A Taxonomic Study of Astragalus sikokianus with a Disjunct ...

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A Taxonomic Study of Astragalus 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 plants 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, taxonomy.

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

Many species of Astragalus L. (Fabaceae; Papilionoideae; Galegeae) are narrow endemics that often prefer marginal habitats or specialized substrates (sanderson and Wojciechowski 2000). Although this diversity largely arises from those narrow endemics, some of those species are often merged with others as synonyms or varieties. Therefore, the number of species belonging to Astragalus ranges from 2,000 to 3,000 (Lock and simpson 1991, Mabberley 1997, Maassoumi 1998). Work by Podlech and Zarre (2013) has led to a revision of Old World Astragalus as having 2,398 taxa that belong to

136 sections. However, the taxonomic status of some regionally endemic species is still unclear. Especially, the Astragalus in East Asia has been poorly examined since that region is peripheral to its center of species diversity.

Astragalus sikokianus Nakai has been known as a perennial herb distributed in coastal areas of Korea and Japan (Kim et al. 2003). This species was first described based on a type specimen collected from Naruto City, Tokushima Prefecture, shikoku District, Japan (Nakai 1953). However, the population in that locality has become extinct and other natural

218 The Journal of Japanese Botany Vol. 91 Centennial Memorial Issue

habitats have not been found in Japan. since then, this species has been considered a Japanese endemic but naturally extinct species (Ohashi 1982, 2001) and is now designated as an extinct species in the wild on the Japanese Plant Red List (Japanese Ministry of the Environment 2012). In Korea, however, Kim et al. (2003) have reported its disjunct distribution within some costal populations. Therefore, because of its classification as extinct in Japan but its newly discovered presence in Korea, the natural habitat of A. sikokianus was thought to be restricted to Korea.

For taxonomic purposes, S. Y. Kim (2004) hypothesized that the Korean endemic species A. koraiensis Y. N. Lee, first described from mountainous area of Gangwon-do (Lee 1981), should be merged to A. sikokianus as a synonym and that the latter (including A. koraiensis) has close affinity to A. bhotanensis Baker, which is distributed from Bhutan to western China. The closeness of A. sikokianus with A. bhotanensis and A. koraiensis has also been re-confirmed from a morphological study based primarily on fruit characteristics (song and Heo 2014). However, the taxonomic revision of Old World Astragalus (Podlech and Zarre 2013) has separately assigned A. sikokianus to sect. Uliginosi A. Gray as an independent species and A. koraiensis to sect. Brachycephalae N. D. simpson as a synonym of A. bhotanensis. According to this treatment, A. bhotanensis and A. sikokianus coexist in Korea but also have disjunct distributions from the Korean Peninsula to western China and shikoku of Japan. However, in our recent taxonomic study of Korean Astragalus, we found no morphological differences between Korean and Japanese plants nor between mountain and coastal plants (i.e., A. koraiensis and A. sikokianus) (Choi et al. 2015). Furthermore, we detected morphological similarities between specimens identified as A. bhotanensis from northwestern China and A. sikokianus (incl. A. koraiensis) in the Korea–Japan region. The results of various

taxonomic examinations (S. Y. Kim 2004, Podlech and Zarre 2013, song and Heo 2014, Choi et al. 2015) are also congruent with close affinity among plants from western China, Korea, and Japan. Nevertheless, the taxonomic circumscription and systematic position of these species remain controversial.

The internal transcribed spacer (ITs) regions of nuclear ribosomal DNA have been used in phylogenetic investigations of Astragalus (Wojciechowski et al. 1999, Kazempour Osaloo et al. 2003, 2005) because they show higher evolutionary rates and are easy to amplify. In addition, the 1,250 ITs sequence accessions (http://www.ncbi.nlm.nih.gov/nuccore/?term=Astragalus) belonging to approximately 400 species of Astragalus represent the most abundant collection of sequences among DNA regions that can be applied to the phylogeny of this genus (see Zarre and Azani 2013). Therefore, we selected the ITS region for conducting the first phylogenetic investigation of A. sikokianus.

Taxonomic study of endangered species is a prerequisite for their conservation. Moreover, the disjunct distributions proposed for such taxonomically problematic species have not previously been confirmed through molecular analysis. Thus, our research objective was to delimit the taxonomic boundary of this species by evaluating several morphological characters and by developing a molecular phylogeny based on ITs regions relevant to A. bhotanensis, A. koraiensis, and A. sikokianus.

