Assessment of Morphological, Anatomical and Palynological Variation in the Medicinal Plant Disporopsis longifolia Craib (Asparagaceae) for Botanical Quality Control

Disporopsis longifolia Craib is an Asian medicinal plant belonging to the Asparagaceae family. The plants are well known for their steroidal saponins and phenolic compounds and are traditionally used as tonics for back pain, bellyache, cough, diabetes, asthma, pneumonia and rheumatism. However, they are challenging to identify to species level using morphology. This raises a serious concern for their medicinal applications where botanical quality control is essential. The most appropriate morphological, anatomical and pollen characters for species diagnosis were therefore determined. Synonyms were identified and lectotypification provided. The morphological characters were described from 76 fresh and dried specimens to include a broad range of materials from differing habitats and locations. Paraffin and peeling methods were applied for anatomical studies of leaves and stems and a modified acetolysis method was undertaken for pollen morphology. This paper compares the new character data to published data from other species in the genus, namely D. aspersa, D. fuscopicta, D. jinfushanensis, D. pernyi and D. undulata. This is the first report of such anatomical and pollen morphology characters for D. longifolia. The results provide accurate morphological, anatomical and palynological characters for quality control and are best applied in combination with each other.


Introduction
Species of the genus Disporopsis Hance including D. longifolia Craib are used as medicinal plants and are popular in China and several other countries in Southeast Asia such as Laos, Myanmar, Thailand and Vietnam [1][2][3][4]. The rhizome or whole plant is used as a tonic for back pain, bellyache, cough, asthma, pneumonia and rheumatism [1][2][3][4]. Moreover, the rhizome of D. longifolia can be eaten with betel nut (Areca catechu L., Arecaceae Juss.) and it is an ingredient in chicken soup [5,6].
Some phytochemical studies have been undertaken on the genus that is characterized by its steroidal saponins and phenolic compounds. Disporopsis aspersa (Hua) Engl. ex Diels, a close relative of D. longifolia, contains the ester (2-isopropyl-5-methylphenyl 2-(naphthalen1-yl) acetate) and furanone ) which has antifungal, neuritogenic and anti-inflammatory activity [7,8]. Several chemical compounds have been isolated from D. longifolia such as a range of known spirostanol glycosides and two new fucose-containing spirostanol glycosides, namely (25R)-spirost-5en-3β-yl O-β-D-glucopyranosyl-(1→4)-β-D-fucopyranoside and (25S)-spirost-5-en-3β-yl O-β-D-glucopyranosyl-(1→4)-β-D-fucopyranoside [9]. However, meaningful interpretation Plants 2023, 12, 259 2 of 14 of these phytochemical data is reliant on accurate biological species delimitation. The species of the genus are very similar at a morphological level and they share many common characters. They are perennial erect herbs with moniliform rhizomes and erect and distal arching aerial stems. Their leaves are simple with alternate arrangement and the flowers are solitary or in a raceme. The solitary flower is single or fascicled. The perianth is campanulate and has a corona. The anther has two thecae and is introrse, and the stigma is capitate or slightly trifid. The fruits are berries [10,11]. Because of this morphological similarity, there is a serious issue with species recognition and identification for medical applications.
Disporopsis longifolia is very commonly distributed in Northern and Northeastern floristic regions of Thailand and its rhizomes are used for food and traditional medicine [7,24]. This paper aimed to use herbarium specimen label information to gather location, phenological and ethnobotanical data. It then determined the most useful morphological, anatomical and palynological characters for species diagnosis to provide the necessary data for quality control in the world's medicinal plant market. To achieve this, studies were undertaken on 76 samples from fresh and herbarium material. The results from D. longifolia are compared with other species in the genus and related genera, using previously published studies, that could potentially be confused with the target species for medical application.

