Korean J. Pl. Taxon. 50(3): 253−261 (2020) pISSN 1225-8318 eISSN 2466-1546 https://doi.org/10.11110/kjpt.2020.50.3.253 Korean Journal of RESEARCH ARTICLE

Phylogenetic position of splendentissima, a Korean endemic sedge ()

Kyong-Sook CHUNG*, Sungyu YANG1 and Bo-Mi NAM2* Department of Medicinal Plant Science, Jungwon University, Goesan 28024, Korea 1Korea Herbal Medicine Resources Research Center, Korea Institute of Oriental Medicine, Naju 58245, Korea 2International Biological Material Research Center, Korea Research Institute of Bioscience & Biotechnology, Daejeon 34141, Korea (Received 17 April 2020; Revised 15 September 2020; Accepted 21 September 2020)

ABSTRACT: Carex splendentissima U. Kang & J. M. Chung, endemic to the Korean peninsula, is characterized by staminate terminal spikes and glabrous elliptic perigynia. Based on its broad leaves, androgynous spikes, and tri-stigmatic features, the species has been placed in Carex sect. Siderostictae Franch. ex Ohwi, an East Asian section and a basal group in the genus. To clarify the monophyly and phylogenetic position of the species, a molecular study using the internal transcribed spacer region of nuclear ribosomal DNA and chloroplast DNA (trnL-F) data was conducted. The DNA sequence data of ten taxa in sect. Siderostictae and closely related taxa (two taxa in sect. Surculosae) with outgroups were analyzed based on maximum parsimony and maximum like- lihood (ML) criteria. In the analyses, C. splendentissima was monophyletic and placed within the Siderostictae clade (sect. Siderostictae + two species of sect. Surculosae), forming a clade with C. ciliatomarginata and C. pachygyna (endemic to Japan). The clade (C. splendentissima + C. ciliatomarginata + C. pachygyna) shows evi- dence of diploidy. Furthermore, C. splendentissima is a sister to C. ciliatomarginata in the ML tree, and the two taxa have staminate terminal spikes. This study also updates the distribution of C. splendentissima and provides keys to the four Korean taxa in sect. Siderostictae. To conserve the endemic species C. splendentissima, further research on its genetic and ecological features should be conducted at the population level. Keywords: Carex, Carex splendentissima, endemic plant, Cyperaceae

Carex splendentissima U. Kang & J. M. Chung (Cyperaceae) 1) (Egorova, 1999; Tang et al., 2010; Hoshino et al., 2011; is an endemic to the Korean peninsula and distinguished by Park et al., 2016). Among four taxa in Korea, two species are long rhizomes, glabrous leaves and perigynia, staminate endemic: C. splendentissima and C. okamotoi Ohwi (Park et terminal spikes, and tri-stigmas (Fig. 1) (Kang et al., 2012). It al., 2016). occurs only in Gangwon-do Province, mountainous areas in Section Siderostictae is a very important group in a the middle east portion of the peninsula, on limestone rocks phylogenetic perspective being the earliest diverging group near streams (Figs. 1, 2) (Park et al., 2016). Based on broad within the Cyperaceae tribe Cariceae (Waterway and Starr, leaves and bisexual lateral inflorescences (androgynous, 2007; Waterway et al., 2009). Because the East Asian section staminate flowers at the tip and pistillate flowers at the base), is basal to the rest of Carex species, to understand high diversity Kang et al. (2012) placed the species in Carex L. sect. in Carex, the most specious genus in temperate zones with Siderostictae Franch. ex Ohwi (Carex subgenus Carex). The more than 2,000 taxa worldwide, it is critical to clarify section occurs in East Asia in such countries as Japan, Korea, evolutionary mechanisms and/or speciation in Asian groups China, Vietnam, and Russia. A few species grow broadly in (Global Carex Group, 2015). High diversity in Carex has been East Asia, but many taxa in the section are geographically postulated by chromosome, genetic rearrangements via fission structured, being endemic to certain country and/or areas (Table and/or fusion events in holocentric chromosomes, possessing

*Author for correspondence: [email protected], [email protected]

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Fig. 1. Photographs of Carex splendentissima in a natural habitat (Jeongseon-gun, Gangwon-do). A, B. Habitats near streams. C. Populations associated with Saussurea chabyoungsanica and Aruncus dioicus var. kamtschaticus. D. Habits showing glabrous broad leaves. E. Inflorescence with terminal staminate and lateral androgynous spikes (staminate at the top and pistillate at the base). non-localized centromeres (Hipp et al., 2009, 2013). Some diversity due to agmatoploidy (chromosome number increases previous investigations such as Chung et al. (2011) and by fission without gene duplications) (Luceño and Guerra, Escudero et al. (2012) demonstrated that great variations in 1996) and/or aneuploidy in various linages. However, chromosome numbers are associated with rapid species polyploidy events are very rare, and are only detected in sect.