Materials and MethodsM o r p h o l o g i c a l o b s e r v a t i o n s a n d distributional surveys

samples from various Astragalus species were collected from 2002 to 2015. Voucher specimens are held in the Herbarium of Inha University (IUI). To investigate their morphological characters and geographical distribution, we also examined specimens of A. bhotanensis, A. koraiensis, and A. sikokianus

December 2016 Choi et al.: Astragalus sikokianus 219

from the following herbaria: Chonnam National University (CNU); National Institute of Biological Resources, Korea (KB); Kyungpook National University (KNU); Kangwon National University (KWNU); Korea National Arboretum (KH); sungkyunkwan University (sKK); seoul National University, College of Agriculture Life sciences (sNUA); Tohoku University (TUs); the University of Tokyo (TI); The New York Botanical Garden (NY); and The Chinese Academy of sciences, Beijing (PE). We also studied photograph of Chinese specimens from the herbaria of Chengdu Institute of Biology (CDBI); south China Botanical Garden (IBsC); Kunming Institute of Botany (KUN); Northwest Institute of Plateau (QTPMB); Chongqing Municipal Academy of Chinese Materia Medica (sM); Northwestern Institute of Botany (WUK) through the Chinese Virtual Herbarium (www.cvh.org.cn). Our analytical methods followed those we have previously described (Choi et al. 2015).

DNA extraction, amplification, and sequencingGenomic DNA was extracted from fresh

or silica gel-dried leaves, using a G-spinTM IIp Kit for plants (Intron). The nuclear ribosomal region, including ITs1, 5.8s, and ITs2, was amplified with primer pair ITS-5/ITS-4 (White et al. 1990). In all, 18 samples were examined

from the A. sikokianus and related species from eastern Asia (Table 1). All PCRs were conducted with a GeneAmp® PCR system 2700 Thermal Cycler (Applied Biosystems). Each reaction mixture contained 200 μM dNTPs (GeneCraft), 1x PCR buffer with 1.5 mM MgCl2, 1 U of Taq DNA polymerase (TaKaRa), 10 ng of DNA, and an appropriate concentration of primers in a total volume of 50 μL. Conditions included an initial denaturation at 94°C for 2 min; followed by 35 cycles at 94°C for 30 s, 52°C for 45 s, and 72°C for 1 min; with a final extension at 72°C for 10 min. The PCR products were visualized on 2% agarose gels, purified by PCRquick-spinTM (Intron), and sequenced with an ABI 3100 Genetic Analyzer and an ABI BigDyeTM Terminator Cycle sequencing Ready Reaction Kit (Applied Biosystems).

Phylogenetic analysesAll sequences determined in this study

were aligned by using ClustalW software (Thompson et al. 1994) implemented in Geneious 7.1.8 (Kearse et al. 2012). Alignments were refined manually and each sequence required the introduction of numerous single and multibase gaps. Each indel was treated as a one-point mutation. The ITs ribotypes were determined based on those aligned sequences. The genealogical degree of relatedness among

Table 1. Details of Astragalus samples sequenced for ITs. Numbers in the parentheses indicate the number of individuals

Taxon Geographic origin RibotypeA. 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)


ribotypes was represented by a statistical parsimony network, generated by the program TCs version 1.21 (Clement et al. 2000).

For our phylogenetic analysis, the ribotypes detected from TCs analysis and 15 ITs sequences obtained from GenBank (Table 2) were realigned as described above. The ingroup comprised representative sectional members related to sect. Uliginosi, as reported by Hardion et al. (2010). As the outgroup, two Oxytropis DC. species were selected as described by Dastpak et al. (2013). Finally, 22 ITs sequences were used to build a phylogeny. A maximum likelihood (ML) analysis of the complete, final alignment with all taxa was conducted with RAxML (Stamatakis 2006, Stamatakis et al. 2008), as implemented with RAxML-HPC2 via the Cipres science Gateway (Miller et al. 2010). All parameters were left on their default settings except for the search in the non-parametric bootstrap analysis, which was done in a single run that included 1000 iterations.