Species Description and Morphological Characters
Morphological data were recorded from 76 plants (listed in Materials and Methods) and summarized into the following description for Disporopsis longifolia: Type material in Kew (K; holotype), Edinburgh (E; isotype) and Paris (P; lectotypes) herbaria were viewed (! notation below) and protologues consulted. This description can be used with the photographs in Figure 1 to accurately identify the species, and it follows the format and notation from the Flora of Thailand Project [25]. Synonyms are listed and lectotypification provided.
Disporopsis longifolia is distinguished by its moniliform rhizome and the flowers that are single or in a fascicle with 2-7 flowers. The flowers are campanulate and white. The perianth has a corona and stamens are inserted on the corona lobe. The stigma is capitate and the fruits are green and white when ripening. According to herbarium labels, the rhizome is used as a tonic for cystitis, diuretics, cough, headaches, cold and fever, and the leaves are used to treat knee pain. It is distributed in China, Myanmar, Thailand, Laos and Vietnam and situated near streams or on limestone areas of evergreen forest at 300-1700 m in altitude. It flowers in May to July and fruits in July to February. Its vernacular names include Chua Mo Hua Cho, and Lo Ma Chor Ju.
Lectotypification notes: Polygonatum laoticum Gagnep. and P. tonkinense Gagnep. were synonymized under Disporopsis longifolia Craib by Sungyun and Tamura [11] but they did not list the type. The type Polygonatum laoticum Gagnep. is C. Thorel 3319. Later, we found three herbarium sheets in P. We selected P00687140 as the lectotype, and selected P00687141 and P00687142 as isolectotypes.

Stem, Petiole and Leaf Anatomy
The stem, petiole, leaf (including midrib, margin, costal and intercostal zones) and leaf surface results are shown in Figures 2 and 3.
Stem anatomy: The stem is rounded with two small ridges ( Figure 2A). The epidermis is rounded or rectangular with echinate cuticle thickening. The cortical region comprises 7-13 layers of parenchyma cells with air spaces and raphides ( Figure 2B). The parenchyma cells are rounded, subrounded or rectangular and there is intercellular space. Pericycular vascular bundles and ground tissue vascular bundles are present; both are the closedcollateral bundle type. The pericycular vascular bundle regions have 28-36 bundles with 3-7 layers of sclerenchyma cells. The sclerenchyma cells form rings and are surrounded with pericycular vascular bundles. The ground tissue has parenchyma cells, intercellular space, air spaces and vascular bundles. The vascular region in the ground tissue has 15-28 bundles with sclerenchyma cells surrounding the bundles.    Petiole anatomy: The petiole is V-or U-shaped ( Figure 2C). The upper and lower epidermis cells have echinate cuticle thickening and papillae that are present only on the lower epidermis. The ground tissue includes parenchyma and collenchyma. The parenchyma cells are rounded, subrounded or polygonal with intercellular space. The collenchyma cells are rounded or subrounded. The vascular bundle regions distributed in the ground tissue have 11-14 bundles that are of the closed-collateral type with sclerenchyma surrounding them.
Lamina anatomy: The lamina comprises midrib, margin, costal and intercostal zones. The midrib is U-shaped ( Figure 2D). The epidermal cells of both leaf surfaces are rounded, subrounded or rectangular with echinate cuticle thickening ( Figure 2E,F). The ground tissues have only rounded or subrounded shaped parenchyma cells. The vascular bundles distributed in the ground tissue are closed-collateral bundle type with sclerenchyma surrounding them. The margin is revolute with large epidermal cells ( Figure 3B). The echinate cuticle is thickened on the upper and lower epidermal cells and papillae are present.
