Asarum pubitessellatum, sp. nov. (sect. Heterotropa, Aristolochiaceae) from Taiwan based on morphological and palynological evidence

Recently, we discovered an unknown Asarum from Taiwan which is closely related to A. crassisepalum S.F. Huang, T.H. Hsieh and T.C. Huang and A. taipingshanianum S.F. Huang, T.H. Hsieh and T.C. Huang by sharing a thick leaf blade, spreading perianth-lobes and a conical to cylindrical perianth-tube. We compared it with other related species and identified this plant as a new species. This new species differs distinctly from the above two related species by having larger plant body, shortened rhizomes and an inner surface of the perianth-tube that is covered with numerous simple trichomes (vs. glandular trichomes). The pollen tectum in this new species is perforate, which differs from the incomplete reticulate with small supratectate granules in A. crassisepalum and the compact rugulate with small supratectate granules in A. taipingshanianum. Furthermore, these three species are geographically separated from one another. Asarum pubitessellatum C.T. Lu & J.C. Wang, a new species is described and illustrated. The trichomes on the inner surface of the perianth-tube and pollen micromorphology were the valuable characters in the low-level classification of Heterotropa species in Taiwan.


Background
Asarum L. (Aristolochiaceae) consists of more than 100 species mainly distributed in the north temperate zone (Cheng and Yang 1983;Kelly 1998Kelly , 2001Huang et al. 2003). Most species are distributed in eastern Asia, some species in North America, and one species is endemic to Europe. On the basis of the infrageneric classification proposed by Kelly (1998), this genus was separated into two distinct subgenera, each with two sections. The subgenus Asarum, composed of the sections Asarum and Geotaenium, is characterized by connate styles with terminal stigmas, inferior ovaries, and the inner surface of perianth-tubes puberulent to strigose; while the subgenus Heterotropa, comprising sections Asiasarum and Heterotropa (including Hexastylis), is characterized by six free styles with lateral stigmas, superior or half-inferior ovaries, and the inner surface of perianth-tubes longitudinal or with a strong network of ridges (Cheng and Yang 1983;Sugawara 1987;Kelly 1997).
Section Heterotropa is morphologically diversified and composed of ca. 70 species mainly distributed in eastern Asia, particularly in the Sino-Japanese region. However, because the perianth-tube is fleshy and brittle, distortion of the flower in pressed specimens makes its structure difficult to recognize. Consequently, herbarium specimens of Heterotropa are difficult to identify reliably leading to underestimates of the species diversity of the section. In Taiwan, the Flora of Taiwan 2 nd ed. recorded only four species in this section; however, our field expeditions in recent years have led to the discovery of 3 new species and 2 new records Lu et al. 2010).
Recently, Ms. Pi-Fong Lu discovered an unknown Asarum in Miaoli, Taiwan. We compared it with other related species and identified this plant as a new species based on the morphological and palynological evidence discussed here.

Methods
The plants examined in this study were collected from native habitats and then transplanted into the greenhouse of National Taiwan Normal University, Taipei, Taiwan. The morphological and palynological data for A. pubitessellatum were based on the voucher specimens: TAIWAN. Miaoli Hsien: Mt. Chialishan, C. T. Lu 812 (TNU) and same loc., P. F. Lu 19112 (TNU). The palynological data of A. crassisepalum were based on the voucher specimens: TAIWAN. Hsinchu Hsien: Yuanyang Lake, C. T. Lu 624 (TNU). The palynological data of A. taipingshanianum were based on the voucher specimens: TAIWAN. Ilan Hsien: Tsuifeng Lake, C. T. Lu 743 (TNU).
Morphological Study-The measurement of floral characters was conducted using a Mitutoyo CD-6″CS digimatic caliper.
Pollen morphology-Pollen grains for scanning electron microscopic (SEM) study were collected from fresh anthers and prepared using the method proposed by Erdtman (1952). The acetolyzed grains were dehydrated through an ethanol series, critical point dried, coated with gold, and examined with a Hitachi SM 2400 scanning electron microscope. Descriptive terminology for pollen morphology follows that of Huang et al. (1995).  C. Huang but differs from the latter two by having larger plant body (20-30 cm vs. less than 10 cm tall); shortened rhizome; larger perianthtube (ca. 1.5 cm vs. 1-1.2 cm in diam.); multicellular simple trichomes (vs. glandular trichomes) on the inner surface of perianth-tube.

Habitat and geographical distribution
To date, this new species is only known from the type locality in Mt. Chialishan, Miaoli Hsien, Taiwan. It was found under a shaded and moist plantation of the coniferous species Cryptomeria japonica (L.f.) D. Don. The habitat is similar to those of its allies, A. crassisepalum and A. taipingshanianum, which grow in mixed coniferousbroadleaf forests, often among mosses. These three species all occur in cloudy forest zones at middle elevation but in geographically different areas. Asarum crassisepalum inhabits the mountainous area around Yuanyang Lake in Hsuehshan Mountain Range, A. taipingshanianum inhabits the mountainous area of Taipingshan in the Central Mountain Range, and A. pubitessellatum can only be found on Mt. Chialishan on the western side of the Hsuehshan Mountain Range (Figure 3).

Conservation assessment
Only two localities with less than 50 plant individuals of A. pubitessellatum have been found. The area of occurrence is estimated to be 10 km 2 . According to the IUCN red list categories (IUCN 2001) criteria, this species is categorized as critically endangered CR (B2abiii, C2a).

Etymology
We name this species A. pubitessellatum based on the inner surface of its perianth-tube being covered with numerous simple trichomes along ridges. This character is different from other Taiwanese, Chinese, and Japanese Heterotropa species, which have perianth-tubes that are covered with glandular trichomes.