ResultsMorphological differences between

Astragalus sikokianus (incl. A. koraiensis) and A. bhotanensis

Leaf and peduncle: The leaves (13−25 cm) and leaflets [15−25(−28) × 6−10(−13) mm] are larger for A. sikokianus than for A. bhotanensis (6−15 cm and 5−15 × 3−6 mm). In contrast, the peduncles are shorter for A. sikokianus [2−6(−8) cm] than for A. bhotanensis (6−17 cm). Those variations somewhat overlap each other when immature specimens are considered. However, the leaves of A. sikokianus are always longer than the subtending peduncle while leaves of A. bhotanensis are more or less sub-equal in length to the peduncle (Table 3, Figs. 1, 2).

Flower: Generally, A. sikokianus and A. bhotanensis have yellowish and purplish corollas, respectively (Table 3). More specifically, the corolla of A. sikokianus tends to be light-yellow and the tip of the standard and keel-petals is purple or reddish while the A. bhotanensis corolla is usually reddish or bluish-purple with whitish tips on the keel and/or wings. Hairiness of the calyx is also very different between these species, being predominantly covered with white hairs and only a few black hairs for A. sikokianus but having only dark brown to black hairs for A. bhotanensis (Table 3).

Legume: Each legume is arranged along a short capitate infructescence (5−10 mm). This trait is common to both A. sikokianus and A. bhotanensis but is distinct from other species in sect. Uliginosi. However, these two species differ in the hairiness and size of their legumes (Table 3). As a critical diagnostic characteristic, the legumes of A. sikokianus are covered with medifixed white hairs that gradually become nearly glabrescent over time while those of A. bhotanensis are always completely glabrous. The peduncles are more elongated for A. bhotanensis than for A. sikokianus but the legumes of A. bhotanensis (1.5−2.5 × 0.4−0.5 cm) are smaller than those of A. sikokianus (2.5−3 × 0.5−0.7 cm).

ITS sequence analysisThe sequence alignments revealed seven

Table 2. species and accession numbers of ITs sequences obtained from GenBank for phylogenetic analysis

Taxon Accession number Outgroup

Oxytropis rechingeri Vassilcz. AB741305O. takhti-soleimanii Vassilcz. AB741306

IngroupAstragalus glycyphyllos L. AB051941A. depressus L. AB231147A. hamosus L. AB051936A. cymbicarpos Brot. AF121678A. edulis Bunge AF121677A. boeticus L. AB051937A. odoratus Lam. AB051968A. fragrans Willd. AB051967A. tragacantha L. GU223902A. griffithii Bunge AB830797A. falcatus Lam. U50488, U50489A. canadensis L. L10770, L10771A. oreganus Torr. & A. Gray AF121687


Fig 1. Holotype of Astragalus bhotanensis Baker (Bhutan. Panga, 7000 ft. alt., Griffith 1000, K000959815).


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. bhotanensisSize of leaflet 15−25(−28) × 6−10(−13) mm 5−15 × 3−6 mmLength of leaf 13−25 cm 6−15 cmLength of peduncle 2−6(−8) cm 6−17 cmColor of corolla mainly yellowish mainly purplishColor of calyx hair predominantly white predominantly dark brown to blackHairs on legume pubescent to glabrescent glabroussize 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 sequences for A. sikokianus and related species (GenBank Accession Numbers KU168596−KU168602) (Table 4, Fig. 3). The related congeners A. uliginosus L. and A. schelichowii Turcz. showed two distinct types: AULI and AsCH, respectively. We also found five types (A through E) for A. bhotanensis, A. koraiensis, and A. sikokianus. Ribotype A was

detected and shared from type localities of A. sikokianus (Tokushima Pref., shikoku, Japan) and A. koraiensis (Jeongseon-gun, Gangwon-do, Korea). Individuals within ribotype B were restricted to Korea and differed from type A by a single substitution and an indel of CG. The ITs sequences from six localities, including coastal and mountainous areas of Korea and Japan, were also examined, but no genetic differentiation


was found in their geographical origin or taxa. Ribotype C was identified from the disjunct population of northwestern China and could be linked to the Korea-Japan type A by only a single mutational step. Our TCs analysis indicated that types A through C of A. sikokianus were closely related to each other and formed a loop. Types D and E of Chinese A. bhotanensis differed from types of A. sikokianus by at least three mutational steps. Moreover, the geographically close Chinese type C was distantly related to the Chinese A. bhotanensis types in the TCs network (Fig. 3).

