J. Jpn. Bot. 91 Suppl.: 217–230 (2016)

A Taxonomic study of sikokianus with a disjunct distribution between northwestern China and the Korea-Japan region

In-Su Choi, Dong-Pil Jin and Byoung-Hee Choi*

Department of Biological Sciences, Inha University, Incheon 22212, KOREA *Corresponding author: [email protected]

(Accepted on January 9, 2016)

Astragalus sikokianus has been known as endemic to the Korea-Japan region but is taxonomically confused with A. koraiensis from Korea and A. bhotanensis from Bhutan and western China. To clarify its taxonomic boundary and distribution, we examined external morphological characters and nrITS sequence data. Morphologically, A. koraiensis and A. sikokianus are hardly distinguishable from each other but they differ from A. bhotanensis of Bhutan and southwestern China in their leaves, leaflets, peduncles, corollas, calyx, and legumes. In contrast, the diagnostic characters of known as A. bhotanensis from northwestern China match those of A. sikokianus. Our TCS network and ML tree based on ITS sequence variations also recognized the plants from northwestern China, Korea, and Japan as being an independent evolutionary lineage that is distinct from the other group of A. bhotanensis from southwestern China. Therefore, this morphological and molecular evidence leads us to conclude that A. koraiensis is synonymized with A. sikokianus and, as delimited here, the latter species also includes plants from northwestern China. It is also possible that this disjunct distribution of newly circumscribed A. sikokianus can be explained by historical geographic changes and long-distance dispersal in East Asia.

Key words: Astragalus koraiensis, Astragalus sikokianus, Astragalus bhotanensis, disjunct distribution, .

Many species of Astragalus L. (; 136 sections. However, the taxonomic status of Papilionoideae; Galegeae) are narrow endemics some regionally endemic species is still unclear. that often prefer marginal habitats or specialized Especially, the Astragalus in East Asia has been substrates (Sanderson and Wojciechowski 2000). poorly examined since that region is peripheral Although this diversity largely arises from to its center of species diversity. those narrow endemics, some of those species Astragalus sikokianus Nakai has been are often merged with others as synonyms or known as a perennial herb distributed in coastal varieties. Therefore, the number of species areas of Korea and Japan (Kim et al. 2003). belonging to Astragalus ranges from 2,000 to This species was first described based on a 3,000 (Lock and Simpson 1991, Mabberley type specimen collected from Naruto City, 1997, Maassoumi 1998). Work by Podlech and Tokushima Prefecture, Shikoku District, Japan Zarre (2013) has led to a revision of Old World (Nakai 1953). However, the population in that Astragalus as having 2,398 taxa that belong to locality has become extinct and other natural

