Phytotaxa 277 (1): 047–058 ISSN 1179-3155 (print edition) http://www.mapress.com/j/pt/ PHYTOTAXA Copyright © 2016 Magnolia Press Article ISSN 1179-3163 (online edition) http://dx.doi.org/10.11646/phytotaxa.277.1.4 A new variety of Panax (Araliaceae) from Lam Vien Plateau, Vietnam and its molecular evidence NONG VAN DUY1, LE NGOC TRIEU2, NGUYEN DUY CHINH2 & VAN TIEN TRAN2* 1Tay Nguyen Institute for Scientific Research, Vietnam Academy of Science and Technology, 116 Xo Viet Nghe Tinh, Dalat City, Lam Dong Province, Vietnam 2Faculty of Biology, Da Lat University, 01 Phu Dong Thien Vuong, Dalat city, Lam Dong Province, Vietnam *Author for correspondence: E-mail: [email protected] Abstract A new variety of Panax, P. vietnamensis var. langbianensis is described from Lang Bian Mountain, Lam Vien Plateau in southern Vietnam. It is visually distinct from other two varieties of P. vietnamensis (var. vietnamensis, var. fuscidiscus) by having the shorter petiolules 8–11 mm long, leaflet apex acuminate, pedicels 0.4–0.6 cm, petals smaller, disk prominent, and styles typically 2 (or rarely 1). The recognition of the new variety is supported by the evidence of molecular sequence data from three markers (ITS1-5.8S-ITS2, 18S rRNA and partial matK) in addition to morphological consideration. The new variety is known only from one population from the locality where the type specimens were collected. The species occurs in an area of only approximately 1 km2 with a population size of approximately 100–200 individuals. Therefore it should be regarded as critically endangered (CBB2acb (ii,iii,v); C2a(i); E) according to the IUCN Red List Categories and criteria. Keywords: IUCN, molecular phylogeny, relationship, taxonomy Introduction Panax Linnaeus (1753: 1058) is a small genus of Araliaceae typified by P. quinquefolium Linnaeus (1753: 1058). It consists of 16–18 species (Wen & Zimmer 1996, Wen 2001, Sharma & Pandit 2011), of which two species, P. quinquefolius Linnaeus (1753: 1058) and P. trifolius Linnaeus (1753: 1059), grow wild in eastern North America (Seemann 1868, Burkill 1902, Graham 1966, Proctor & Bailey 1987), whereas other 14–16 species are distributed in eastern central and eastern Asia (Burkill 1902, Hara 1970, Hoo & Tseng 1973, Wen & Zimmer 1996). The greatest diversity of Panax is recognized in the eastern, southwestern and central China, the central to eastern Himalayas (Nepal, Bhutan and India) and some parts of South-East Asia (Burkill 1902, Hara 1970, Hoo & Tseng 1973, Wen & Zimmer 1996). The genus is distinguished from other genera of Araliaceae by its perennial herbaceous habit, stout rootstock, simple stem with scales at the base, palmate compound leaves in whorls of 3–5, solitary terminal umbellate inflorescence, shortly 5-toothed calyx, 5 imbricate petals, and globose, sometimes slightly compressed or triangular drupaceous fruit (Hara 1970, Hoo & Tseng 1973, Xiang & Lowry 2007). As is generally known, genotype rather than phenotype is influenced neither by physiological stage of the plant nor by environmental conditions (Komatsu & al. 2001). At the present, 18S rRNA and partial matK were powerful in resolving the phylogenetic relationship within angiosperm families (Plunkett et al. 1996, Soltis et al. 1997), and indentifying the botanical origins herbal drugs (Fushimi et al. 1996, 1997). ITS can help better elucidate the delimitation and relations of species within the genus. In the study of angiosperms, ITS reveals high variability of nucleotide sequence and conservation of the length, and is able to solve the problems of plant phylogeny at lower taxonomic category (Tian & Li 2002). Past attempt using various molecular markers to clarify relationships among Panax species has had ambiguous results. However, ITS has been applied to resolve phylogenetic relationships in Panax (Wen et al. 1996); 18S rRNA and partial matK have been applied to resolve phylogenetic relationships between P. vietnamensis Ha & Grushvitzky (1985: 519) and five related species and have been approved by Fushimi et al. (2003, ), Zuo et al. (2011). Taxonomy of Vietnamese Panax has been studied by relatively few authors (Ho 1970, 2000, Ha & Grushvitzky 1985). P. schinseng Nees von Esenbeck (1833: 70) var. japonicum (Nees von Esenbeck 1833: 70) Makino (1910: 223) is the first taxon in Panax reported from Vietnam by Ho (1970), who indicated that it grows under dense and Accepted by Yunfei Deng: 9 Sept. 2016; published: 23 Sept. 2016 47 moist broadleaf forest near the top of Lang Bian Mountain, Lam Dong Province. Ha & Grushvitzky (1985) described P. vietnamensis based on specimens from Ngoc Linh Mountain, Kon Tum Province. Ho (2003) listed four species from Vietnam, namely P. bipinnatifidus Seeman (1868: 54), P. japonicus (Nees) Meyer (1842: 525), P. pseudoginseng Wallich (1829: 117) and P. vietnamensis. However, Tap (2005) has recognized the genus Panax as containing only three species, P. bipinnatifidus, P. stipuleanatus H.T. Tsai & K.M. Feng in Yunnan Institute of Botany (1975: 44), P. vietnamensis. Long & al. (2013) newly