We constructed an ML tree based on a matrix

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 high bootstrap value of 95.8% and was separate from the A. sikokianus clade. Ribotypes A through C also clustered together as subdivisions A and C (originated from China, Korea, and Japan) and B (restricted to Korea). However, those groupings were only weakly supported.

DistributionThe distributions of A. sikokianus (incl.

A. koraiensis) and A. bhotanensis were surveyed from published literature, herbarium specimens, and field investigations. Japanese A. sikokianus was once reported from its type locality (Shikoku) but is now extinct. The most Korean populations of A. sikokianus are dense along mountain streams at Gangwon-do. The rest of the populations have been sporadically recorded from eastern and western coastal areas that range from Incheon City to Gangwon-do, Gyeongsangbuk-do, and Jeollanam-do. In addition to this Korea-Japan distribution, a new and disjunct distribution has been proposed for A. sikokianus (incl. A. koraiensis) (Fig. 5), based on morphological and molecular data. such populations occur in northwestern China, approximately 1,500 km away from the Korean locality. These Chinese A. sikokianus grow along the upper streams of the Yellow River and on grasslands in the northern region of the Qinling and Hengduan Mountains (i.e., northwestern Sichuan, western Shaanxi, and southern Gansu), an area that represents the most northern boundary of the sichuan Basin. Overall, this species shows the most highly

Fig 3. Relatedness among ITs ribotypes detected in Astragalus sikokianus and related species, represented in statistical parsimony network. AULI. A. uliginosus. AsCH. A. schelichowii. White circles (A−C) indicate A. sikokianus (incl. A. koraiensis); black circles (D−E), A. bhotanensis; small, unlabeled circles, hypothetical missing ribotype.

Table 4. Polymorphic sites and ITs ribotypes based on sequences of A. sikokianus and related species

Ribotypes Accession number

Aligned position0 0 1 1 1 1 1 2 2 5 55 8 0 0 0 2 2 1 3 5 99 5 0 8 9 3 6 1 0 2 3

AULI KU168602 C C C C G C A T G T GAsCH KU168598 G G ∙ ∙ ∙ ∙ ∙ ∙ ∙ G ∙

A KU168596 G G ∙ ∙ ∙ ∙ ∙ ∙ A G ∙B KU168600 G G ∙ - - T ∙ ∙ A G ∙C KU168597 G G T ∙ ∙ ∙ ∙ ∙ A G ∙D KU168599 G G ∙ ∙ ∙ ∙ C C A G TE KU168601 G G ∙ - - ∙ C C A G T


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 China (101−108°E) to Korea (126−129°E), and Japan (134°E), while being somewhat sequential in its latitude (31−37°N).

Plants of A. bhotanensis grow from Bhutan to southwestern China. Although both A. bhotanensis and A. sikokianus exist in China, they are separated by the sichuan Basin. Most Chinese populations of A. bhotanensis are concentrated in Yunnan as well as in nearby southern sichuan and western Guizhou. The distribution at Xizang is almost adjacent to Bhutan. Overall, this species exists from Eastern

Himalaya to the southern Hengduan Mountains, i.e., the southern region of the Qinghai-Tibetan Plateau (QTP).

DiscussionTaxonomic identity of Astragalus koraiensis Y. N. Lee

Korean plants from the mountainous region of Gangwon-do, Korea were initially labeled as A. henryi Oliv., which was a misidentification of Lee and An (1963). They were later re-identified by Chung et al. (1982) and Chung and shin (1990), who reported those plants as being A.


bhotanensis, a new addition to the Korean flora. However, Lee (1981) designated this species as a new Korean endemic, A. koraiensis, based on morphological differences such as yellow flowers and a floral axis that was shorter than that attributed to A. bhotanensis. After Kim et al. (2003) described a Korean seashore population of A. sikokianus, the coastal plants tended to be identified as A. sikokianus and the mountainous plants as A. koraiensis in Korea. Likewise, Podlech and Zarre (2013) treated the mountainous plants of A. bhotanensis and A. koraiensis as conspecific species and the coastal A. sikokianus plants as a distinct species. From our extensive morphological examination, however, we found no morphological differences between Korean and Japanese plants or those from the mountains versus the coast (i.e., A. koraiensis and A. sikokianus). Furthermore, genetic variations in the ITs sequences did not reflect their habitat type or taxon (Table 1). The results of previous analyses of RAPD band patterns (s. Y. Kim 2004) from 10 Korean localities also concurred with our findings. Although we were unable to find the type specimen of A. koraiensis (Choi et al. 2015), the

protologue (Lee 1981) and all morphological variations known from Korea were identical to the description for A. sikokianus. Therefore, we conclude that A. koraiensis must be treated as a synonym of A. sikokianus.

