Notes on Allium section Rhizirideum (Amaryllidaceae) in South Korea and northeastern China: with a new species from Ulleungdo Island

Abstract Allium section Rhizirideum is reviewed for South Korea and neighboring northeastern China based on critical observation of wild populations and herbarium materials. Species delimitations are re-evaluated on the basis of morphological and somatic chromosome numbers, resulting in the recognition of five species. Allium dumebuchum from Ulleungdo Island, South Korea, is described as a new species. This species is most similar to A. senescens due to its habits, but is clearly distinguished particularly by its rhomboid scapes in cross-secion, light purple perianth color, entire and narrowly triangular inner filaments, and flowering season from late September. One previously recognized species is placed into synonymy: A. pseudosenescens (under A. senescens). Photographs and a key to species of Allium section Rhizirideum in South Korea and northeastern China are provided in addition to information on nomenclatural types, synonymies, chromosome numbers, distribution, and specimens examined.


Introduction
With over 900 species (Seregin et al. 2015), Allium L. is one of the largest genera in the Amaryllidaceae (Friesen et al. 2006;Fritsch et al. 2010;Li et al. 2010). It is characterized by bulbs enclosed in membranous to fibrous tunics, free or almost free tepals, and often a subgynobasic style (Friesen et al. 2006). Most taxa produce remarkable amounts of cysteine sulphoxides causing the well-known characteristic odor and taste (Friesen et al. 2006). Allium is distributed naturally in the northern hemisphere and in South Africa, mostly in regions with dry seasons (De Sarker et al. 1997;Friesen et al. 2006;Nguyen et al. 2008;Neshati and Fritsch 2009). The classification of Allium by Friesen et al. (2006) based on molecular phylogenetic analyses includes 15 subgenera and 56 sections. About 23 taxa, excluding cultivated species, are known from the Korean peninsula and neighboring northeastern China (Choi and Oh 2011;Shukherdorj et al. 2018;Choi et al. 2019).
Allium section Rhizirideum G.Don ex W.D.J.Koch is the typical section of subgenus Rhizirideum (G.Don ex W.D.J.Koch) Wendelbo and characterized by having bulbs enclosed in membranous tunics and attached to horizontal rhizomes, a leaf shape ranging from hemicylidrical to plain, and a flower color from white to purple (Sinitsyna et al. 2016). Section Rhizirideum consists of 24 species and is part of the third of three main evolutionary lines of Allium (Fritsch 2001;Fritsch and Friesen 2002;Friesen et al. 2006;Li et al. 2010;Choi et al. 2012b). The distribution area of section Rhizirideum reaches from Europe to East Asia (Sinitsyna et al. 2016). There is a distinct narrowing of the distribution area east of the Ural Mountains approximately along 70° eastern longitude, and most species of section Rhizirideum are distributed in temperate Asia (Sinitsyna et al. 2016). The center of species diversity is situated in the mountain steppes of South Siberia and Mongolia (Sinitsyna et al. 2016). The species of section Rhizirideum share a basic chromosome number of x = 8, and four ploidy levels were found: di-, tetra-penta-, and hexaploids (Sinitsyna et al. 2016).
The taxonomy of the section is complicated because of morphological diversity and hybridization involving polyploidy (Friesen 1988(Friesen , 1992Kamelin 2004). Additionally, the nomenclature is confusing, which may be explained by similar morphology of some species and disappearance of many morphological characters in the voucher specimens in herbaria (Sinitsyna et al. 2016;Sinitsyna and Friesen 2018). Recently, Sinitsyna et al. (2016) and Sinitsyna and Friesen (2018) investigated the phylogenetic relationships in the section Rhizirideum based on molecular markers, and organized nomenclature, distribution maps and identification key for all known species of the section. Although there is general agreement regarding the Allium species of section Rhizirideum in South Korea and its neighboring northeastern China (Choi and Oh 2011) studies on materials from these regions are still limited.
Here, we have combined morphological, cytological, and molecular characters to address the taxonomy of Allium section Rhizirideum, and organized nomenclature, distribution maps and identification key for species in South Korea and north-eastern China. The goals of this study are: 1) to review and expand the current knowledge on general morphology (in addition to Choi and Oh 2011;Sinitsyna et al. 2016), somatic chromosome numbers (in addition to Choi and Oh 2011), DNA barcoding, and distribution (in addition to Choi and Oh 2011;Sinitsyna et al. 2016) especially with a focus on the materials from South Korea and north-eastern China, and 2) to describe a new species of section Rhizirideum from Ulleungdo Island, South Korea, A. dumebuchum H.J.Choi. This study together with that of Sinitsyna et al. (2016) and Sinitsyna and Friesen (2018) will provide a sound foundation for a global monograph and the systematic understanding of Allium section Rhizirideum.

