Journal Pre-proof Cytotaxonomy of endangered species Orobanche filicicola in Korea, and its closely related species, O. coerulescens (Orobanchaceae) (I) Bokyung Choi, Tae-Soo Jang, Jeong-Mi Park, Ji Hoon Kim, Sunhee Sim, Chang Woo Hyun, Soonok Kim, Minsu Park, Nuree Na, Do Keun Lee PII: S2287-884X(20)30048-0 DOI: https://doi.org/10.1016/j.japb.2020.04.001 Reference: JAPB 506 To appear in: Journal of Asia-Pacific Biodiversity Received Date: 25 February 2020 Revised Date: 22 March 2020 Accepted Date: 6 April 2020 Please cite this article as: Choi B, Jang T-S, Park J-M, Kim JH, Sim S, Hyun CW, Kim S, Park M, Na N, Lee DK, Cytotaxonomy of endangered species Orobanche filicicola in Korea, and its closely related species, O. coerulescens (Orobanchaceae) (I), Journal of Asia-Pacific Biodiversity, https:// doi.org/10.1016/j.japb.2020.04.001. 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Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 National Science Museum of Korea (NSMK) and Korea National Arboretum (KNA), Publishing Services by Elsevier. 1 Title page 2 3 Cytotaxonomy of endangered species Orobanche filicicola in Korea, and its closely 4 related species, O. coerulescens (Orobanchaceae) (I) 5 6 Authors: Bokyung Choia,† · Tae-Soo Janga,† · Jeong-Mi Parkb* · Ji Hoon Kimc · Sunhee 7 Simb · Chang Woo Hyunb · Soonok Kimb · Minsu Parkd · Nuree Nad · Do Keun Leee 8 † These authors contributed equally to this work. 9 10 a 11 National University, Daejeon 34134, Korea 12 b 13 Incheon 22689, Korea 14 c 15 d 16 e Department of Biological Science, College of Bioscience and Biotechnology, Chungnam Department of Biological Resources Research of National Institute of Biological Resources, IRUDAIN Inc. Jeju 63145, Korea Department of Biology Education, Kongju National University, Gongju 32588, Korea Wild plant research society on Mt. Baekdu, Seoul 07247, Korea 17 18 E-mail address: jmpark99@korea.kr (J-M. Park). 19 20 21 22 Running title: Cytotaxonomy of some Orobanche species native to Korea (I) 23 Abstract 24 25 The Orobanche is the largest group, which are mainly distributed in the northern hemisphere and 26 consisted of about 170 species. The present study is focused on a cytological study on Korean 27 endangered species Orobanche filicicola as well as morphologically similar species O. coerulescens 28 and their morphotype, which was previously referred to O. coerulescens f. alba but it was 29 taxonomically treated as a synonym of O. coerulescens. Chromosome numbers of 13 individuals in 30 six populations of two Korean Orobanche taxa (O. coerulescens and O. filicicola) as well as 31 morphotype of O. coerulescens were examined to understand the karyotype diversity. A chromosome 32 number of 2n = 2x = 38 was uniformly observed for all the Orobanche taxa in Korea despite that 33 individuals of two different color types (typical purple and whitish yellow) were included. The 34 karyotypes of O. filicicola were from metacentric to submetacentric and the length of chromosomes 35 ranged from 2.08 to 4.74 µm, resulting in a Haploid Karyotype Length (HKL) of 60.92 µm. 36 Investigations of meiotic configuration of O. filicicola, O. coerulescens, and its morphotype were 37 constantly stable. In those species all homologous chromosome pairs have formed regular bivalents as 38 previously documented in other species of the genus Orobanche. 39 40 41 Keywords: Chromosome number; Endangered species; Meiosis; Orobanche; Taxonomy 42 Introduction 43 44 Orobanche L., is the largest genus among the holoparasitic genera in the family 45 Orobanchaceae Vent. and comprised of ca. 170 species that are distributed predominantly in the 46 Northern Hemisphere (Zhang and Tzvelev 1998; Schneeweiss et al 2004a, b). The genus includes 47 annual, biennial and perennial herbs and are characterized by a fleshy body with spike or raceme 48 inflorescences and possesses haustoria which the plant can obtain water and nutrients from their host 49 plants (Zhang and Tzvelev 1998). The genus Orobanche is divided into four sections as the following, 50 which is well supported by the base chromosome numbers: sect. Orobanche L. (x = 19); sects. 