Taxonomic circumscription and sectional position of Astragalus sikokianus

A recent revision of Old World Astragalus (Podlech and Zarre 2013) has indicated that A. bhotanensis is highly variable with respect to almost all of its morphological characteristics. Furthermore, its populations range disjunctively from Bhutan to China and Korea. However, our survey showed that the specimens, matched with the morphological variations of type specimen (Fig. 1) and protologue (Baker 1876), apparently are geographically restricted to only the southern region of the QTP (Fig. 5). Plants in the northeastern part of the QTP have larger leaves and leaflets, a shorter peduncle, a calyx predominantly covered with white hairs, yellowish flowers, and larger legumes that are similar to those of A. sikokianus (Fig. 2). Likewise the ITs ribotype C detected from that region is distantly related to Chinese types

Fig. 5. Distribution of Astragalus sikokianus (incl. A. koraiensis) and A. bhotanensis, modified from the PE Herbarium Website (http://pe.ibcas.ac.cn/).


D and E of A. bhotanensis, and can be directly linked to the Korean-Japanese type A by a single mutational step (Fig. 3). The topology of its ML tree did not differ greatly from our TCs analysis, even though the results were only weakly supported (Fig. 4). Based on these morphological and molecular variations and geographical patterns, we can argue that plants occurring in northwestern China and the Korea-Japan region are A. sikokianus (incl. A. koraiensis). Furthermore, we consider that A. sikokianus is an independent evolutionary lineage and is a sister species to A. bhotanensis, which occurs from Bhutan to southwestern China.

In the protologue, Nakai (1953) assigned A. sikokianus to sect. Euodmus Bunge, a synonym of sect. Uliginosi A. Gray. Hence, A. sikokianus is currently positioned in that section, which contains 10 species distributed from the Old to the New World (Podlech and Zarre 2013). Recent analyses of nitro compounds (Na et al. 2015) have provided similar chemotaxonomic evidence for A. sikokianus and A. canadensis of sect. Uliginosi. Nevertheless, Podlech and Zarre (2013) have stated that molecular analysis is necessary because A. sikokianus appears to be very similar to A. bhotanensis, a type species of monotypic sect. Brachycephalae. As anticipated from morphological examinations (S. Y. Kim 2004, Podlech and Zarre 2013, song and Heo 2014, Choi et al. 2015), A. sikokianus forms a monophyletic group with A. bhotanensis (Fig. 4). However, this monophyletic group itself is nested within sect. Uliginosi. Thus, it is questionable whether the sect. Brachycephalae as a taxon is independent from sect. Uliginosi even though the latter is polyphyletic. Moreover, the intermediate morphology shown from A. sikokianus between sect. Uliginosi and sect. Brachycephalae suggests that the two sections have merged. Nonetheless, the sectional revision of polyphyletic sect. Uliginosi is a prerequisite for formal integration with sect. Brachycephalae. This will also require more

morphological and molecular data that covers all of the sect. Uliginosi species. For now, the placement of A. sikokianus within sect. Uliginosi seems adequate.

The disjunct distribution pattern of A. sikokianus

Astragalus sikokianus is disjunctly distributed between the shikoku Island of Japanese Islands and the Korean Peninsula (Kim et al. 2003). This pattern is acknowledged in the flora of Korea (Choi 2007) as well as the Old World revision of that genus (Podlech and Zarre 2013). Kim et al. (2003) have speculated that the key mechanism for this distribution is long-distance dispersal by ocean currents, but no research has yet focused on that aspect. However, a continuous distribution from mountain streams to the seashore plus the sporadic occurrence of coastal populations over long distances in Korea tend to suggest the possibility of dispersal by floating legumes or seeds. Although definitive evidence is lacking, the fact that ribotype A is shared among mountain streams of Gangwon-do, the seashore at Gyeongsangbuk-do of Korea, and on shikoku of Japan (Table 1) supports the conclusion that this species is capable of long-distance dispersal.