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C. splendentissima and C. okamotoi, are diploids (2n = 12) (Chung et al., 2013, 2017). To clarify monophyly and the phylogenetic position of C. splendentissima, molecular phylogenetic analyses were conducted using ITS and trnL-F sequence data. Furthermore, distribution of the species was updated based on voucher specimens, and keys to the four Korean members in sect. Siderostictae were presented.

Materials and Methods

Plant leaves were sampled from herbarium specimens or living at the Korea National Arboretum herbarium (KH), National Institute of Biological Resources herbarium (KB), and through field work at Jeongseon-gun, Gangwon-do Province. Voucher specimen information and accession numbers of DNA data are provided in Appendixes 1 and 2. Sequence data of some ingroups and outgroups were obtained from GenBank (Appendix 2). Total DNA were extracted from leaves using DNeasy Plant Mini Kit (Qiagen, Valencia, California, USA). ITS and trnL-F regions were amplified by polymerase chain reaction (PCR) using (Solg Taq DNA polymerase, SolGent Co., Fig. 2. The distribution of Carex splendentissima based on voucher Daejeon, Korea). ITS-1 (Urbatsch et al., 2000) and ITS4 (White specimens. et al., 1990) primers were utilized for the ITS region, and trnL- F region primers followed Tablet et al. (1991). PCR reaction Siderostictae (Waterway et al., 2009). Polyploidy contributes conditions were modified from Yano et al. (2014). PCR to early lineage diversity whereas agmatoploidy and/or products were checked in 1% agarose gels, and then purified aneuploidy play an important role in recent diverging lineages PCR products were sequenced in SolGent Co. in Carex (Waterway et al., 2009; Yano et al., 2014). Raw sequence data were edited in Sequencher (version 4.8, Within the section, terminal spike sexuality, perigynium Gene Codes Corporation, Ann Arbor, MI, USA) and aligned shapes and sizes, distribution areas, and ploidy levels are using MUSCLE (Edgar, 2004). Gaps were not used as various (Table 1). The basal phylogenetic position of the characters, and tree searches were performed using parsimony section indicates primitive character states of morphological, and maximum likelihood (ML) methods. Most maximum cytological, and geographic characters (Global Carex Group, parsimonious trees were obtained by PAUP* (Swofford, 2002) 2015). In the analyses of the internal transcribed spacer region using the heuristic search option with 100 random addition and 5.8S gene of nuclear ribosomal DNA (hereafter, ITS) and sequence, tree-bisection-reconnection (TBR) branch swapping, trnL intron and trnL-F intergenic spacer of chloroplast DNA and accelerated transformation (ACCTRAN) character-state sequences (hereafter, trnL-F), traditional sect. Siderostictae optimization. RAxML v. 8 (Stamatakis, 2014) was used for formed the Siderostictae clade with two sections Carex the ML analyses using the GTRGAMAMA model, which was subgenus Vigneastra, sections Hemiscaposae C. B. Clarke and strongly supported (Chung et al., 2012). Tree searching for ITS Surculosae Raymond, which are previously classified in (Yano and trnL-F data was conducted separately, and then a combined et al., 2014). Chromosomes in these sections are larger in size data set was analyzed. Prior to the analyses of combined data, and lower in number than in recently derived lineages the incongruence length difference (ILD) test evaluated ITS (Waterway et al., 2009; Chung et al., 2013). Tetraploidy in the and trnL-F data for congruence with the heuristic searchers clade has been observed in the species occurring in relatively using random-sequence addition replicates in PAUP* (Farris et broader geographic areas, for examples, C. glossostigma al., 1994, 1995). Branch support values were estimated by Hand.-Mazz., C. grandiligulata Kük., and C. siderosticta 1,000 heuristic bootstrap replicates with simple sequence Hance (Table 1) (Yano et al., 2014). Both Korean endemics, addition and TBR branch swapping (Felsenstein, 1985).

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Table 1. Diagnostic characters of species in the Carex section Siderostictae.