Morphological characters
This revision is based on the use of living and herbarium material, including photographs of type specimens, from the following herbaria: B, CBU, KB, KH, KWNU, LE, LINN, PE (abbreviations are according to Thiers 2020+), and the herbarium of Changwon National University (CWNU). Field surveys were carried out mainly in South Korea and north-eastern China from July 2014 to October 2020. We also observed populations from Far Eastern Russia and Mongolia especially for Allium spirale Willd. and A. senescens L. Materials preserved in 70% ethanol were used especially for observation and measurement of floral parts, cross-sections of leaf and scape. Segments from the middle third of the leaf blade and scape were stained with 2% aceto carmin for observation of the crosssection. Measurements were based on at least 30 samples for quantitative characters.

Principal component analysis
To analyze floral morphology known as a key character to distinguish Allium species (Choi and Oh 2010;Choi and Oh 2011), principal components analysis (PCA) was performed based on 14 characters: flower number per inflorescence, inflorescence length, inflorescence width, pedicel length, inner tepal length, inner tepal width, outer tepal length, outer tepal width, inner filament length, inner filament width, outer filament length, anther length, anther width, and pistil length. The principal components analysis used the {ggfortify} and {ggplot2} packages of the R-project (Tang et al. 2016;Wickham 2016; R Core Team 2020). The specimens used for principal components analysis were indicated with an asterisk (*) in specimens examined for each species.

Somatic chromosome numbers
Root tips were pre-treated in distilled water on ice for 24 h in total darkness at 4 °C and then fixed in Carnoy's fluid (3 parts absolute ethanol: 1 part glacial acetic acid, v/v) overnight at 4 °C. The root tips were macerated in 1M hydrochloric acid at 60 °C for 3-5 min. After washing 3-5 times to eliminate residual hydrochloric acid and staining with feulgen for 5 min, the material was squashed for observation in 2% aceto carmin. Observations and photographing of chromosome micrographs were made using an Olympus BX43 (Tokyo, Japan).

DNA barcoding
In this study, we investigated the application of concatenated cpDNA regions of ndhJ-trnF, trnH-psbA, psbD-trnT, and psbJ-petA in barcoding analyses of Allium section Rhizirideum and related taxa (Table 1). In order to analyze the relationship among the species using these four cpDNA regions, we extracted each cpDNA region from complete chloroplast genome sequences stored in NCBI GenBank (https://www.ncbi.nlm.nih.gov/; Fig. 6). The species in subgen. Butomissa were selected as outgroup referred from wide phylogenetic study of Allium (Li et al. 2010). Detailed information on sample collection, voucher specimens and Genbank accession numbers of each sample is provided in Table 2.
Total genomic DNA was extracted from silica gel-dried leaf materials using the DNeasy Plant Mini Kit (Qiagen, Seoul, South Korea). We conducted PCR with a ProFlex 96-Well PCR System (Applied Biosystems, Foster City, CA, USA). Each reaction mixture contained AccuPower PCR PreMix (Bioneer, Daejeon, South Korea), ca. 10 ng (1μL) of genomic DNA, and 100 pM of primers in a total volume of 20 μL. Conditions included an initial denaturation at 94 °C for 5 min, followed by 30 amplification cycles comprising 94 °C for 1 min, 54 °C for 1 min, and 72 °C for 1 min, with a final extension at 72 °C for 7 min. After the PCR products were visualized on 2% agarose gels, they were treated with a MG PCR Purification kit (MGmed), and sequenced with the ABI 3730xl Analyzer, using the ABI BigDye Terminator v3.1 Cycle Sequencing Kits (Applied Biosystems, Foster City, CA, USA). The obtained sequences were manually determined and aligned by using MAFFT with Geneious Prime 2019.2.3 (Biomatters Ltd., Auckland, NZ). The DNA sequences generated in this study have been deposited in GenBank ( Table 2).
The phylogenetic analyses were conducted using Maximum Likelihood (ML) by using W-IQ-TREE (Trifinopoulos et al. 2016), based on user-friendly web servers for IQ-TREE (Nguyen et al. 2015). The concatenated sequence dataset was tested to find the best-fit model by using W-IQ-TREE with the Akaike criterion and new model selection procedures. TIM+R3+F were confirmed as best-fit models for the sequences. Maximum likelihood analysis was performed with default settings in W-IQ-TREE (Fig. 6).

