Journal of Species Research 6(1):51-58, 2017 Wangsania, a new genus of Apiaceae endemic to Korea Byoung Yoon Lee1,*, Jina Lim1, Jaram Hong1, Myounghai Kwak1 and Jin-Oh Hyun2 1Division of Plant Resources, National Institute of Biological Resources, Hwangyeong-ro 42, Seo-gu, Incheon 22689, Republic of Korea 2Northeastern Asia Biology Institute, Songpa dae-ro 34-15, Songpa-gu, Seoul 05677, Republic of Korea *Correspondent: [email protected] Wangsania, a new genus of Apiaceae from limestone areas and typified by W. insolens (Kitagawa) B.Y. Lee & J.O. Hyun, is described from Gangwon-do, Korea. Wangsania insolens was initially treated as Peucedanum insolens Kitagawa due to similarity with Peucedaunm cervaria, but it differs markedly in its unique morphological characters, such as rarely branched roots and a single basal leaf with 3 or -4 ternately dissected leaflets. These characters apparently represent apomorphies for Wangsania. The parsimony analysis of nuclaer ribosomal DNA ITS sequences revealed Wangsania to be not closely related to either Peucedanum or to any of 14 different tribes or clades within subfamily Apioideae. Keywords: Apiaceae, New genus, Peucedaum insolens, Wangsania insolens Ⓒ 2017 National Institute of Biological Resources DOI:10.12651/JSR.2017.6.1.051 INTRODUCTION ternately dissected leaflets, and mainly basal peduncles arising from a root crown. We treat these two charac- Peucedanum, Apiaceae, Umbellales occurs in Euraisa ters as being unique features that separate it from other and Africa (Pimenov and Leonov, 1993; Spalik et al., species of Peucedanum. The goal of our research was to 2004). Members of the genus are characterized by flat- confirm the taxonomic identity and determine the phylo- tened fruits without prominent dorsal ridges and with genetic position of P. insolens by phylogenetic analyses more or less developed lateral wings (Spalik et al., 2004). using nuclear ribosomal DNA ITS sequences. Peucedanum was regarded as an unnatural group and separated into several genera by Pimenov (1987), Frey (1989), and Magee et al. (2009). For example, most spe- MATERIALS AND METHODS cies of Peucedaum in North America are now treated in Lomatium. Furthermore, some European members were The unique morphological characters of P. insolens established as a new genus, Holandrea, based on mor- make its position within the genus Peucedanum uncer- phological and phytochemical information (Reduron et tain. We therefore selected representatives of subfamily al., 1997), while two species of Peucedaum from Mad- Apioideae for analysis. On the basis of previous phylo- agascar were described as the new genus, Billburttia geneic analyses of chloroplast genes (rbcL, matK), in- (Magee et al., 2009). The taxonomic complexity of tron (rpl16, rps16, rpoC1) and nuclear ribosomal DNA Peucedaum is reflected in molecular evidence that show­ internal transcribed spacer (ITS) sequences and chloro- ed the genus was not monophyletic and its members were plast DNA restriction sites, Downie et al. (2001) recon- scattered throughout molecular phylogeny trees (Downie firmed the taxonomy of subfamily Apioideae at the tribal et al., 2001; Spalik et al., 2004). Peucedaum was placed levels and recognized Aciphylleae, Bupleureae, Careae, within the ‘Angelica’ clade sensu stricto (Downie et al., Echinophoreae, Heteromorpheae, Oenantheae, Pleuros- 2001), which was traditionally treated as Drude’s (1897- permeae, Pyramidopterae, Scandiceae, and Smyrnieae. 1898) tribe Peucedaneae. Six species of Peucedanum, P. Downie et al. (2001) also recognized seven additional coreanum, P. japonicum, P. terebinthaceum, P. elegans, clades that had previously been taxonomically untreat- P. hakuunense, and P. insolens (Lee et al., 2011) have ed. Among the ten tribes and seven clades, we did not been treated in Korea. Among them, P. insolens is unique include members of the Echinophoreae due to a lack of in its leaf morphology due to single basal leaf with 3-4 molecular information and did not incorporate DNA se- 52 JOURNAL OF SPECIES RESEARCH Vol. 6, No. 1 Table 1. Twenty three accessions of the family Apiaceae examined for nuclear ribosomal DNA internal transcribed spacer (ITS) sequences variation. Accessions from NCBI Genbank were started with single or two letters while newly investigated sequences started with letters of WIGIS (Wildlife Integrated Genetic Information System; National Institute of Biological Resources, Korea) or WBN (WIGIS Barcode Numbers). Asterisk (*) means that the DNA sequences were submitted to the NCBI Genbank, but their publication could not be checked by authors of the current paper. Tribe or clade Taxon Source or voucher information Genbank or WIGIS accession number Aciphylleae Aciphylla aurea Michell et al., 1998 U72377 Careae Aegopodium alpestre Ansan, Seorak mt. Gangwon-do (July 2. 