—217— 218 The Journal of Japanese Botany Vol. 91 Centennial Memorial Issue habitats have not been found in Japan. Since taxonomic examinations (S. Y. Kim 2004, then, this species has been considered a Japanese Podlech and Zarre 2013, Song and Heo 2014, endemic but naturally extinct species (Ohashi Choi et al. 2015) are also congruent with close 1982, 2001) and is now designated as an extinct affinity among plants from western China, species in the wild on the Japanese Red Korea, and Japan. Nevertheless, the taxonomic List (Japanese Ministry of the Environment circumscription and systematic position of these 2012). In Korea, however, Kim et al. (2003) species remain controversial. have reported its disjunct distribution within The internal transcribed spacer (ITS) some costal populations. Therefore, because of regions of nuclear ribosomal DNA have its classification as extinct in Japan but its newly been used in phylogenetic investigations discovered presence in Korea, the natural habitat of Astragalus (Wojciechowski et al. 1999, of A. sikokianus was thought to be restricted to Kazempour Osaloo et al. 2003, 2005) because Korea. they show higher evolutionary rates and are For taxonomic purposes, S. Y. Kim (2004) easy to amplify. In addition, the 1,250 ITS hypothesized that the Korean endemic species sequence accessions (http://www.ncbi.nlm. A. koraiensis Y. N. Lee, first described from nih.gov/nuccore/?term=Astragalus) belonging mountainous area of Gangwon-do (Lee 1981), to approximately 400 species of Astragalus should be merged to A. sikokianus as a synonym represent the most abundant collection of and that the latter (including A. koraiensis) sequences among DNA regions that can be has close affinity to A. bhotanensis Baker, applied to the phylogeny of this genus (see Zarre which is distributed from Bhutan to western and Azani 2013). Therefore, we selected the China. The closeness of A. sikokianus with A. ITS region for conducting the first phylogenetic bhotanensis and A. koraiensis has also been re- investigation of A. sikokianus. confirmed from a morphological study based Taxonomic study of endangered species is primarily on fruit characteristics (Song and Heo a prerequisite for their conservation. Moreover, 2014). However, the taxonomic revision of Old the disjunct distributions proposed for such World Astragalus (Podlech and Zarre 2013) taxonomically problematic species have not has separately assigned A. sikokianus to sect. previously been confirmed through molecular Uliginosi A. Gray as an independent species analysis. Thus, our research objective was to and A. koraiensis to sect. Brachycephalae N. delimit the taxonomic boundary of this species D. Simpson as a synonym of A. bhotanensis. by evaluating several morphological characters According to this treatment, A. bhotanensis and and by developing a molecular phylogeny based A. sikokianus coexist in Korea but also have on ITS regions relevant to A. bhotanensis, A. disjunct distributions from the Korean Peninsula koraiensis, and A. sikokianus. to western China and Shikoku of Japan. However, in our recent taxonomic study of Materials and Methods Korean Astragalus, we found no morphological Morphological observations and differences between Korean and Japanese distributional surveys plants nor between mountain and coastal Samples from various Astragalus species plants (i.e., A. koraiensis and A. sikokianus) were collected from 2002 to 2015. Voucher (Choi et al. 2015). Furthermore, we detected specimens are held in the Herbarium of morphological similarities between specimens Inha University (IUI). To investigate their identified as A. bhotanensis from northwestern morphological characters and geographical China and A. sikokianus (incl. A. koraiensis) in distribution, we also examined specimens of A. the Korea–Japan region. The results of various bhotanensis, A. koraiensis, and A. sikokianus December 2016 Choi et al.: Astragalus sikokianus 219

Table 1. Details of Astragalus samples sequenced for ITS. Numbers in the parentheses indicate the number of individuals Taxon Geographic origin Ribotype A. uliginosus Mt. Baekdu, Korea AULI (1) A. schelichowii Mt. Baekdu, Korea ASCH (1) A. sikokianus Tokushima Pref., Shikoku, Japan A (3) A. sikokianus Pohang-si, Gyeongsangbuk-do, Korea A (1) A. koraiensis Jeongseon-gun, Gangwon-do, Korea A (1), B (2) A. sikokianus Samcheok-si, Gangwon-do, Korea B (1) A. koraiensis Taebaek-si, Gangwon-do, Korea B (2) A. sikokianus Isl. Ganghwado, Incheon, Korea B (1) A. sikokianus Mao County, Sichuan, China C (1) A. sikokianus Jinchuan County, Sichuan, China C (1) A. bhotanensis Zhaojue County, Sichuan, China D (1) A. bhotanensis Weining County, Guizou, China E (1) A. bhotanensis Dafang County, Guizou, China E (1) from the following herbaria: Chonnam National from the A. sikokianus and related species from University (CNU); National Institute of eastern Asia (Table 1). All PCRs were conducted Biological Resources, Korea (KB); Kyungpook with a GeneAmp® PCR System 2700 Thermal National University (KNU); Kangwon National Cycler (Applied Biosystems). Each reaction University (KWNU); Korea National Arboretum mixture contained 200 μM dNTPs (GeneCraft), (KH); Sungkyunkwan University (SKK); Seoul 1x PCR buffer with 1.5 mM MgCl2, 1 U of Taq National University, College of Agriculture DNA polymerase (TaKaRa), 10 ng of DNA, Life Sciences (SNUA); Tohoku University and an appropriate concentration of primers in a (TUS); the University of Tokyo (TI); The New total volume of 50 μL. Conditions included an York Botanical Garden (NY); and The Chinese initial denaturation at 94°C for 2 min; followed Academy of Sciences, Beijing (PE). We also by 35 cycles at 94°C for 30 s, 52°C for 45 s, and studied photograph of Chinese specimens from 72°C for 1 min; with a final extension at 72°C the herbaria of Chengdu Institute of Biology for 10 min. The PCR products were visualized (CDBI); South China Botanical Garden (IBSC); on 2% agarose gels, purified by PCRquick- Kunming Institute of Botany (KUN); Northwest spinTM (Intron), and sequenced with an ABI Institute of Plateau (QTPMB); Chongqing 3100 Genetic Analyzer and an ABI BigDyeTM Municipal Academy of Chinese Materia Medica Terminator Cycle Sequencing Ready Reaction (SM); Northwestern Institute of Botany (WUK) Kit (Applied Biosystems). through the Chinese Virtual Herbarium (www. cvh.org.cn). Our analytical methods followed Phylogenetic analyses those we have previously described (Choi et al. All sequences determined in this study 2015). were aligned by using ClustalW software (Thompson et al. 1994) implemented in DNA extraction, amplification, and sequencing Geneious 7.1.8 (Kearse et al. 2012). Alignments Genomic DNA was extracted from fresh were refined manually and each sequence or silica gel-dried leaves, using a G-spinTM IIp required the introduction of numerous single Kit for plants (Intron). The nuclear ribosomal and multibase gaps. Each indel was treated as region, including ITS1, 5.8S, and ITS2, was a one-point mutation. The ITS ribotypes were amplified with primer pair ITS-5/ITS-4 (White determined based on those aligned sequences. et al. 1990). In all, 18 samples were examined The genealogical degree of relatedness among 220 The Journal of Japanese Botany Vol. 91 Centennial Memorial Issue

