Revision of Polygonatum (Asparagaceae, Nolinoideae, Polygonateae) of Taiwan

Abstract Polygonatum is widely distributed in the northern hemisphere, especially in eastern Asia. There has been no comprehensive taxonomic study of Taiwanese taxa for some time, and researchers could not agree on a consistent treatment of the genus. Therefore, we revised the genus by literature review and type specimen examination along with comparison of morphology, karyotype and pollen characteristics. Only one species P.arisanense Hayata was recognized in this study. P.chingshuishanianum Ying and P.formosanum (Hayata) Masamune & Simada are regarded as varieties of P.arisanense and are here presented as two new combinations, P.arisanensevar.chingshuishanianum and P.arisanensevar.formosanum.


Introduction
The genus Polygonatum comprises ca. 60 species, distributed in the northern hemisphere, mainly from southwest China to Japan (Chen et al. 2000). Some species, such as P. sibiricum Redoute and P. odoratum (Mill.) Druce have been cultivated as medical or horticultural crops (Wujisguleng et al. 2012, Tang et al. 2018. The diagnostic characters of the genus include the presence of rhizomes, single and often arching stem, alternate, opposite, or whorled phyllotaxis, axillary inflorescences with one to several flowers, perianth tubes longer than segments, and berries (Wang et al. of the Department of Forestry, National Chung Hsing University. Voucher specimens (include living ones) were deposited in the herbarium of the National Chung Hsing University (TCF). The following herbaria were examined: CHIA, HAST, KYO, NTUF, PPI, TAI, TAIE, TAIF, TCF, TI, TNM, and TNU. We also retrieved type specimen images of Polygonatum from the website of the Muséum National d'Histoire Naturelle (P). Pollen observation and karyotype analysis materials are listed in Table 1.
The floral morphology is important in the taxonomy of Polygonatum, and therefore we had to check the flower morphology of living plants, especially P. chingshuishanianum, whose floral part was lacking on the type specimen. In order to do this, we visited the type population of P. chingshuishanianum for several years, and were only able to capture two anthesis individuals.

Pollen morphology
The pretreatment of the anther followed the method of Halbritter (1998). The anther was dried with a critical point dryer (Quoram E3100). The pollen was taped on to the stub with copper tape, after sputter-coating with gold (Quoram SC7620) and observed by SEM (Hitachi S-3400N). The terminology and description of pollen morphology were in accordance with Punt et al. (2007) and Hesse et al. (2009) while the aperture type classification followed the procedure of Halbritter and Hesse (1993).

Karyotype analysis
Plant materials for karyotype analysis were cultivated in the greenhouse of the Department of Forestry. Root tips were collected in the morning on a sunny day and preserved in 0.002M 8-hydroxyquinoline solution below 10 °C for eight hours. Afterwards, the roots were fixed in Carnoy's solution (glacial acetic acid: 99.5% EtOH, 1:3) at 4 °C overnight. The fixed roots were stained with acetic-orcein overnight, squashed and observed under a light microscope (Accu-Scope 3025 Series). Cells showing good chromosome spreading were photographed with a CCD camera (ProgRes C14 Plus). Karyotype analysis was done according to the procedures of Levan et al. (1964) and Stebbins (1971).

Distribution and conservation rank evaluation
The distribution maps were made by the location of herbaria specimens and our field work. Older specimens were geo-referenced using the study of Huang et al. (1993), and points proximal to written locality were mapped. Classification of geographical climatic regions and altitudinal vegetation zones followed the guidelines of Su (1984Su ( , 1985. We used the protocol of the red list of vascular plants of Taiwan, 2017 (editorial committee of red list of vascular plants of Taiwan, 2017) for evaluation of conservation ranks.

Diagnostic characteristics of Polygonatum
Leaf Leaf morphology is an important character for identification of intrageneric taxa of Polygonatum (Chen et al. 2000). The leaf shape of P. arisanense var. chingshuishanianum is lanceolate to oblong lanceolate with obtuse apex (Fig. 7C, D), whereas the leaves of P. arisanense var. arisanense and P. arisanense var. formosanum are ovate to ovate-lanceolate with acute and obtuse apexes respectively (Fig. 6C, D, 8C, D). The texture also differs among these taxa. The leaf of P. arisanense var. arisanense is chartaceous, whereas P. arisanense var. chingshuishanianum and P. arisanense var. formosanum have chartaceous to coriaceous leaves.

Rhizome
The Taiwanese taxa all have tuberous rhizomes that are similar in appearance to one another, rather than the terete rhizomes found in P. odoratum (Fig. 6B, 7B, 8B).