The leaf is hypostomatic with raised stomata ( Figure 3A). The coastal zone of the upper and lower epidermis has smooth cuticle thickening ( Figure 3C,E) and the cells are rounded, square or elongated rectangular. The intercostal zone of upper and lower epidermis also has smooth cuticle thickening ( Figure 3D,F) and the cells are also rounded, square or elongated rectangular. The palisade mesophyll is columnar or subrounded with 1-2 layers. The spongy mesophyll is rounded or irregular. The leaf surface of upper and lower epidermis has elongated rectangular and polygonal cells ( Figure 3G,H). The anticlinal cell wall is strongly straight and the periclinal cell wall is straight to slightly undulate. The lower epidermis has anomocytic stomata ( Figure 3H).
Our studies using information from the literature and herbarium labels indicates that the rhizome of D. longifolia was used for traditional medicine by boiling it three times and the last water used as a tonic for cystitis, diuretics, cough, headaches, cold and fever. Moreover, the leaves are used as medicine to treat knee pain. In the northern parts of Thailand, the rhizome of D. longifolia is used for eating with betel nut [7]. This paper studied a wide range of samples across its natural distribution and has been able to describe key characteristics of this plant based on gross morphology, anatomy and pollen morphology. It is possible, but difficult, to identify the species using the morphological characters described here, especially if flowering material is unavailable. Therefore, it is advisable to include anatomical and pollen morphological studies to confirm taxonomic identity. In summary, based on previous studies [11,23,24] and observations in this paper, D. longifolia has underground moniliform rhizomes and its aerial stem is erect to distally arching. The flowers are solitary or fascicled with 2-7 florets in the axil of a leaf. The flowers are of campanulate shape and white and the perianth is connate with six lobes. The tepal apex is ciliate. The stamens are introrse and longitudinally split and they are inserted on the perianth lobes. The stigma is capitate and the fruits are green to white berries.
Care must also be taken not to confuse Disporopsis with its close relative Polygonatum, both in tribe Polygonateae [19,20,26], because they have similar morphology. They share moniliform rhizomes and the leaves have alternate arrangement. The flowers are cylindrical to campanulate and are in axils of leaves, and the fruits of both are berries [10,20,26]. Both genera have been used as medicinal plants in Thailand [7,8] as whole plants or especially rhizome [1][2][3][4][5][6][7][8]. Therefore, it is important to distinguish the two correctly. A corona is present in Disporopsis but absent in Polygonatum. The filament of Disporopsis is inserted on the corona but in Polygonatum, it is inserted on the perianth.
Charoenphol [24] reported D. longifolia in Thailand and described the flower characters with fascicles of 5-6 florets or more. Our study determined that the flowers are solitary to fascicled with 2-7 florets in the same area and populations. Therefore, this paper confirmed that the D. longifolia has solitary flowers or fascicled inflorescences with 2-7 florets. In addition, variation in the colour of the lower stem was recorded, which is green, purplish red or green with purple maculate in Thai D. longifolia depending on habitat. The number of florets per fascicle has been used for species identification and distinction by others. For example, Songyun and Tamura [11] distinguished D. longifolia by its fascicles with 5-10 florets compared to D. aspersa, D. fuscopicta, D. jinfushanensis, D. pernyi and D. undulata which have solitary flowers or fascicles with 2-3 florets. In this paper, the number of flowers per fascicled inflorescence shows high variability and it is recommended that this characteristic should be avoided for species identification.
Species of Disporopsis can be divided into two groups. One group has moniliform rhizomes, including D. longifolia and D. fuscopicta, and the other has terete rhizomes, including D. aspersa, D. jinfushanensis, D. pernyi and D. undulata. The first group, D. longifolia and D. fuscopicta, also have sagittate anthers [11,12,23] but different corona and fruit. In D. longifolia, the corona is fleshy and the lobes do not exceed the anthers. This differs in D. fuscopicta, where the corona is membranous and the lobes exceed the anthers. The berry fruits are white in D. longifolia or purplish in D. fuscopicta [27]. Therefore, the diagnostic characteristics of D. longifolia are the moniliform rhizomes, the fleshy corona, the corona lobes not exceeding anthers, and the white berries.
