crystals can be found in the leaf blade epidermal cells. The leaves are amphistomatic (have stomata on both surfaces) and are confined to the intercostal zones. The stomata are typically paracytic, with two lateral subsidiary cells placed parallel to the guard cells. The subsidiary cells are dome-shaped, and the guard cells are dumbbell-shaped. The size of stomata (including subsidiary cells) is 23.86-37.12 (30.67

Discussion
In its natural habitats in Pakistan, Arundo donax can reach 5 m tall [17], in China, it grows 2-6 m tall [20], and in Japan and its neighbouring regions, it grows 2-5(-8) m tall [18]. This species is naturalised in warm temperate and tropical parts of the Americas; it grows up to 8 m tall [32]. It was introduced and cultivated in Peninsular Malaysia and Singapore; it grows 1.5-3 m tall [14,19], and in Java, it grows 1.5-4 m tall [13]. In addition, from our field observations and examination of specimens in Thailand, it grows 3-8.2 m tall. Plant growth and geographical distribution are affected by environmental factors.
According to Backer and Bakhuizen van den Brink [13] and Watson and Dallwitz [7], the branches of the genus Arundo are extravaginal; however, from our observations, we found the branches of Arundo donax are intravaginal ( Figure 4B,C).
The fruit of Arundo is a caryopsis and oblong [10,15], but we have not seen it in this study. A discussion about the anatomy is mentioned in the result.
According to previous studies, the sizes of pollens are 12 × 12 µm [33] and in the range of 29-40 µm in diameter [34]. Furthermore, from our study, we found the pollen diameter in the equatorial axis to be in the range of 24.95-38.13 (29.52 ± 3.79) µm, consistent with Trigo and Fernández [34].
According to Sanghi and Sarna [33], the exine sculpturing is psilate; however, from our observations, we found it to be granular.

Materials and Methods
Plant specimens of Arundo donax were observed and collected in the Northern (Mae Hong Son, Chiang Mai, Lamphun, and Nakhon Sawan), Eastern (Nakhon Ratchasima), and Central (Chai Nat and Bangkok) regions of Thailand (Table A1, Figure A1). Herbarium specimens deposited in BK, BKF, QBG, and those included in the digital herbarium databases of BM, C, E, JSTOR, K, K-W, L, and P were examined by consulting the taxonomic literature (acronyms follow the study by Thiers [35]). The herbarium accession number can be seen on the specimens examined. The taxonomic history of this species was compiled using the taxonomic literature and online databases [5,36]. The morphological characters, distribution, habitat, phenology, and uses were described from our observations and interviews during field work and from label information on the specimens examined. The vernacular names were compiled from the specimens examined and the literature [18,24,37,38].
The preparation of plant samples was for anatomical observation. Transverse and longitudinal sections of the culm internodes, as well as transverse sections of the leaf blades, were through the midribs. The culm internode samples were sectioned with a sliding microtome at 16-20 µm thickness, stained with safranin and fast green, and mounted in DePex mounting media. The leaf samples were dehydrated in an increasing ethanol concentration series of 30%, 50%, 70%, 95%, and absolute ethanol, embedded in paraffin, sectioned with a rotary microtome at 16-20 µm thickness with Haupt's adhesive affixing paraffin sections to slides, stained in safranin and fast green, cleared with xylene, and mounted in DePeX mounting media. Leaf epidermal preparations were made by peeling and mounting on slides. The anatomical characteristics were investigated and recorded photographically with an Olympus BX53 microscope and an Olympus DP74 microscope digital camera at the Department of Botany, Faculty of Science, Kasetsart University (KU). The anatomy terminologies follow the study by Metcalfe [39].
The samples of pollen grains were taken from the herbarium specimen collected from Suan Luang Rama IX Park, Bangkok (C. Ngernsaengsaruay et al., Ad01-15122021). They were examined and recorded photographically with an Olympus BX53 microscope and an Olympus DP74 microscope digital camera. Materials were prepared for scanning electron microscopy (SEM) at the Scientific Equipment Centre, Faculty of Science, KU by mounting pollen grains on stubs using double-sided sellotape, sputter-coated with gold, and examined by a FEI Quanta 450 SEM (Hillsboro, OR, USA) at 15.00 KV. The characteristics of pollen grains (size, shape, symmetry, aperture, exine thickness, and sculpturing) were examined and measured. The pollen morphology terminologies follow Punt et al. [40].

Conclusions
The morphology, taxonomy, culm internode and leaf blade anatomy, and palynology of Arundo donax are reported. Arundo is related to Phragmites but differs in having its lemma with long white hairs outside below the middle part (vs. glabrous); rachilla glabrous (vs. with long white hairs); glumes subequal, as long as spikelet (vs. unequal, shorter than spikelet); and the lowest floret bisexual (vs. male or sterile). Two names in Arundo are lectotypified: A. bifaria and A. bengalensis, which are synonyms of A. donax. It is mostly found in wet habitats such as open areas along riverbanks and streams up to elevations of 620 m above mean sea level, especially in the northern region of Thailand. The culms are locally used for light construction, building rough fences, walls, and benches, and are also used in the traditional Lanna rituals of "Suep Chata". A. donax is also cultivated as a medicinal grass and is used in the treatment of itching rashes. It is also cultivated in the garden of Suan Luang Rama IX as an ornamental grass to provide botanical education for people. Dr. Nattapon Banjatammanon, a lecturer and clarinet player from the Department of Music, Faculty of Humanities, Kasetsart University, and a co-author in this paper, has been purchasing dried culms of A. donax from France for making clarinet reeds himself. The anatomical characteristics of the culm internodes affect the musical performance of clarinet reeds made from A. donax. The chloroplasts in the transverse section of the leaf blades are not found in the bundle sheath cells, which indicates that it is a C3 grass. Generally, the pollen grains in Poaceae are spheroidal with a single pore (monoporate) surrounded by an annulus, and the exine sculpturing is granular.
Arundo donax can be promoted as the next cash crop due to its fast growth and diverse usage. Reeds for woodwind musical instruments are a possible market. Moreover, traditional medicines and light construction also have economic benefits. Plantations of A. donax would be under consideration in other countries where it is naturally distributed, including Thailand as well.