Table 2. Species and accession numbers of ITS sequences Astragalus sikokianus (incl. A. koraiensis) obtained from GenBank for phylogenetic analysis and A. bhotanensis Taxon Accession number Leaf and peduncle: The leaves (13−25 cm) Outgroup and leaflets [15−25(−28) × 6−10(−13) mm] are Oxytropis rechingeri Vassilcz. AB741305 larger for A. sikokianus than for A. bhotanensis O. takhti-soleimanii Vassilcz. AB741306 (6−15 cm and 5−15 × 3−6 mm). In contrast, the Ingroup peduncles are shorter for A. sikokianus [2−6(−8) Astragalus glycyphyllos L. AB051941 cm] than for A. bhotanensis (6−17 cm). Those A. depressus L. AB231147 variations somewhat overlap each other when A. hamosus L. AB051936 immature specimens are considered. However, A. cymbicarpos Brot. AF121678 the leaves of A. sikokianus are always longer A. edulis Bunge AF121677 than the subtending peduncle while leaves of A. A. boeticus L. AB051937 A. odoratus Lam. AB051968 bhotanensis are more or less sub-equal in length A. fragrans Willd. AB051967 to the peduncle (Table 3, Figs. 1, 2). A. tragacantha L. GU223902 Flower: Generally, A. sikokianus and A. A. griffithiiBunge AB830797 bhotanensis have yellowish and purplish A. falcatus Lam. U50488, U50489 corollas, respectively (Table 3). More A. canadensis L. L10770, L10771 specifically, the corolla of A. sikokianus tends A. oreganus Torr. & A. Gray AF121687 to be light-yellow and the tip of the standard and keel-petals is purple or reddish while the A. ribotypes was represented by a statistical bhotanensis corolla is usually reddish or bluish- parsimony network, generated by the program purple with whitish tips on the keel and/or TCS version 1.21 (Clement et al. 2000). wings. Hairiness of the calyx is also very different For our phylogenetic analysis, the ribotypes between these species, being predominantly detected from TCS analysis and 15 ITS covered with white hairs and only a few black sequences obtained from GenBank (Table 2) hairs for A. sikokianus but having only dark were realigned as described above. The ingroup brown to black hairs for A. bhotanensis (Table 3). comprised representative sectional members Legume: Each legume is arranged along a related to sect. Uliginosi, as reported by Hardion short capitate infructescence (5−10 mm). This et al. (2010). As the outgroup, two Oxytropis trait is common to both A. sikokianus and A. DC. species were selected as described by bhotanensis but is distinct from other species Dastpak et al. (2013). Finally, 22 ITS sequences in sect. Uliginosi. However, these two species were used to build a phylogeny. A maximum differ in the hairiness and size of their legumes likelihood (ML) analysis of the complete, final (Table 3). As a critical diagnostic characteristic, alignment with all taxa was conducted with the legumes of A. sikokianus are covered with RAxML (Stamatakis 2006, Stamatakis et al. medifixed white hairs that gradually become 2008), as implemented with RAxML-HPC2 via nearly glabrescent over time while those of A. the Cipres Science Gateway (Miller et al. 2010). bhotanensis are always completely glabrous. The All parameters were left on their default settings peduncles are more elongated for A. bhotanensis except for the search in the non-parametric than for A. sikokianus but the legumes of A. bootstrap analysis, which was done in a single bhotanensis (1.5−2.5 × 0.4−0.5 cm) are smaller run that included 1000 iterations. than those of A. sikokianus (2.5−3 × 0.5−0.7 cm).