The disjunct distribution of A. bhotanensis between western China and Korea has been argued by Chung et al. (1982), Chung and shin (1990), and Podlech and Zarre (2013). Nevertheless, our findings imply that this applies not to A. bhotanensis but, instead, to A. sikokianus. Even though those two regions are 1,500 km apart, their common morphology, latitudinal range of distribution, and habitats with close genetic affinity for plants from northwestern China to Japan indicate a unique pattern of distribution for this species. The historical biogeographic diversification from the QTP plus a range shift or expansion to or from other regions has been previously described (see Wen et al. 2014 and references therein). To the best of our knowledge, this disjunct


distribution between northwestern China and the Korea-Japan region has, until recently, been poorly recognized. Hence, we argue that plants of A. sikokianus growing in northwestern China, Korea, and Japan have a distributional pattern distinct from that of the sino-Japanese flora. Moreover, we speculate that, when one considers their common habitats, the long-distance dispersal of this species could primarily have been mediated by historical changes to the Yellow River in China as well as sea currents around Korea and Japan.

Taxonomic treatmentAstragalus sikokianus Nakai in Bull. Natl.

sci. Mus. Tokyo no. 33: 15 (1953); Kim & al. in J. Jpn. Bot. 78: 168 (2003); Podlech & Zarre, Tax. Rev. Gen. Astragalus (Leguminosae) Old World 2: 1548 (2013) [Type: JAPAN. shikoku, Tokushima Pref., Naruto City, Douno-ura, Awaikoyoin. Aug. 1950. A. Yoshiyuki s.n., TNs, n.v.].

A. koraiensis Y. N. Lee in Korean J. Bot. 24: 27 (1981). – A. sikokianus var. koraiensis (Y. N. Lee) M. Kim, Korean Endemic Plants: 115 (2004), syn. nov. [Type: KOREA. Kangwon-do, Jeongseon-gun, Kollchari. 1 Aug. 1977. Y. N. Lee s.n., n.v.].

A. bhotanensis sensu Ho, Fl. Reip. Popul. sin. 42(1): 274 (1993), p.p., quoad pl. ex Gansu, Shaanxi et N Sichuan; sensu Xu & Podlech, Fl. China 10: 406 (2010), p.p., quoad pl. ex Gansu, Shaanxi, N Sichuan et Korea; sensu Podlech & Zarre, Tax. Rev. Gen. Astragalus (Leguminosae) Old World 2: 1550 (2013), p.p., quoad pl. ex Gansu, Shaanxi, N Sichuan et Korea.

Distribution: China (Gansu, Shaanxi, sichuan), Korea (Incheon, Gangwon-do, Gyeongsangbuk-do, Jeollanam-do) and Japan [Shikoku (extinct)] (Fig. 5).

Habitat: Growing on seashores, roadsides, river banks, seashore embankments, streams.

Taxonomic note: Astragalus koraiensis Y. N. Lee was once treated as a variety of A. sikokianus (M. Y. Kim 2004), without any

diagnostic characters when compared with A. sikokianus. However, multiple studies (s. Y. Kim 2004, song and Heo 2014, Choi et al. 2015) as well as our investigation have revealed no difference between those two species. Hence, we synonymize A. koraiensis with A. sikokianus.