Terminal Perigynium Chromosome Number Taxon Distribution spike Shape Length (mm) (2n) Carex ciliatomarginata Nakai Staminate Obovate 2.8–4.5 Japan, Korea, China 12 (Chung et al., 2013) C. esquiroliana H. Lév. Staminate Narrowly elliptic ca. 3.5 China, Vietnam Unknown C. glossostigma Hand.-Mazz. Androgynous Ovate-elliptic ca. 3 China 24 (Yano et al., 2014) C. grandiligulata Kük. Androgynous Elliptic 4–5 China 12, 24 (Yano et al., 2014) C. longshengensis Y. C. Yang & S. Yun Liang Androgynous Oblong-elliptic 4.5–5.5 China 12 (Yano et al., 2014) C. oblanceolata T. Koyama Androgynous Oblong to elliptic ca. 3 China Unknown C. okamotoi Ohwi Staminate Broadly obovate ca. 2.5 Korea 12 (Lee and Kim, 2008, Chung et al., 2013; Yano et al., 2014) C. pachygyna Franch. & Sav. Androgynous Obovate 2–2.8 Japan 12 (Yano et al., 2014) C. siderosticta Hance Androgynous Obovate or elliptic 3–4 Japan, China, Korea, 12, 24 (Hoshino and Russia Tanaka, 1977; Hoshino, 1981; Yano et al., 2014) 12 (Chung et al., 2013, 2016, 2017) 24 (Tang and Xiang, 1989; Starodubtsev, 1989; Hoshino et al., 1993; Probatova et al., 1998, 2000) C. splendentissima U. Kang & J. M. Chung Staminate Narrowly elliptic or 2.8–3.2 Korea 12 (Chung et al., 2017) elliptic C. subcapitata X. F. Jin, C. Z. Zheng Androgynous Broadly elliptic ca. 4 China 12 (Yano et al., 2014) & B. Y. Ding C. tumidula Ohwi Staminate or Ovate ca. 3 Japan 12 (Hoshino, 1981) androgynous C. wuyishanensis S. Yun Liang Androgynous Broadly ovate ca. 3 China Unknown

Table 2. Statistics of nuclear rDNA ITS and chloroplast trnL-F sequences. ITS trnL-F Combined No. of species and accessions 19, 33 19, 33 19, 33 No. of sequences aligned 636 1,052 1,688 No. of variable sites 178 152 330 No. of parsimony-informative sites 117 65 182 No. of MP trees 203 264 208 Tree length 355 189 552 Consistency Index 0.6535 0.8889 0.7220 Retention Index 0.7092 0.8966 0.7540 ITS, internal transcribed spacer; MP, maximum parsimony.

Results and Discussion data of ITS (636 bp) and trnL-F (1,052 bp) were aligned and analyzed under maximum parsimony (MP) and ML Statistics of ITS, trnL-F, and combined data were assumptions. Number of parsimony-informative sites were summarized in Table 2. The combined 1,688 bp DNA sequence higher in ITS (18%) than in chloroplast trnL-F data (6%). The

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Fig. 3. Phylogenetic trees of combined internal transcribed spacer and trnL-F data. A. One of parsimony trees (tree length = 552, Consistency Index = 0.7220, Retention Index = 0.7540). B. Maximum likelihood tree. Numbers on branches indicate bootstrap values more than 69%. In both analyses, Carex splendentissima forms a monophyletic group (dotted box) and the clade with C. pachygyna and C. ciliatomarginata within the Siderostictae clade.