Results ITS sequence analysis Morphological differences between The sequence alignments revealed seven December 2016 Choi et al.: Astragalus sikokianus 221

Fig 1. Holotype of Astragalus bhotanensis Baker (Bhutan. Panga, 7000 ft. alt., Griffith 1000, K000959815). 222 The Journal of Japanese Botany Vol. 91 Centennial Memorial Issue

Fig 2. Astragalus sikokianus Nakai from Shaanxi, China (A: Yellow River Investigation Team 944, PE) and Gangwon- do, Korea (B: S. Y. Kim & al. s.n., IUI) showing similarities in sizes of leaf, leaflet, peduncle and legume.

Table 3. Distinguishing morphological characters for Astragalus sikokianus (incl. A. koraiensis) and A. bhotanensis Character A. sikokianus A. bhotanensis Size of leaflet 15−25(−28) × 6−10(−13) mm 5−15 × 3−6 mm Length of leaf 13−25 cm 6−15 cm Length of peduncle 2−6(−8) cm 6−17 cm Color of corolla mainly yellowish mainly purplish Color of calyx hair predominantly white predominantly dark brown to black Hairs on legume pubescent to glabrescent glabrous Size of legume 2.5−3 × 0.5−0.7 cm 1.5−2.5 × 0.4−0.5 cm ribotypes from a 603 bp matrix of ITS detected and shared from type localities of A. sequences for A. sikokianus and related species sikokianus (Tokushima Pref., Shikoku, Japan) (GenBank Accession Numbers KU168596− and A. koraiensis (Jeongseon-gun, Gangwon- KU168602) (Table 4, Fig. 3). The related do, Korea). Individuals within ribotype B were congeners A. uliginosus L. and A. schelichowii restricted to Korea and differed from type A by a Turcz. showed two distinct types: AULI single substitution and an indel of CG. The ITS and ASCH, respectively. We also found five sequences from six localities, including coastal types (A through E) for A. bhotanensis, A. and mountainous areas of Korea and Japan, were koraiensis, and A. sikokianus. Ribotype A was also examined, but no genetic differentiation December 2016 Choi et al.: Astragalus sikokianus 223

of 22 ITS sequences (Fig. 4). Apart from A. odoratus, the majority of sect. Uliginosi was gathered with its type species A. uliginosus as well as the North American A. canadensis and A. oreganus. However, this clade of species was also paraphyletic because it included sect. Brachycephalae (i.e., A. bhotanensis). Within this clade, A. bhotanensis and A. sikokianus formed a monophyletic group with a bootstrap value of 59.5%. The clade of A. bhotanensis (ribotypes D and E) was strongly supported by a Fig 3. Relatedness among ITS ribotypes detected high bootstrap value of 95.8% and was separate in Astragalus sikokianus and related species, represented in statistical parsimony network. AULI. from the A. sikokianus clade. Ribotypes A A. uliginosus. ASCH. A. schelichowii. White circles through C also clustered together as subdivisions (A−C) indicate A. sikokianus (incl. A. koraiensis); A and C (originated from China, Korea, and black circles (D−E), A. bhotanensis; small, unlabeled Japan) and B (restricted to Korea). However, circles, hypothetical missing ribotype. those groupings were only weakly supported.