The anatomy of stem, petiole, midrib, leaf margin and leaf surfaces of D. longifolia were investigated to detect additional characters for identification. Both D. fuscopicta and D. longifolia have hypostomatic leaves [27] but they differ by the anticlinal cell walls. The anticlinal cell walls are sinuous in D. fuscopicta [27] whereas strongly straight in D. longifolia. Therefore, the anticlinal cell walls are significant characters for species identification. Some samples were collected from limestone areas and were determined to have raphides in their stems, whereas some samples collected from other locations such as near streams, on mountain slopes or on mountain summit in evergreen forest lacked raphides. Therefore, raphides in stem are a plastic characteristic that varies based on locality and they cannot be used reliably for species delimitation.
Anatomical characters of genus Disporopsis are poorly known and no anatomical studies of D. longifolia have previously been published. In summary, the stem of D. longifolia has two small ridges, the epidermal cells of stem, petiole, midrib and leaf margin all have echinate cuticle thickening, and the upper and lower epidermis in costal and intercostal zones have smooth cuticle thickening. Papillae are present on the lower epidermis of the petiole and leaf margin. The cortical region has 7-13 layers of parenchyma cells that sometimes accumulate raphides. There are pericycular vascular bundles and vascular bundles in the ground tissue, and the bundles in stem, petiole and leaves are of the closedcollateral bundle type. The petiole is V-or U-shaped and midrib is U-shaped. The petiole has 11-14 vascular bundles. Raised stomata are present on hypostomatic leaves. The palisade mesophyll is columnar or subrounded with 1-2 layers. The spongy mesophyll is rounded or irregular. The leaf margin is revolute and has large epidermal cells. In addition, the upper and lower epidermis of the leaf have elongated rectangular and polygonal cells. The anticlinal cell wall is strongly straight and periclinal cell wall is straight to slightly undulate. The lower epidermis is of anomocytic stomata type.
Pollen morphological characters of Disporopsis are also poorly documented and this paper is the first report on pollen morphological characters of D. longifolia. Pollen grains of D. longifolia are heteropolar monads of medium size, have bilateral symmetry, and are prolate and monosulcate. The ornamentation is perforate and areolate at the aperture areas. Pollen morphologies have been described for D. aspersa, D. fuscopicta and D. pernyi in China [28]. They shared common characters including the monads, bilateral symmetry, monosulcate aperture and reticulate ornamentation. In general, it was determined that the pollen morphological evidence did not strongly support species identification [28]. In contrast, it was determined that the D. longifolia has perforate ornamentation in general areas and areolate ornamentation in the aperture area. These two types of pollen ornamentation, perforate and areolate, are useful to distinguish D. longifolia from the aforementioned species. In addition, Wang et al. [28] reported medium pollen size of D. aspersa, D. fuscopicta and D. pernyi, including polar axis of 20.3-25.9 µm in length and equatorial axis of 41.4-44.3 µm in length. In contrast, D. longifolia had a longer polar axis of 40-42 ± 0.79 µm and shorter equatorial axis of 29-31 ± 0.73 µm. The differences are again significant for taxonomic separation of D. longifolia from D. aspersa, D. fuscopicta and D. pernyi.
Given the potential value of D. longifolia and its use in traditional medicine, it is important to conserve its wild populations for future utilization. No IUCN (Standard and Petitions Committee 2019) conservation status category had previously been proposed for this species, so we here designate the status of Least Concern (LC). This is because Disporopsis longiflolia has a wide distribution in China, South-Eastern Asia and in Thailand, and it is distributed in Northern and North-Eastern protected areas.
Our studies have provided essential baseline data for future phytochemical assessments where accurate species identification is essential. We have also provided essential lectotypification of synonyms. There are several other methods available for botanical quality control of medicinal plants and including molecular DNA barcoding and chemical characterisation [29,30], but these studies are not always the most appropriate and they rely on accurate species identification using morphology and anatomy in the first instance.