Morphological characters
Our data indicate that several morphological characters are of taxonomic utility in Allium section Rhizirideum. Among these, the shape and size of leaf, scape and various floral parts are useful diagnostic traits at the specific level (Table 3; Fig. 1; Choi and Oh 2010;Choi and Oh 2011). According to the PCA results, first combined five principal components accounted for 83.65% of the total variation among traits in the studied taxa. The PC1 accounted for 52.94% of variance, while PC2 accounted for 13.54% of total variability. The first two principal components were strongly associated with the inflorescence length, outer tepal length and inner filament width. The anther length and inner tepal width were mostly contributed to PC1, while the pedicel length and flower number were contributed only to PC2. PC1 versus PC2 in scatter plot showed that A. dumebuchum and A. minus were distinctly separated from A. senescens, A. spirale, and A. spurium (Fig. 2).

Key to the species of Allium section Rhizirideum in South Korea and northeastern China
Phenology. Flowering from late September to October; fruiting from late October to November. Distribution and habitat. Endemic to South Korea (Ulleung-do Island; Fig. 5). Open slope of rocky area.
Etymology. The specific epithet, "dumebuchum" is based on the name of traditional vegetable for this species in South Korea. Vernacular name. The Korean name of the new species is "Du-me-bu-chu (두메 부추)".
Conservation status. The new species is endemic to Ulleungdo Island, and usually grows along the coast at altitudes of -23-171m a.s.l. From the present study, the extent of occurrence (EOO) and the area of occupancy (AOO) of this species have been calculated to be 47,683 km 2 and 48 km 2 , respectively. Currently, there is no information on population size and trend data. However, this new species is only known from a single location of Ulleungdo Island, and mainly occurs on the coast which is critically threatened by extensive construction and repair of coastal roads (Choi et al. 2012a). Therefore, decline in habitat area, habitat extent, and quality of habitat for this species have been continuously observed. Thus, Allium dumebuchum should be considered as Critically Endangered [CR B1ab(iii)] according to the IUCN Red List categories and criteria (IUCN 2021).
Notes. Allium dumebuchum, occurring in Ulleungdo Island of South Korea, has usually been misidentified as A. senescens (Choi and Oh 2010;Choi and Oh 2011). However, this new species remarkably distinguished itself from its related species of section Rhizirideum (e.g., A. spirale, A. spurium, A. minus, and A. senescens) in having clearly bigger floral parts that bloom from late September (Table  3; Fig. 1). The PCA results based on quantitative floral characters of five related species in section Rhizirideum clearly identified A. dumebuchum from others (Fig.  2). This new species is a tetraploid (2n = 4x = 32) taxon along with A. senescens, and A. minus is a diploid (2n = 2x = 16), whereas A. spirale and A. spurium showed polyploidy (Table 3; Fig. 3). Moreover, molecular phylogenetic analyses using chloroplast markers (ndhJ-trnF, trnH-psbA, psbD-trnT, and psbJ-petA) also clearly indicate that A. dumebuchum is genetically distinct from other species of section Rhizirideum (Fig. 6).