Table 4. Polymorphic sites and ITS ribotypes based on Distribution sequences of A. sikokianus and related species The distributions of A. sikokianus (incl. Aligned position A. koraiensis) and A. bhotanensis were Accession Ribotypes 0 0 1 1 1 1 1 2 2 5 5 number 5 8 0 0 0 2 2 1 3 5 9 surveyed from published literature, herbarium 9 5 0 8 9 3 6 1 0 2 3 specimens, and field investigations. Japanese AULI KU168602 C C C C G C A T G T G A. sikokianus was once reported from its type ASCH KU168598 G G ∙ ∙ ∙ ∙ ∙ ∙ ∙ G ∙ locality (Shikoku) but is now extinct. The most A KU168596 G G ∙ ∙ ∙ ∙ ∙ ∙ A G ∙ Korean populations of A. sikokianus are dense B KU168600 G G ∙ - - T ∙ ∙ A G ∙ along mountain streams at Gangwon-do. The C KU168597 G G T ∙ ∙ ∙ ∙ ∙ A G ∙ rest of the populations have been sporadically D KU168599 G G ∙ ∙ ∙ ∙ C C A G T recorded from eastern and western coastal areas E KU168601 G G ∙ - - ∙ C C A G T that range from Incheon City to Gangwon- do, Gyeongsangbuk-do, and Jeollanam-do. In was found in their geographical origin or taxa. addition to this Korea-Japan distribution, a new Ribotype C was identified from the disjunct and disjunct distribution has been proposed population of northwestern China and could be for A. sikokianus (incl. A. koraiensis) (Fig. linked to the Korea-Japan type A by only a single 5), based on morphological and molecular mutational step. Our TCS analysis indicated that data. Such populations occur in northwestern types A through C of A. sikokianus were closely China, approximately 1,500 km away from the related to each other and formed a loop. Types Korean locality. These Chinese A. sikokianus D and E of Chinese A. bhotanensis differed grow along the upper streams of the Yellow from types of A. sikokianus by at least three River and on grasslands in the northern region mutational steps. Moreover, the geographically of the Qinling and Hengduan Mountains (i.e., close Chinese type C was distantly related to northwestern Sichuan, western Shaanxi, and the Chinese A. bhotanensis types in the TCS southern Gansu), an area that represents the network (Fig. 3). most northern boundary of the Sichuan Basin. We constructed an ML tree based on a matrix Overall, this species shows the most highly 224 The Journal of Japanese Botany Vol. 91 Centennial Memorial Issue

Fig. 4. Maximum likelihood tree inferred from ITS regions of Astragalus sikokianus and related species. Numbers above nodes indicate bootstrap values (>50%) resulting from 1000 replicates. Ribotypes are those featured in Tables 1, 4 and Fig. 3. discontinuous distribution in longitude, from Himalaya to the southern Hengduan Mountains, China (101−108°E) to Korea (126−129°E), and i.e., the southern region of the Qinghai-Tibetan Japan (134°E), while being somewhat sequential Plateau (QTP). in its latitude (31−37°N). Plants of A. bhotanensis grow from Bhutan Discussion to southwestern China. Although both A. Taxonomic identity of Astragalus koraiensis bhotanensis and A. sikokianus exist in China, Y. N. Lee they are separated by the Sichuan Basin. Most Korean plants from the mountainous region Chinese populations of A. bhotanensis are of Gangwon-do, Korea were initially labeled as concentrated in Yunnan as well as in nearby A. henryi Oliv., which was a misidentification of southern Sichuan and western Guizhou. The Lee and An (1963). They were later re-identified distribution at Xizang is almost adjacent to by Chung et al. (1982) and Chung and Shin Bhutan. Overall, this species exists from Eastern (1990), who reported those plants as being A. December 2016 Choi et al.: Astragalus sikokianus 225