Inflorescence
The inflorescences of Polygonatum species are solitary to multi-flowered umbels, axillary and often pendulous. The three taxa of Taiwan have similar inflorescence forms, but can be distinguished by the number of flowers within an inflorescence. The inflorescences of P. arisanense var. chingshuishanianum have only one or two flowers, while P. arisanense var. arisanense and P. arisanense var. formosanum have (3-)5-7 and 2-3(-5) flowers, respectively (Fig. 6E, 7E, 8E).

Flower
The flowers of the three taxa are typical for Polygonatum. The perianth tube of P. arisanense var. chingshuishanianum is smaller (6-8 mm × 4-5 mm) than the others. Besides, two forms of perianth tube are found among the taxa of Taiwan. The first one is found in P. arisanense var. arisanense, which have an acute base (less than 90°) (Fig. 6F). The second type is found in P. arisanense var. chingshuishanianum and P. arisanense var. formosanum, which have an obtuse to truncate base (more than 90°) (Fig. 7E, 8F).

Pollen morphology
The pollen grains of Taiwanese Polygonatum are monosulcate monads of medium size, spheroidal, simple-sulcate aperture type, and the proximal polar view is perforate. The widths of the lumina and the muri are similar among the three taxa. No conspicuous difference is found among them (Table 2, Fig. 1).

Distribution
The taxa of Polygonatum of Taiwan are all endemic, and were found from low to medium altitude in mountainous regions. Compared to other varieties, P. arisanense var. arisanense has the widest distribution. It grows in most geographical climate zones except for the Lanyu region, the Southeast, the south section of the East region, and oth-  Su (1984), which had more precipitation and higher relative humidity (Fig. 3). The habitats are often shady with high moisture, under, or in the margin, of a forest. In contrast, P. arisanense var. chingshuishanianum and P. arisanense var. formosanum have very narrow distributions, especially the former. P. arisanense var. chingshuishanianum is only found in one location, near the summit of Mt. Chingshuishan in Hualien county of eastern Taiwan (Fig. 4). The plants are growing on a limestone slope with full sunlight and thin soil. On the other hand, P. arisanense var. formosanum is only found in the region of Yangmingshan National Park, a volcanic area in northern Taiwan (Fig. 5). The populations were found from the exposed roadside to the forest habitat.

Conservation rank
In the red list of vascular plants of Taiwan 2017 (editorial committee of the red list of vascular plants of Taiwan, 2017), the conservative rank of P. chingshuishanianum (=P. arisanense var. chingshuishanianum) and P. odoratum var. pluriflorum (=P. arisanense var. arisanense and P. arisanense var. formosanum) was evaluated as data deficient (DD) and of least concern (LC). Here we reevaluate three taxa of Polygonatum of Taiwan. As a result, P. arisanense var. arisanense is evaluated at LC rank. The population was still large in the island and the plants are common, so there is no immediate threat to this taxon. Polygonatum arisanense var. chingshuishanianum is ranked as critical endangered (CR)(A1(a), B1(bii+biii), D1), because there are few fertile individuals and there is a hiking trail that crosses the population area. Polygonatum arisanense var. formosanum is evaluated as near threatened (NT)(C, C1). This variety is only found in Yangmingshan National Park, but there are more than ca. 10,000 fertile individuals and they are regenerating well. However, considering the narrow distribution and the fact that some of the populations are near places of human activity, we evaluate the conservative rank as near threatened.