Representative specimens examined: CHINA. Gansu: Lanzhou-shi, Mt. Xinglongshan, 4 Aug. 1959, Y. Q. He 5612 (PE); Tianshui-shi, Liziyuan, 6 Jul. 1963, Q. X. Li 1178 (PE); Diebu-xian, 30 Jul. 1998, Bailong River Expedition 1075 (PE); Heshui-xian, Near the town of Taibai, 14 Jul. 1954, Yellow River Investigation Team 555 (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, Tao Ping, 7 Jul. 1991, X. L. Chen & Y. F. Wang 910068 (PE); Lintan-xian, Mt. Lianhuashan, 26 Sep. 1940, W. Y. Hsia 8802 (PE); Qingyang-xian, Mt. Xiaofangshan, 10 Sep. 1953, s. Q. Zhong & P. G. Zhang 203 (PE); Yongdeng-xian, 12 Jul. 1991, T. N. He 2070 (QTPMB); Yuzhong-xian, Mt. Xinglongshan, 12 Aug. 2006, B. Li & Z. L. Liu 2006039 (WUK); schimu to Paloutzai, south Kansu, no data, Fenzel & Pai 2746 (PE); Dacaotan, 7 Aug. 1940, W. Y. Hsia 8252 (PE); Shaanxi: Taibai-xian, Jutouzhen, 10 Aug. 1958, ? 0624 (PE); Taibai-xian, Jutouzhen, 17 Jul. 1958, 520 commando 2-0003 (PE); Yanan-shi, Naninwan, santaizhuangcun, 30 Aug. 1954, Yellow River Investigation Team 944 (PE); Sichuan: Aba, Mao-xian, 18 Aug. 1978, Z. Mao s.n. (SM); Heishui-xian, 21 Jul. 1957, X. Li 73705 (PE); Maerkang-xian, 10 Jul. 1957, Z. Y. Zhang & H. F. Zhou 22745 (PE); Nanping-xian, Heihe-xiang, 2 Aug. 1962, Nanpaing Team 0969 (CDBI); Nanping-xian, Yuwa-xiang, 2 Jul. 1959, Nanpaing Team 4125 (CDBI); Pingwu-xian, no data, H. L. Tsiang 11007 (PE); Xiazhai, Mao-xian, 27 Jul. 1983, Z. X. Tang 1533 (PE). JAPAN. Shikoku: Tokushima Pref., Naruto city, Kawashima-cho, 7 Jun. 1984, T. Yamazaki 5272 (TI). KOREA. Incheon-si: Ganghwa-gun, Gyodong-myeon, Eumnae-ri, Wolseon Port, 2 Jul. 2013, J.-H. Kim & al. Gyodong130652 (KB); Ganghwa-gun, Isl. Gyodong, 27 Jun. 2015, I. S. Choi 1506001−1506003 (IUI); Ganghwa-gun, Isl. seongmodo, Temp. Bomunsa, 2 Jul. 2003, s. Y. Kim & al. 307021−3070210 (IUI); Gangwon-do: Gangneung-si, Okgye-myeon, Mt. seokbyungsan, 13 Aug. 2006, J. O. Hyun & al. 1004042 (KH); Gangneung-si, Wangsan-myeon, Mt. Nochu, 3 Oct. 2008, B. K. Kwon 081003-061 (KB); Donghae-si, swaeun-dong, road side, 8 Jun. 2011, G.-H. Nam & al. sHY2-611 (KB); Jeongseon-gun, Buk-myeon, Mt. Ballon, From Goyang-ri to Mountain, 14 Jun. 2012, J.-H. Kim & J.-s. Kim sHY3-922 (KB); Jeongseon-gun, Buk-myeon, Goyang-ri, Mt. Goyang, Keungol, 17 Jul. 2012, G.-H. Nam & al. sHY3-1237 (KB); Jeongseon-gun, Buk-myeon, Gujeol-ri, Mt. Nochu, Around the suuidonggyegok, 24 sep. 2010, Y.-D. Kim & s.-H.