Fig. 5. Distribution of Astragalus sikokianus (incl. A. koraiensis) and A. bhotanensis, modified from the PE Herbarium Website (http://pe.ibcas.ac.cn/). bhotanensis, a new addition to the Korean flora. protologue (Lee 1981) and all morphological However, Lee (1981) designated this species variations known from Korea were identical to as a new Korean endemic, A. koraiensis, based the description for A. sikokianus. Therefore, we on morphological differences such as yellow conclude that A. koraiensis must be treated as a flowers and a floral axis that was shorter than synonym of A. sikokianus. that attributed to A. bhotanensis. After Kim et al. (2003) described a Korean seashore Taxonomic circumscription and sectional population of A. sikokianus, the coastal plants position of Astragalus sikokianus tended to be identified as A. sikokianus and the A recent revision of Old World Astragalus mountainous plants as A. koraiensis in Korea. (Podlech and Zarre 2013) has indicated that A. Likewise, Podlech and Zarre (2013) treated the bhotanensis is highly variable with respect to mountainous plants of A. bhotanensis and A. almost all of its morphological characteristics. koraiensis as conspecific species and the coastal Furthermore, its populations range disjunctively A. sikokianus plants as a distinct species. From from Bhutan to China and Korea. However, our extensive morphological examination, our survey showed that the specimens, matched however, we found no morphological differences with the morphological variations of type between Korean and Japanese plants or those specimen (Fig. 1) and protologue (Baker 1876), from the mountains versus the coast (i.e., A. apparently are geographically restricted to koraiensis and A. sikokianus). Furthermore, only the southern region of the QTP (Fig. 5). genetic variations in the ITS sequences did Plants in the northeastern part of the QTP have not reflect their habitat type or taxon (Table larger leaves and leaflets, a shorter peduncle, 1). The results of previous analyses of RAPD a calyx predominantly covered with white band patterns (S. Y. Kim 2004) from 10 Korean hairs, yellowish flowers, and larger legumes localities also concurred with our findings. that are similar to those of A. sikokianus (Fig. Although we were unable to find the type 2). Likewise the ITS ribotype C detected from specimen of A. koraiensis (Choi et al. 2015), the that region is distantly related to Chinese types 226 The Journal of Japanese Botany Vol. 91 Centennial Memorial Issue