Discussion
Here we present evidence that leads us to conclude that there is but one species and three taxa of Polygonatum of Taiwan. These are enumerated below.
The differences between Polygonatum arisanense and related taxa Polygonatum arisanense were identified as P. odoratum, P. odoratum var. pluriflorum or P. cyrtonema by several authors (Ying 1969(Ying , 1990(Ying , 2000Wang et al. 1978;Jeffery 1980;Wang 1997;Boufford et al. 2003). These species all have similar appearances with alternate leaves, pendulous axillary inflorescences, white perianth tubes, and purplish berries. Identification of species often relies on the rhizome and stamen morphology (Tamura 1993;Chen et al. 2000), but such parts are often neglected when collecting the samples, or are hard to observe in herbarium sheets. The morphology of the rhizome provides significant value in determining the taxonomy of Polygonatum, but this character is usually lacking on the herbarium specimens, even on the type specimens. Different forms can be recognized among the taxa; for example, P. hookeri Baker has terete rhizomes, while P. franchetii Hua and P. filipes Merr. ex C. Jeffery & McEwan have moniliform ones. Tuberous rhizomes are found in all Taiwanese taxa, and are different from the terete rhizomes of P. odoratum. Therefore, misidentification is relatively common in Polygonatum species. According to literature review, and examination of living and herbarium materials, P. arisanense is distinguished from P. odoratum by having tuberous rhizome, which is terete in the latter. On the other hand, P. arisanense was distinguished from P. cyrtonema by its filaments being adnate to the middle of the perianth tube, and the anther being without spurs, whereas the filaments are adnate near the apex of the perianth, and the anthers are spurred in the latter. Such a result was also supported by the molecular study (Floden and Schilling 2018) The fact that the chromosome number of P. odoratum was 2n = 20 (Tamura 1990), and of P. arisanense was 2n = 22, revealed that the difference between them was not only on the morphological, but also on the genetic level (Table 4). Besides, Hsu (1971) also recorded a tetraploid individual (2n = 44) of P. arisanense from Taitung county, implying that the polyploidy events may be found in some populations. Such events were often found in the populations of Polygonatum (Therman 1950;Tamura 1990). Further study of the distribution and frequency of polyploidy event of P. arisanense population is needed.
The taxonomic status of P. chingshuishanianum and P. formosanum In order to determine the taxonomic status of P. chingshuishanianum, the floral morphology is the key character due to the lack of type specimen. The observed flower morphology was generally identical to that of Ying (1988Ying ( , 2000, and the morphol-ogy of other parts matched those of the type specimen. Therefore, the plant we found was identified as P. chingshuishanianum, and was the first record of floral morphology after the original publication of Ying (1988). Generally, the floral morphology was similar to P. arisanense, but minor distinctions included the fact that there were fewflowered inflorescences, smaller flowers, truncate to obtuse perianth tube base, dwarf plant size and thicker leaves. These were all different from typical P. arisaensense. The morphology of the pollen and karyotype were also similar to P. arisanense, but the floral morphology was distinct from the lower altitude population of P. arisanense var. arisanense. Moreover, the habitat of this taxon was on the limestone region of eastern Taiwan, which had abundant endemic species found in this region, such as Dianthus seisuimontanus Masamune (Caryophyllaceae), Berberis chingshuiensis T. Shimizu (Berberidaceae), and Rhamnus chingshuiensis T. shimizu (Rhamnaceae). Such a unique geological environment may lead to unique evolution events. Therefore, we treated this taxon as a variety of P. arisanense rather than an independent species as Ying (1988Ying ( , 2000 (Table 5). Polygonatum officinale var. formosanum was originally described by Hayata (1920). and elevated to P. formosanum Masamune and Simada (1936). From nearly that time henceforth it has been treated as a synonym of P. odoratum, P. odoratum var. pluriflorum, or as P. cyrtonema (Liu and Ying 1978;Wang 1997;Chen et al. 2000;Ying 2000). After careful observation of the types and living plants from the type locality, this taxon was found to be more similar to P. arisanense rather than the species and taxa mentioned above. This taxon differs from var. arisanense by having thicker leaves, larger flowers, basal flattened perianth tubes, and fewer-flowered inflorescences, but the pollen morphology and karyotype are similar to those of P. arisanense, so we prefer to treat this taxon as a variety of P. arisanense (Table 5).  Endemic. Distributed from low to ca. 2000 m mountains through the island (Fig. 3). Flowers pendulous, pedicels 5.0-6.5 mm long, glabrous, articulated close to the flower, perianth tube 1-1.5 cm long, ca. 8 mm wide, base flattened, perianth segments 6, arranged into 2 whorled, each 3, triangular, ca. 5 mm long, ca. 5 mm wide, green with dark green strip, apex obtuse, floccose. Stamens 6, base expansion and flattened, inserted at middle of perianth, filaments 5.5-6.5 mm long, anthers oblong-lanceolate, 1.5-2.0 mm long, ca. 1 mm wide. Ovary superior, globose, 4.5-5.5 mm long, 3.5-4.5 mm in diam., glabrous, style filiform, ca. 1 cm long, glabrous, stigma entire, pubescent. Fruits berry, globose, glabrous. 2n=22. Endemic. Known only from the summit of Mt. Chingshuishan, ca. 2300-2400 m, on exposed limestone slopes (Fig. 4). Perennial herbs. Rhizome tuberous. Stem straight to arching, 30-80 cm long, green or purplish, glabrous, covered with scale leaf at the base, caducous. Leaves deciduous, alternate, thick chartaceous to coriaceous, ovate to lanceolate ovate 3-multiple nerved, apex attenuate, base obtuse, 8.0-10.5 cm long, 3.5-5.5 cm wide, sessile or short-petioled, ca. 3 mm long, often purplish. Inflorescences axillary, solitary to umbels with 3-5 flowers, peduncle subequal to pedicels, 1.5-2.0 cm long, articulated close to the flower, bracteoles very minutely, caducous. Flowers pendulous, perianth tube, 2.5-3.5 cm long, 0.5-1.0 cm