ITS data resulted in 203 parsimony trees, and the trnL-F data with C. ciliatomarginata in Korea (Park et al., 2016). C. produced 264 parsimony trees. Consistency Index (CI) and okamotoi, the other Korean endemic, was nested with C. Retention Index (RI) were higher in the trnL-F than in the ITS siderostacta with a high bootstrap value (≥ 80%). C. data. ILD test p-value (0.04) allowed to conduct combined data siderostacta is the most widely distributed species in the analyses of the two data sets. The combined data of CI and section. In Korea, the species grows commonly throughout the RI were 0.7220 and 0.7540, respectively. country, but C. okamotoi occurs mainly in Southern areas of Both MP and ML analyses supported monophyly of C. the peninsula (Park et al., 2016). Diploidy has been reported splendentissima and placed it in the Siderostictae clade. C. in C. okamotoi (2n = 12), whereas diploidy and tetraploidy splendentissima was included in the Siderostictae clade with have been reported in C. siderostacta (2n = 12, 24) (Table 1). sections Siderostictae and Surculosae and formed a clade with Polyploidy events in C. siderosticta may have caused complex C. ciliatomarginata and C. pachygyna in sect. Siderostictae in genetic relationships. Further investigations with broad taxon all analyses. One of parsimony trees and an ML tree obtained and gene sampling are required to resolve the relationship from combined data set are presented in Fig. 3. Because all between C. siderosticata and C. okamotoi. The analyses the parsimony trees and a strict consensus tree have similar suggest that two Korean endemics, C. splendentissima and C. topologies on the relationships and positions of C. okamotoi, have evolved independently in the peninsula. splendentissima, one of parsimony tree is presented. The The clade of C. splendentissima, C. ciliatomarginata, and monophyly of C. splendentissima was strongly supported with C. pachygyna is diploidy (Table 1, Fig. 3). Two members of high bootstrap values (≥ 99%) in MP and ML analyses. the clade, C. splendentissima (Korean endemic) and C. However, sister species were different between the two trees. pachygyna (Japanese endemic), exhibit a narrow distribution C. ciliatomarginata was sister to C. splendentissima in ML pattern, supporting the association of distribution patterns with analyses, while C. pachygyna in the maximum parsimony tree. ploidy levels in Carex (Yano et al., 2014). Genome sizes (1C) The results supported the treatment of the species in sect. of C. splendentissima and C. ciliatomarginata are 0.643 pg Siderostictae by Kang et al. (2012). Because of broad leaves DNA and 0.742 pg DNA, respectively (unpublished data) and staminate terminal spikes, the species was often compared whereas C. siderosticta, tetraploidy (2n = 24), has 1C = 1.20

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pg DNA (Nishikawa et al., 1984). Polyploidization is broad leaves, and glabrous perigynia and grows in limestone significant in the clade of C. siderosticta and C. okamotoi, areas. Four members of sect. Siderostictae occur in Korea, but whereas C. splendentissima is in the diploid clade. Contrasting a key to the four species is not available. Two versions of keys to the basal lineage (Siderostictae clade), C. scoparia var. to four taxa are provided here. Depending on characters scoparia in C. subgenus Vignea sect. Ovales has low genome available, the keys can be used. The phylogenetic analyses size, 1C = 0.342–0.361 pg DNA (Chung et al., 2011). Genome concluded that C. splendentissima, a Korean endemic sedge, sizes and chromosome numbers are not associated in the is monophyletic and belongs to the Siderostictae clade with subgenus (Chung et al., 2012). Dynamic mechanisms of the close relationship with C. ciliatomarginata and C. holocentric chromosomes, such as polyploidization in early pachygyna. The sister species of C. splendentissima needs lineages and agmatoploidy in recently diverging lineages, have to be investigated with a more appropriate dataset contributed complex, diverse evolutionary history in the genus. comprehensive sampling of the Siderostictae clade. To The evolution of unisexual and bisexual spikes is challenging conserve the endemic and ecological specialist species, to understand in Carex. In Carex, unisexual inflorescences are further research on genetic and ecological structures at a diverse in sexuality, branching patterns, and positions; and the population level is required. pistillate spikes with tri-stigmatic gynoecia and closed perigynia are present in early diverging lineages (Starr and Key to the four Korean species Ford, 2009). In the Siderostictae clade, two types of terminal in Carex sect. Siderostictae spike arrangements (staminate and androgynous) and two levels of ploidy levels (diploidy and tetraploidy) are present. 1. Leaf margins ciliolate; perigynia surfaces pubescent ····· Both characters are very important to understand evolution of ···························································· C. ciliatomarginata Carex. Evaluation of the character evolution should be 1. Leaf margins not ciliolate; perigynia surfaces glabrous conducted with robust, well-resolved phylogenetic trees of the 2. Leaves less than 1 cm in width; perigynia ovate ······ Siderostictae clade. ···································································· C. okamotoi C. splendentissima is a specialist in ecological habitat 2. Leaves more than 1 cm in width; perigynia narrowly preference, growing mainly on limestones. The distribution of elliptic or elliptic the species was updated based on the voucher specimens 3. Leaf abaxial surfaces pubescent; terminal spikes examined in KB and KH (Fig. 2). In the Jeongseon habitat, bisexual (androgynous) ·················· C. siderosticta the species was associated with other limestone lovers and/or 3. Leaf abaxial surfaces glabrous; terminal spikes ecological generalists such as Saussurea chabyoungsanica H. unisexual (staminate) ··············· C. splendentissima T. Im, Spiraea trichocarpa Nakai, Aruncus dioicus var. kamtschaticus (Maxim.) H. Hara, Polystichum sp., Lespedeza Key modified from Park et al. (2016) maximowiczii C. K. Schneid., and Lindera obtusiloba Blume. 1. Terminal spikes bisexual (androgynous); lateral spikes Limestone areas cover about 20% of South Korea mainly in unisexual (staminate) ································ C. siderosticta middle East of the country (Korea National Arboretum, 2012). 1. Terminal spikes unisexual (staminate); lateral spikes Limestone in Gangwon-do province formed in Cambrian- bisexual (androgynous) Ordovician; and the Jeongseon-gun site, the type locality of C. 2. Culms, leaves, perigynia pubescent ····························· splendentissima, is a well-known, large limestone areas hosting ······················································· C. ciliatomarginata Trichophorum dioicum J. Jung & H. K. Choi and Scirpus 2. Culms, leaves, perigynia glabrous sylvaticus L. var. maximowiczii Regel (Korea National 3. Leaves broad lanceolate or linear lanceolate, 4– Arboretum, 2012). How to manage the limestone areas are 8 mm wide; perigynia obovate ········· C. okamotoi critical to conserving many valuable natural resources, 3. Leaves oblanceolate or obovate, 14–46 mm wide; including C. splendentissima. Furthermore, although the perigynia narrowly elliptic or elliptic ····················· limestone preference of the species is understood, evaluations ···················································· C. splendentissima of population size, structures, and rarity have not been conducted. To establish proper, efficient conservation strategy ORCID: Kyong-Sook CHUNG https://orcid.org/0000-0002- of the species, ecological, phylogenetic, taxonomic features of 4464-4698; Sungyu YANG https://orcid.org/0000-0001- the species should be understood. 5081-0296; Bo-Mi NAM https://orcid.org/0000-0002-6769- C. splendentissima is distinct with staminate terminal spikes, 9317