D and E of A. bhotanensis, and can be directly morphological and molecular data that covers linked to the Korean-Japanese type A by a all of the sect. Uliginosi species. For now, the single mutational step (Fig. 3). The topology placement of A. sikokianus within sect. Uliginosi of its ML tree did not differ greatly from our seems adequate. TCS analysis, even though the results were only weakly supported (Fig. 4). Based on The disjunct distribution pattern of A. these morphological and molecular variations sikokianus and geographical patterns, we can argue that Astragalus sikokianus is disjunctly distributed plants occurring in northwestern China and between the Shikoku Island of Japanese Islands the Korea-Japan region are A. sikokianus (incl. and the Korean Peninsula (Kim et al. 2003). A. koraiensis). Furthermore, we consider that This pattern is acknowledged in the flora of A. sikokianus is an independent evolutionary Korea (Choi 2007) as well as the Old World lineage and is a sister species to A. bhotanensis, revision of that genus (Podlech and Zarre 2013). which occurs from Bhutan to southwestern Kim et al. (2003) have speculated that the key China. mechanism for this distribution is long-distance In the protologue, Nakai (1953) assigned A. dispersal by ocean currents, but no research sikokianus to sect. Euodmus Bunge, a synonym has yet focused on that aspect. However, a of sect. Uliginosi A. Gray. Hence, A. sikokianus continuous distribution from mountain streams is currently positioned in that section, which to the seashore plus the sporadic occurrence of contains 10 species distributed from the Old coastal populations over long distances in Korea to the New World (Podlech and Zarre 2013). tend to suggest the possibility of dispersal by Recent analyses of nitro compounds (Na et al. floating legumes or seeds. Although definitive 2015) have provided similar chemotaxonomic evidence is lacking, the fact that ribotype A is evidence for A. sikokianus and A. canadensis of shared among mountain streams of Gangwon- sect. Uliginosi. Nevertheless, Podlech and Zarre do, the seashore at Gyeongsangbuk-do of Korea, (2013) have stated that molecular analysis is and on Shikoku of Japan (Table 1) supports the necessary because A. sikokianus appears to be conclusion that this species is capable of long- very similar to A. bhotanensis, a type species of distance dispersal. monotypic sect. Brachycephalae. As anticipated The disjunct distribution of A. bhotanensis from morphological examinations (S. Y. Kim between western China and Korea has been 2004, Podlech and Zarre 2013, Song and Heo argued by Chung et al. (1982), Chung and 2014, Choi et al. 2015), A. sikokianus forms Shin (1990), and Podlech and Zarre (2013). a monophyletic group with A. bhotanensis Nevertheless, our findings imply that this (Fig. 4). However, this monophyletic group applies not to A. bhotanensis but, instead, to A. itself is nested within sect. Uliginosi. Thus, it is sikokianus. Even though those two regions are questionable whether the sect. Brachycephalae 1,500 km apart, their common morphology, as a taxon is independent from sect. Uliginosi latitudinal range of distribution, and habitats even though the latter is polyphyletic. Moreover, with close genetic affinity for plants from the intermediate morphology shown from A. northwestern China to Japan indicate a unique sikokianus between sect. Uliginosi and sect. pattern of distribution for this species. The Brachycephalae suggests that the two sections historical biogeographic diversification from the have merged. Nonetheless, the sectional QTP plus a range shift or expansion to or from revision of polyphyletic sect. Uliginosi is a other regions has been previously described prerequisite for formal integration with sect. (see Wen et al. 2014 and references therein). Brachycephalae. This will also require more To the best of our knowledge, this disjunct December 2016 Choi et al.: Astragalus sikokianus 227 distribution between northwestern China and diagnostic characters when compared with the Korea-Japan region has, until recently, A. sikokianus. However, multiple studies (S. been poorly recognized. Hence, we argue that Y. Kim 2004, Song and Heo 2014, Choi et al. plants of A. sikokianus growing in northwestern 2015) as well as our investigation have revealed China, Korea, and Japan have a distributional no difference between those two species. Hence, pattern distinct from that of the Sino-Japanese we synonymize A. koraiensis with A. sikokianus. flora. Moreover, we speculate that, when one Representative specimens examined: China. Gansu: considers their common habitats, the long- Lanzhou-shi, Mt. Xinglongshan, 4 Aug. 1959, Y. Q. He 5612 (PE); Tianshui-shi, Liziyuan, 6 Jul. 1963, Q. X. distance dispersal of this species could primarily Li 1178 (PE); Diebu-xian, 30 Jul. 1998, Bailong River have been mediated by historical changes to the Expedition 1075 (PE); Heshui-xian, Near the town of Yellow River in China as well as sea currents Taibai, 14 Jul. 1954, Yellow River Investigation Team 555 around Korea and Japan. (PE); Heshui-xian, Near the town of Taibai, 28 Jul. 1954, Yellow River Investigation Team 790 (PE); Kangle-xian, Penglu, 20 Aug. 1996, Y. S. Lian et al. 96839 (PE); Li-xian, Taxonomic treatment Tao Ping, 7 Jul. 1991, X. L. Chen & Y. F. Wang 910068 Astragalus sikokianus Nakai in Bull. Natl. (PE); Lintan-xian, Mt. Lianhuashan, 26 Sep. 1940, W. Y. Sci. Mus. Tokyo no. 33: 15 (1953); Kim & al. Hsia 8802 (PE); Qingyang-xian, Mt. Xiaofangshan, 10 Sep. in J. Jpn. Bot. 78: 168 (2003); Podlech & Zarre, 1953, S. Q. Zhong & P. G. Zhang 203 (PE); Yongdeng- xian, 12 Jul. 1991, T. N. He 2070 (QTPMB); Yuzhong- Tax. Rev. Gen. Astragalus (Leguminosae) Old xian, Mt. Xinglongshan, 12 Aug. 2006, B. Li & Z. L. Liu World 2: 1548 (2013) [Type: JAPAN. Shikoku, 2006039 (WUK); Schimu to Paloutzai, South Kansu, no Tokushima Pref., Naruto City, Douno-ura, data, Fenzel & Pai 2746 (PE); Dacaotan, 7 Aug. 1940, W. Awaikoyoin. Aug. 1950. A. Yoshiyuki s.n., TNS, Y. Hsia 8252 (PE); Shaanxi: Taibai-xian, Jutouzhen, 10 Aug. 1958, ? 0624 (PE); Taibai-xian, Jutouzhen, 17 Jul. n.v.]. 1958, 520 commando 2-0003 (PE); Yanan-shi, Naninwan, A. koraiensis Y. N. Lee in Korean J. Bot. 24: Santaizhuangcun, 30 Aug. 1954, Yellow River Investigation 27 (1981). – A. sikokianus var. koraiensis (Y. Team 944 (PE); Sichuan: Aba, Mao-xian, 18 Aug. 1978, N. Lee) M. Kim, Korean Endemic Plants: 115 Z. Mao s.n. (SM); Heishui-xian, 21 Jul. 1957, X. Li (2004), syn. nov. [Type: KOREA. Kangwon- 73705 (PE); Maerkang-xian, 10 Jul. 1957, Z. Y. Zhang & H. F. Zhou 22745 (PE); Nanping-xian, Heihe-xiang, 2 do, Jeongseon-gun, Kollchari. 1 Aug. 1977. Y. Aug. 1962, Nanpaing Team 0969 (CDBI); Nanping-xian, N. Lee s.n., n.v.]. Yuwa-xiang, 2 Jul. 1959, Nanpaing Team 4125 (CDBI); A. bhotanensis sensu Ho, Fl. Reip. Popul. Pingwu-xian, no data, H. L. Tsiang 11007 (PE); Xiazhai, Sin. 42(1): 274 (1993), p.p., quoad pl. ex Gansu, Mao-xian, 27 Jul. 1983, Z. X. Tang 1533 (PE). Japan. 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[Shikoku (extinct)] (Fig. 5). 2011, G.-H. Nam & al. SHY2-611 (KB); Jeongseon-gun, Habitat: Growing on seashores, roadsides, Buk-myeon, Mt. Ballon, From Goyang-ri to Mountain, river banks, seashore embankments, streams. 14 Jun. 2012, J.-H. Kim & J.-S. Kim SHY3-922 (KB); Taxonomic note: Astragalus koraiensis Jeongseon-gun, Buk-myeon, Goyang-ri, Mt. Goyang, Keungol, 17 Jul. 2012, G.-H. Nam & al. SHY3-1237 (KB); Y. N. Lee was once treated as a variety of A. Jeongseon-gun, Buk-myeon, Gujeol-ri, Mt. Nochu, Around sikokianus (M. Y. Kim 2004), without any the Suuidonggyegok, 24 Sep. 2010, Y.-D. Kim & S.-H. 228 The Journal of Japanese Botany Vol. 91 Centennial Memorial Issue