Materials and Methods
The morphological characters were described from fresh and dried specimens as listed under specimens examined below. The fresh specimens were collected from 10 field sites in the Northern and North-Eastern Floristic Regions of Thailand [31], Figure 5. Voucher specimens were prepared and deposited in BK, BKF, KKU and QBG (acronyms of herbaria follow Thiers [32] Index Herbariorum). Herbarium specimens were consulted from BK, BKF, KKU, QBG and the online herbarium databases including BM, C, E, G, K, K-W, L, P, S, W and WU to gain additional samples.

Materials and Methods
The morphological characters were described from fresh and dried specimens as listed under specimens examined below. The fresh specimens were collected from 10 field sites in the Northern and North-Eastern Floristic Regions of Thailand [31], Figure 5. Voucher specimens were prepared and deposited in BK, BKF, KKU and QBG (acronyms of herbaria follow Thiers [32] Index Herbariorum). Herbarium specimens were consulted from BK, BKF, KKU, QBG and the online herbarium databases including BM, C, E, G, K, K-W, L, P, S, W and WU to gain additional samples. Ethnobotanical data were collected from label information on herbarium sheets, the literature reviews and interviews with local tribesman and local people of the Karen ethnolinguistic group (Sino-Tibetan language) also known as the Kayin, Kariang or Kawthoolese.
Anatomical characters were studied using the paraffin method following Thammathaworn [33] and permanent slides of stem, petiole and leaf were prepared following Kermanee [34]. The permanent slides were prepared using the paraffin method. Fresh stem and leaves were fixed in 50% formalin acetic acid (FAA) for 24 h and then washed in 50% ethanol. The samples were dehydrated in a graded Tertiary Butyl Alcohol (TBA) series (50, 70, 85, 95 and 100%, respectively). In addition, samples were immersed in a mixture of pure TBA and liquid paraffin at 60 • C for 12 h and embedded in paraffin at 60 • C. The samples in paraffin blocks were sectioned with 15-25 µm thickness using a rotary microtome (Leica RM2165, Germany). The sections were affixed on slides. These slides were stained with a combination of safranin O and fast green. The stained samples were washed with 95% ethanol and dehydrated with absolute ethanol. The samples were cleared continuously in xylene and absolute ethanol (1:1) and immersed in pure xylene. The slides were mounted with DPex and investigated under a light microscope (Carl Zeiss Primo Star).
Permanent slides of the epidermis were prepared using a peeling method [34]. The method is applicable to fresh and spirit preserved leaves. The epidermis was scraped off with a razor blade and washed with water. Then, it was soaked in 0.1% chlorine, washed with water and stained with 1% safranin for 3-5 min. Samples were then dehydrated by ethanol series (30%, 50%, 70%, and 95% respectively) and cleared with xylene. Finally, the samples were mounted on slides with DPex and photographed under a light microscope (Carl Zeiss Primo Star).
A modified acetolysis technique was applied for the preparation of the pollen morphological characters [35] and the pollen slides were investigated under a light microscope (Carl Zeiss Primo Star) and a scanning electron microscope (FEI, Quanta 450) following Halbritter et al. [36]. The anatomical and pollen permanent slides were stored at the Palynology Special Research Unit, Department of Botany, Faculty of Science, Kasetsart University, Bangkok.

Conclusions
The detailed data and descriptions presented here allow for accurate identification of D. longifolia. These characters are best used in combination and can be applied to living and herbarium material and material with different parts missing. The anatomical and palynological characteristics are reported here for the first time. DNA barcoding and chemical characterisation can also be used for species identification, but these techniques rely on accurate baseline species identification using morphology and anatomy in the first instance. Our study therefore provides the essential information required for further molecular and phytochemical evaluation where species identification is of paramount importance. Data Availability Statement: Data sharing not applicable. No new data were created or analyzed in this study. Data sharing is not applicable to this article.