Discussion
Morphologically, Asarum pubitessellatum appears to resemble A. crassisepalum and A. taipingshanianum. These three species share the following common characters: lustrous, thick leaves, perianth-lobes spreading horizontally, perianth-tube only slightly constricted at the throat, inner surface with tessellated ridges, base of perianth-lobes smooth (lacking tubercles) or scarred by only a few lines (Figure 4). Despite these similarities, A. pubitessellatum is clearly distinguished from A. crassisepalum and A. taipingshanianum by the following characteristics: (1) larger plant body (20-30 cm high vs. usually less than 10 cm) with shorter rhizomes; (2) larger perianth-tube (ca. 1.5 cm vs. 1-1.2 cm) ( Figure 4); and (3) multicellular simple trichomes on the inner surface of perianth-tube (vs. sessile unicellular glandular trichomes in A. crassisepalum and unicellular-stalked glandular trichomes in A. taipingshanianum) ( Figure 5). A more detailed comparison between A. pubitessellatum and its allies, A. crassisepalum and A. taipingshanianum is given in Table 1 in aid of their identification. Species in sect. Heterotropa usually bear short-stalked, unicellular glandular trichomes on the inner surface of the perianth-tube (Sugawara 1987;Kelly 1997). However, it is normally not possible to preserve trichomes in herbarium specimens; therefore, this character has often been ignored by taxonomists. According to this study, we consider that the trichome type on the inner surface of perianth-tube can be a valuable character in the low-level classification of sect. Heterotropa.
Pollen micromorphology is another valuable character in the low-level classification of the genus Asarum (Mi and Yang 1981;Huang et al. 1995;). Pollen exine ornamentation in sect. Heterotropa has been described as cerebelloid under verrucae for the Chinese species (Mi and Yang 1981), with rugulateperforate, perforated subunits, incomplete reticulate or compact rugulate with large warts to small granules or none on supratectum for the Taiwanese species (Huang et al. 1995;Lu et al. 2010) and microreticulate or microporate with gammae or verrucae or none for the North American species (as Hexastylis) (Niedenberger 2010).
The perforate pollen tectum without gammae or verrucae on the supertectum in A. pubitessellatum is quite different from its related allies: incomplete reticulate with small supratectate granules in A. crassisepalum (Figure 2A,D) and compact rugulate with small supratectate granules in A. taipingshanianum ( Figure 2B,E). This type of pollen is similar to that of A. hypogynum and A. chatienshanianum from Taiwan (Huang et al. 1995;) and A. naniflora from North America (Niedenberger 2010). However, due to a lack of palynological information from the most diversified area, Japan, we cannot validate the significance of pollen morphology in the infra-section classification of sect. Heterotropa.
Geographically, A. pubitessellatum, A. crassisepalum, and A. taipingshanianum are currently distributed allopatrically, though they all occur in a similar habitat, under cypress forest [mainly composed of Chamaecyparis formosensis Matsum. or C. obtusa Sieb. & Zucc. var. formosana (Hayata) Rehder or both]. Their similar gross morphologies, pollen features, and habitat suggest that they are closely related. The contemporary geographic isolation of these three species may result from a decreased forest range or forest fragmentation. Further phylogeographical study is in progress to test this hypothesis.
The two editions of Flora of Taiwan (1975-1979; 1994-2003) and the Supplement to the Flora of Taiwan, 2nd ed. (Wang and Lu 2012) completely described Taiwan's flora up to 2009. However, most recent findings, including new generic records, e.g., Ypsilandra (Hsu et al. 2011) and Phacellanthus (Chung et al. 2010), new species, e.g., Cotoneaster rosiflorus and C. chingshuiensis (Chang et al. 2011a, b), Pouzolzia taiwaniana (Peng et al. 2012), Thismia huangii (Chiang and Hsieh 2011), and Tripterospermum hualiense , and numerous newly recorded species, indicate that the documentation of the island's vast and unique biodiversity is incomplete. This study echoes the suggestion of Hsu et al. (2011) andPeng et al. (2012) that the continuation of the botanical inventories is needed, especially those areas rarely botanized.
A. tawushanianum 4. Leaves triangular-ovate, margin entire; throat slightly constricted, perianth-tube length longer than width. 5 5. Leaf apex acute; outer surface of perianth-tube hairy, tubercles on base of perianth-lobes present, bar-like; filament attached to the base of perianth-tube.
A. macranthum 8. Perianth-lobes shorter than perianth-tube, with few tubercles on the base; orifice larger than 10 mm in diam.; orifice rim narrow; inner surface of perianthtube irregularly tessellated on the upper half, but only longitudinal ribs on the lower half, longitudinal ridges more than 24. A. satsumense 9. Perianth-tube tubiform, length equal to width, inner surface of perianth-tube regularly tessellated, longitudinal ridges 12. A. albomaculatum 9. Perianth-tube obconical or tubiform, length longer than width, inner surface of perianth-tube irregularly tessellated, longitudinal ridges 12-24. 10 10.Perianth-tube tubiform, upper part slightly inflated; lobes yellow-greenish or purple-greenish, adaxial surface pubescent; outer surface yellow-greenish, inner surface purple. A. chatienshanianum 10.Perianth-tube obconical; lobes maroon, adaxial surface densely villous; flowers all maroon. A. villisepalum Conclusions Asarum pubitessellatum C.T. Lu & J.C. Wang, a new species is described and illustrated based on the morphological and palynological evidence. The present study showed the trichomes on the inner surface of the perianthtube and pollen micromorphology were the valuable characters to distinguish the closely related Heterotropa species in Taiwan.