reported P. vietnamensis var. fuscidiscus Komatsu et al. (2003: 91) from Lai Chau Province, northern Vietnam. Thus, 3 species and 1 variety are currently recognized from Vietnam. However, the taxonomic status of ginseng from the Lang Bian Mountain (Ho 1970) had not yet been reviewed. In May and August 2010, we conducted expeditions to Lang Bian Mountain, Lam Dong Province, in southern Vietnam, where Ho had collected specimens of Panax several years earlier. The authors found a population of Panax growing scattered sparsely on slopes in dense forest at the elevation of 1880–1900 m. We confirmed the presence of plants from their rhizomes and inflorescence an umbel of 40–80 flowers, peduncle long, and 5sepals, the features that basically corresponded to the Panax recognized by Ho (1970). The aim of the present study investigated the taxonomic status of this Lang Bian mountain ginseng. We employed molecular sequence data from the complete ITS1-5.8S-ITS2 region, 18S rRNA gene and partial matK sequences to explore its phylogenetic relationships, using the maximum- likelihood and, neiborgh-joining methods. We also investigated morphological characteristics between putatively related taxa. Vegatative and productive characteridtics were recorded from living specimens throughout Vietnam, and we also have studied specimens from various herbaria (HN, IBSC, K, KUN and P). Materials and methods Taxon sampling The fresh leaves of the new variety and its allies, P. bipinnatifidus, P. stipuleanatus, P. vietnamensis var. vietnamensis, P. vietnamensis var. fuscidiscus and Polyscias fruticosa (Linnaeus 1763: 1513) Harms (1894: 45) (outgroup), were collected throughout Vietnam and the voucher specimens are deposited in the Dalat University herbarium (DLU) and Tay Nguyen Institute for Scientific Research (VTN). The plant specimens examined in present study are summarized in Table 1. TABLE 1. Plant materials of Panax collected in Vietnam. GenBank accession number Taxon Locality of voucher Voucher No. ITS1– 8S rRNA matK 5,8SrRNA–ITS2 P. vietnamensis var. vietnamensis Dak Gley, Kon Tum Prov. VTN 994 KX768319 KX768325 KX768331 P. vietnamensis var. fussidiscus Phong Tho, Lai Chau Prov. VTN 990 KX768320 KX768326 KX768332 P. bipinnatifidus Ho Thau, Lai Chau Prov. VTN 992 KX768318 KX768324 KX768330 P. stipuleanatus Ho Thau, Lai Chau Prov. VTN 991 KX768317 KX768323 KX768329 Lang Bian ginseng Lac Duong, Lam Dong Prov. VTN 520 KX768316 KX768322 KX768328 Polyscias fruticosa Da Lat, Lam Dong Prov. VTN 993 KX768321 KX768327 KX768333 The ITS1-5.8S-ITS2, 18S rRNA and partial matK DNA of some allied species are available for download from GenBank. Sample information is shown in Table 2. TABLE 2. DNA barcodes of Panax from GenBank used in current study. Sequence Taxon Source/Collector/ Voucher Locality of voucher GenBank accession number matK P. quinquefolius Wen 6243(US) Virginia, Giles County, USA HQ113077 P. japonicus J1(PE) Tottori, Japan HQ113071.1 P. stipuleanatus D1(PE) Yunnan, Maguan, China HQ113079.1 P. bipinnatifidus D8(PE) Yunnan, Lijiang, China HQ113050.1 P. pseudoginseng Wen 4900(US) Gotehola, Nepal HQ113076.1 18S rRNA P. quinquefolius TMPU, H. Fushimi, 15 British Columbia, Canada D85172.1 P. japonicus var. Kading, Sichuan AB044901.1 bipinnatifidus P. japonicus TMPW 12022 Toyama, Japan D84100.1 P. stipuleanatus K. Komatsu & al., Y277-1 Pingbian, Yunnan, China AB088025.1 P. pseudoginseng K. Komatsu & A. Takano, I–42 Arunachal Bang Pradesh, India AB088026.1 ...continued on the next page 48 • Phytotaxa 277 (1) © 2016 Magnolia Press DUY ET AL. TABLE 2. (Continued) Sequence Taxon Source/Collector/ Voucher Locality of voucher GenBank accession number ITS1–5.8S P. bipinnatifidus Wen 1166(CS) Sichuan, China U41678.1 –ITS2 P. quinquefolius Wen 6243(US) Virginia, Giles County, USA HQ112440.1 P. japonicus J1 (PE) Tottori, Japan HQ112432.1 P.stipuleanatus Wen 1204(CS) Yunnan, China U41695 P. stipuleanatus D1(PE) Yunnan, China HQ112441 P. pseudoginseng Wen 4900(US) Gotehola, Nepal HQ112438.1 DNA extraction, PCA amplification, DNA purification and sequencing Total genomic DNA was extracted using CTAB protocol I (Weising & al. 2005) with a modification of adding 10% SDS to the extraction buffer, which was then dissolved in water for subsequent use. Complete gene amplifications of 18S rRNA gene, ITS1-5.8S-ITS2 region and partial matK gene amplifications were done via the polymerase chain reaction (PCR) and wereperformed in 50µl reactions containing approximately 80 ng genomic DNA, 10 mM Tris buffer (pH 8.3) with 50 mM KCl, 1.8 mM MgCl2, and 0.1% BSA, 0.25 mM of each dNTP, 1 µM of each primer, and 1.5 U Taq DNA polymerase (ThermoScientific). For 18S rRNA gene amplification, the primers used were 18S5’F (5’-CAA CCT GGT TGA TCC TGC CAG T- 3’) and 18S3’R (5’-CTG ATC CTT CTG CAG GTT CAC CTA-3’) and thermal cycling conditions were as follows: 1 cycle consisting of 940C for 3 min, 650C for 8 min, followed by 30 cycles of 940C for 45 sec and 650C for 8 min, and final extension at 720C for 30 min, modified from protocol of Komatsu & al.