Cho 378083-270 (KB); Jeongseon-gun, Dong-myeon, Bukdong-ri, Olmullaegiyaksu, 18 Jul. 2012, G.-H. Nam & al. sHY3-1425 (KB); Jeongseon-gun, Imgye-myeon, Mt. Muraesan, 22 Jul. 2011, G. Y. Chung & al. 378121-0144 (KB); Jeongseon-gun, Jeongseon-eup, Deogu-ri, Deoksangi valley, 18 Jul. 2012, G.-H. Nam & al. sHY3-1388 (KB); Jeongseon-gun, Jeongseon-eup, Hoedong-ri, Mt. Gariwang, Eoeungol, Near Gariwangsan Recreational Forest, 16 Jul. 2008, J. N. Lim, G.-H. Nam & M. H. Yoo P0972 (KB); Jeongseon-gun, Jeongseon-eup, River Donggang, 8 Jun. 1999, Y.-M. Lee & Y.-H. An s-1822 (KH); Jeongseon-gun, sinwol-ri, 11 Jul. 2003, s. Y. Kim & al. 30711 (IUI); Jeongseon-gun, Hwaamyaksu, Buramsa, 11 Jul. 2003, s. Y. Kim & G. J. Kim 30711 (IUI); samcheok-si, Geundeok-myeon, Jangho-ri, 25 Jun. 2011, I.-H. Kim & J.-s. Yoo 2011-0158 (KB); samcheok-si, Hajang-myeon, 5 May 1999, W. C. Lee 9207 (KWNU); samcheok-si, Mapyeong-dong, Mt. Geunsan, 25 Jun. 2011, B. W. Han 379092-087 (KB); samcheok-si, Miro-myeon, Dongsan-ri, Mt. Dutasan, 24 Jun. 2011, H. R. Na & al. 379091-083 (KB); samcheok-si, Miro-myeon, sanggeono-ri, Forest road, 21 Jun. 2011, G.-H. Nam & al. sHY2-646 (KB); samcheok-si, Mt. Baekbyeongsan, 3 Jul. 2004, J.-O. Hyun & H.-K. Park 2004076 (KH); samcheok-si, Mt. Daeseongsan, 20 May 1983, W. C. Lee 9215 (KWNU); samcheok-si, samcheok beach, 21 Jun. 2003, s. Y. Kim 30621 (IUI); Taebaek-si, Mt. Geumdaebong, 5 Jun. 2004, J.-O. Hyun & H.-K. Park 2004073 (KH); Taebaek-si, Changjuk-dong, Mt. Daedeoksan, 18 Jun. 2005, K. I. Heo & J. E. Koh s.n. (sKK); Taebaek-si, Cheolam-dong, Mt. Myeonsan, 27 Jun. 2009, J. O. Hyun & al. 802003 (KH); Taebaek-si, Hyeol-dong, Mt. Hambaeksan, 21 Jun. 2012, Y. I. Kim & al. 378162-143 (KB); Taebaek-si, Manhangjae, 20 Jun. 2003, S. Y. Kim & al. 306201−306207 (IUI); Taebaek-si, Mt. Taebaeksan, 22 Jul. 1986, T. B. Lee 27313 (sNUA); Youngwol-gun, Youngwol-eup, Youngheung-ri, Jangneung, 6 Oct. 2009, J.-O. Hyun & H.-J. Kwon NAPI-2009-1076 (KH); Gyeongsangbuk-do: Goryeong-gun, Gotan, 12 Jul. 1980, W. C. Lee 9213, 9214 (KWNU); Pohang-si, Nam-gu, Guryongpo-eup, Guryongpo-ri, 10 Jul. 1965, I. s. Yang 8364, 8365 (KNU); Pohang-si, Nam-gu, Daebo-myeon, Daedongbae-ri, 11 Oct. 2002, s. G. Kwon & s. Y. Kim 210111−210112 (IUI, TUS); Yeongdeok-gun, Yeongdeok-eup, 13 Jul. 2003, S. Y. Kim & al. 307131−307133 (IUI); Jeollanam-do: sinan-gun, Jeungdo-myeon, Daejo-ri, Isl. Saemi, 28 Jun. 2000, H. T. Im 006680-1−6 (CNU).

We wish to express our sincere thanks to curators of CNU, KB, KH, KNU, KWNU, SKK, SNUA, TUS, NY, and PE for examination of specimens used for the present study. We also thanks to Prof. seung-Chul Kim (sungkyunkwan University) for providing a material of Astragalus uliginosus, and curator

of Tokushima Prefectural Museum for seed of Japanese Astragalus sikokianus in 2004. The plant materials from so-Young Kim, who receive a Ms degree at Inha University, have contributed greatly to this study. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEsT) (No. 2015R1D1A1A01059886).

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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・日本の固有種として知られているが，朝鮮の A. koraiensis とブータン・中国西部の A. bhotanensis に類似しており，分類が混乱している．これら 3 種の関係をはっきりさせるため，形態と nrITs とで比較研究を行った．形態的にはナルトオウギと A. koraiensis は区別できなかった．これ

ら 2 種と中国南西部の A. bhotanensis は葉，小葉，花序柄，花冠，萼および豆果で異なっていたが，中国北西部の A. bhotanensis にはナルトオウギと一致する特徴もあった．ITs 解析の結果，中国の A. bhotanensis は北西部と南西部のものは系統的に異なり，北西部のものはナルトオウギと単系統群をなすことが認められた．これらの


慶尚北道，および全羅南道にあり，さらに隔離分布して，中国では甘粛省，陝西省および四川省にあることが明らかになった．

（韓国・Inha University, Department of Biological sciences）

結果から，ナルトオウギ，朝鮮の A. koraiensis，および中国北西部の A. bhotanensis は A. sikokianus となり，A. bhotanensis はブータン・中国南西部に分布することが分かった．また，ナルトオウギは韓国では仁川，江原道，

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