2011) WBN0329459 Carum carvi Valiejo-Roman et al., 1998 AF077878 Grammosciadium macrodon Downie et al., 2000 AF073553 Oenantheae Conium maculatum Ajani et al., 2008 EU 169251 Pleurospermeae Pleurospermum yunnanense Zhou et al., 2008 EU236202 Pyramidoptereae Elaeosticta ramosissima Degtjareva et al., 2013 HM229376 Scandiceae Scandix australis Wojewodzka et al., 2008 FJ415104* Daucus carota L. Lee et al., 2004 AY552527* Smyrnieae Smyrnium olusatrum Downie et al., 1998 U30594 Angelica clade Angelica dahuria Saryeoni forests, Jeju-do (Aug. 10. 2011) WBN0329461 Peucedanum terebinantheum Daekwanryoung, Gangwon-do (Sep. 8. 2009) WBN0329462 Peucedanum insolens Singi-myeon, Samcheok-si, Gangwon-do WBN0329463 Apium clade Foeniculum vulgare Tabanca et al., 2005 AY581806 Anethum graveolens Reduron et al., 2009 GQ148795 Petroselinum crispum Reduron et al., 2009 GQ148798 Arracacia clade Arracacia aegopodioides Danderson et al., 2009 GQ862378* Rhodosciadium purpureum Danderson et al., 2011 JQ305012* Conioselinum clade Conioselinum vaginatum Zhou et al., 2009 FJ385041 Heracleum clade Heracleum moellendorffi Lee et al., 2012 WIGIS004981 Pastinaca sativa Ajani et al., 2008 EU169305 Komarovia clade Komarovia anisosperma Valiejo-Roman et al., 1998 AF077897 Pimpinella clade Pimpinella brachycarpa Daeamsan, Gangwon-do (Sep. 19. 2009) WBN0329464 quences from the tribes Bupleureae and Heteromorpheae RESULTS because of their unique morphological features. We there- fore chose 22 representative taxa from seven tribes and 1) ITS sequence analysis seven clades within the Apioideae as proposed by Downie et al. (2001) to clarify the relationships of P. insolens Among 23 complete ITS sequences, the ITS 2 region (Table 1). The ITS sequences of 16 taxa were obtained was shorter than ITS 1. The length of ITS 1 region in from Genbank, seven additional taxa were sequenced the taxa investigated ranged from 211 bp in Foeniculum in this study. Total genomic DNAs from the seven taxa vulgare to 221 bp in Pimpinella brachycarpa and were extracted from fresh leaves and tissue from herb- Pastinaca sativa (mean 216 bp). The length of ITS 2 arium specimens using the modified CTAB procedure of region of the taxa surveyed ranged from 217 bp in Doyle and Doyle (1987). Double stranded DNAs of the Elaeosticta ramosissima to 225 bp in Cicuta maculatum complete ITS regions in each genome were amplified (mean 222 bp). Overall length variation of both spacer by the polymerae chain reaction (PCR) using primers regions across all 23 accessions ranged from 432 bp to “ITS 5” and “ITS 4” (White et al., 1990). Details of the 444 bp. These sizes are comparable to the values reported amplification reactions, purification, and alignment were for other Apiaceae (Downie et al., 1998; Lee and Downie, the same as described in Lee (1998). Pairwise nucleotide 1999). Of the 656 initial alignment positions, 73 positions differences of unambiguously aligned positions were de- were deleted due to alignment ambiguities. Of the re- termined from the distance matrix option in Phylogenetic maining 583 unambiguously aligned positions, 195 (33.4 Analysis Using Parsimony (PAUP*, Swofford, 2002). %) were potentially parsimony informative, 288 (49.4%) Phylogenetic analyses of the ITS sequences data sets were constant, and 100 (17.2%) were autapomorphic. were carried out using the heuristic search strategies of Both spacers contributed comparable numbers of infor- PAUP*. All searches were conducted with 100 random- mative nucleotide substitutions to the phylogenetic analy- addition replicates using tree bisection-reconnection sis. Values of direct pairwise ITS sequence divergence of (TBR) branch swapping. A bootstrap analysis was done the examined 23 accessions (using PAUP’s DISTANCE using 100 resampled data sets. All trees were rooted MATRIX option) are presented in Table 2. The sequence with Pleurospermum yunnanense, revealed as the most divergence values ranged from 2.2% (between Foeniculum basal species within the Apioideae (Downie et al., 2001). vulgare and Anethum graveolens) to 23.4% (between February 2017 Table 2. Pairwise sequence divergence of ITS 1, ITS 2, and 5.8S ribosomal DNA regions among taxa included in the study. Mean distances (100×values) are calculated above the diagonal, and actual numbers of unambiguous divergence sites from pairwise sequence comparisons appear below the diagonal. The orders of taxa listed on the table were followed from the strict con- sensus tree shown in the Fig. 1. Taxa 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 1 Foeniculum vulgare - 2.21 6.31 8.36 12.34 13.27 11.05 12.69 12.71 12.31 11.19 15.86 15.57 13.94 15.50 12.22 18.35 22.66 16.57 17.84 14.26 16.48 14.76 L 2 Anethum graveolens 12 - 6.64 8.27 12.75 13.10 10.93 11.81 12.20 11.81 10.52 15.50 15.21 13.41 15.33 12.08 18.52 21.85 17.13 18.20 14.66 15.93 14.60 EE 3 Petroselinum crispum 34 35 - 8.70 12.48 12.66 10.82 12.64 12.85 12.45 11.15 14.68 14.95 13.35 14.31 12.17 18.88 22.06 17.07 17.42 14.39 16.45 14.73 E - T 4 Conioselinum vaginatum 45 45 47 9.41 9.43 6.84 7.37 7.38 7.55 6.26 11.99 12.05 11.00 12.33 10.78 15.79 19.33 14.52 15.78 11.87 14.12 12.43 AL - 5 Heracleum moellendorffii 66 69 67 51 4.79 9.43 10.54 10.00 10.72 9.80 14.97 14.71 12.15 16.51 13.19 17.35 21.64 15.51 16.00 14.33 15.48 14.71 .