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Appendix 1. Voucher specimens of C. splendentissima examined for the distribution map. KOREA. Gangwon-do: Jeongseon-gun, Jeongseon-eup, 8 Jun 2018, Chung et al. 5159 (KH); Pyeongchang-gun, Daehwa- myeon, Haanmi-ri, 8 May 2012, Nam s.n. (barcode number, NIBRVP0000356733) (KB); Yeongwol-gun, Hanbando-myeon, Ongjeong-ri, 13 May 2013, Kim and Chae s.n. (barcode number, NIBRVP0000612118) (KB).

Appendix 2. GenBank accession numbers for DNA data. ITS: Carex albursina USA AY757626; C. ciliatomarginata KOR1 JX644818, KOR2 MT811777, CHN AB725706, JPN AB725707; C. gibba JPN DQ998917; C. glossostigma CHN AB725708; C. grandiligulata CHN AB725709; C. kwangsiensis CHN AB725730; C. longshengensis CHN AB725710; C. okamotoi KOR1 AB725711, KOR2 JX644835, KOR3 MT811778; C. omiana JPN DQ998935; C. pachygyna JPN AB725712; C. plantaginea CAN AY757613; C. siderosticta CHN1 AB725713, CHN2 AB725714, JPN1 AB725715, JPN2 AB725716, KOR1 AB725717, KOR2 AB725718, KOR3 MT811783; C. splendentissima KOR1 MT274323, KOR2 MT811781, KOR3 MT811782; C. subcapitata CHN1 AB725719, CHN2 AB725720; C. tsiangii CHN AB725731; C. tumidula JPN AB725721; Scirpus microcarpus AF284961; Trichophorum pumilum AB643647; T. subcapitatum AB679909. trnL-F: Carex albursina USA AY757554; C. ciliatomarginata KOR1 JX644730, KOR2 MT826862, CHN AB725732, JPN AB725733; C. gibba JPN DQ998970; C. glossostigma CHN AB725734; C. grandiligulata CHN AB725735; C. kwangsiensis

Korean Journal of Plant Taxonomy Vol. 50 No. 3 (2020) Carex splendentissima phylogenetic position 261

CHN AB725756; C. longshengensis CHN AB725736; C. okamotoi KOR1 AB725737, KOR2 JX644729, KOR3 MT826872; C. omiana JPN DQ998988; C. pachygyna JPN AB725738; C. plantaginea CAN AY757540; C. siderosticta CHN1 AB725739, CHN2 AB725740, JPN1 AB725742, JPN2 AB725742, KOR1 AB725743, KOR2 AB725744, KOR3 MT826873; C. splendentissima KOR1 MT316043, KOR2 MT826880, KOR3 MT826882; C. subcapitata CHN1 AB725745, CHN2 AB725746; C. tsiangii CHN AB725757; C. tumidula JPN AB725747; Scirpus microcarpus AF284859; Trichophorum pumilum AB643656; T. subcapitatum AB679914.

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