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I.-S. Choi，D.-P. Jin，B.-H. Choi：マメ科ナルトオウギ Astragalus sikokianus の分類と分布 ナルトオウギ Astragalus sikokianus は韓国・日本の固 ら 2 種と中国南西部の A. bhotanensis は葉，小葉，花序 有種として知られているが，朝鮮の A. koraiensis とブー 柄，花冠，萼および豆果で異なっていたが，中国北西部 タン・中国西部の A. bhotanensis に類似しており，分類 の A. bhotanensis にはナルトオウギと一致する特徴もあ が混乱している．これら 3 種の関係をはっきりさせる った．ITS 解析の結果，中国の A. bhotanensis は北西部 ため，形態と nrITS とで比較研究を行った．形態的には と南西部のものは系統的に異なり，北西部のものはナル ナルトオウギと A. koraiensis は区別できなかった．これ トオウギと単系統群をなすことが認められた．これらの 230 The Journal of Japanese Botany Vol. 91 Centennial Memorial Issue

結果から，ナルトオウギ，朝鮮の A. koraiensis，および 慶尚北道，および全羅南道にあり，さらに隔離分布して， 中国北西部の A. bhotanensis は A. sikokianus となり，A. 中国では甘粛省，陝西省および四川省にあることが明ら bhotanensis はブータン・中国南西部に分布することが かになった． 分かった．また，ナルトオウギは韓国では仁川，江原道， （韓国・Inha University, Department of Biological Sciences）