51 Trionychon Wallr. and Myzorrhiza (Phil.) Beck (x = 12); sect. Gymnocaulis Nutt. (x = 24; 52 Schneeweiss et al 2004a). While sects. Orobanche and Trionychon are found both in the Old World 53 and the New World, sects. Gymnocaulis and Myzorrhiza are restricted to the New World (Schneeweiss 54 et al 2004b). In Korea, along with one recently recognized species, Orobanche filicicola Nakai ex 55 Hyun, H.C. Lim & Shin, two other species (O. coerulescens Stephan and O. pycnostachya Hance) are 56 currently recognized (Hyun et al 2003; Hong 2007). Due to habitat destruction, degradation, and 57 fragmentation, Orobanche species in Korea are threatened, and O. filicicola is endangered among the 58 species in Korea (Hyun et al 2003; Kim 2006). Orobanche pycnostachya is distributed in North Korea 59 (Hong 2007). Thus, the taxon was not included for this study. 60 The unique parasitic lifestyle of Orobanche with its host plants makes the genus a valuable 61 group to study various aspects of its biology including taxonomy and co-evolution with host plants 62 (Piwowarczyk et al 2018, 2019). Despite the evolutionary and ecological importance of the Korean 63 Orobanche species, no comprehensive taxonomic and chromosomal analyses have been performed to 64 date. Although Orobanche filicicola has been nomenclaturally validated and clearly distinguished 65 from other Korean Orobanche species based on corolla color, style pubescence, and overall plant size 66 (Hyun et al 2003; Kim 2006), but the infraspecific taxonomy of O. coerulescens is complicated 67 because the plants have different color types (typical purple: O. coerulescens Stephan f. coerulescens, 68 whitish yellow: O. coerulescens Stephan f. alba Y. Lee; Lee and Kim 2005) and the species can be 69 divided in two types based on presence/absence of trichomes (G-type vs. P-type; Lee et al 2006). 70 Chromosome studies employing both classical and molecular cytogenetic methods using 71 FISH (fluorescence in situ hybridization) have proved to be an important source for taxonomic and 72 systematical analyses in angiosperms (Weiss-Schneeweiss and Schneeweiss 2013; Jang et al 2018a, b; 73 Choi et al 2019). However, chromosomal studies of the Orobanche are still limited. For example, 74 Chromosomal counts of only 98 species out of approximately 170 species are available in the genus 75 (Chromosome Counts Database, CCDB, ver. 1.45, < http://ccdb.tau.ac.il/search/>, Rice et al 2015). 76 Chromosomal studies of Orobanche have rarely been carried out and chromosome number 77 information for the Korean Orobanche species is not available to date (Schneeweiss et al 2004a; Li et 78 al 2017). Thus, the aims of the current study are 1) to report base chromosome number of Orobanche 79 filicicola and O. coerulescens including its infraspecific morphotype (syn. O. coerulescens for. alba), 80 and 2) to provide karyotype information and characterize the meiotic behavior of Orobanche filicicola. 81 82 Material and methods 83 Materials 84 A total of seven individuals of Orobanche filicicola from two populations in Island Je-ju in 85 Korea were investigated cytologically (Table 1 and Appendix A). In order to compare chromosome 86 number as well as meiotic behavior, five individuals from four populations of O. coerulescens 87 including one individual of the its morphotype (syn. O. coerulescens for. alba) were also examined 88 (Table 1). Orobanche pycnostachya was not included in this study because there was no access to any 89 samples due to its distribution (North Korea; Hong 2007). Due to the lack of typical root meristems, 90 chromosome numbers were determined from meiotic divisions in pollen mother cells (PMCs). Young 91 flower buds were fixed in the field in 3 : 1 = ethanol : glacial acetic acid for at least 24 hours at room 92 temperature and stored at –20˚C until use for meiotic analyses (Li et al 2017). Voucher specimens of 93 the examined individuals used in this study are deposited at the herbarium of the Chungnam National 94 University, Daejeon, Korea (CNUK). All investigated specimens are indicated in Appendix A. 95 96 Chromosome counts and karyotype analysis 97 Feulgen staining with Schiff’s reagent was done following the standard method (Jang et al 98 2013). Briefly, material was hydrolyzed in 5N HCl for 30 minutes at room temperature, washed 99 briefly with tap water, and stained with Schiff’s reagent in darkness for an hour. Squash preparations 100 were made in a drop of 60% acetic acid. Preparations with a minimum of ten good quality 101 chromosome spreads were analyzed for each individual with a light microscope (Olympus BX-53) 102 and photographed with digital camera (Olympus DP-74). 103 For measurements of chromosome length of O. filicicola, microspores in metaphase were 104 chosen. Chromosomes were cut out and arranged in Corel Photo-Paint 12.0. The size of chromosomes 105 was measured using Micromeasure ver. 3.3 (<https://sites.biology.colostate.edu/Micromeasure/>) 106 following Jang et al (2013). Chromosome arm and haploid karyotype length were measured on 107 chromosomal spreads with a medium degree of chromosome condensation per each individual. 108 109 Results and discussion 110 111 The karyotypes and chromosome numbers of the Korean endemic plants Orobanche 112 filicicola were reported here for the first time. The chromosome number of all individuals of the two 113 species were diploids, 2n = 38 (n = 19), despite that two different color types (i.e., typical purple vs. 114 whitish yellow) of O. coerulescens were included (Figure 1 and Table 1). The chromosome number of 115 O. coerulescens in Korea was in agreement with previous reports of O. coerulescens occurring in 116 central Europe (Weber 1976) although n = 20 was also found in Rice et al (2015). However, it is 117 possible that the deviant number of O. coerulescens, is likely due to species misidentification because 118 no vouchers were cited in the previous publication (Fedorov 1969). In addition, flower colors might 119 be usually affected by different environmental conditions (Eaton et al 2012; Arista et al 2013; Sobral 120 et al 2015; Sobel et al 2019), therefore, no differences in the chromosome number between two color 121 phenotypes (i.e., typical purple vs. whitish yellow) of O. coerulescens are not surprising (Figure 1) 122 although latter morphotype (i.e., whitish yellow of flower) was previously treated as a separate taxon 123 named as O. coerulescens f. alba (Lee and Kim 2005). 124 Hyun et al (2003) validated O. filicicola as a separate species based on glandular stems and 125 flowers, densely villous anthers along the sutures, blue violet corolla with white lower part, and 126 deeply two-parted calyx in comparison with other Korean Orobanche taxa (Figure 1A). Particularly, 127 Orobanchaceae is cytologically diverse with chromosome numbers of n = 12, 19, 24, which have been 128 mostly derived from descending dysploidy and polyploidy (Schneeweiss et al 2014b). Sectional 129 circumscriptions of Orobanche were characterized by different chromosome numbers (Schneeweiss et 130 al 2004a), and based solely on the chromosome number, therefore, it is possible that O. filicicola and 131 O. coerulescens may be included in the sect. Orobanche (Figures 2 and 4). Nevertheless, it is not 132 sufficient to make any taxonomic decisions for O. coerulescens and O. filicicola (Figures 1–4). 133 Further comprehensive studies on morphological variations as well as molecular analyses based on 134 DNA sequences of more samples from different populations are required to elucidate the taxonomic 135 position/relationships of the Orobanche species (Hyun et al 2003; Schneeweiss et al 2004b; Lee et al 136 2016; Li et al 2017). 137 All chromosomes were metacentric to submetacentric (Figure 2B) and the size in length 138 ranged from 2.08 to 4.74 µm, resulting in a Haploid Karyotype Length (HKL) of 60.92 µm (Figure 139 2A–B). It was shown that normal bivalent formation was observed in relatively high frequencies from 140 meiotic behavior of the investigated Orobanche filicicola (Figure 3C–D). No meiotic irregularities 141 such as laggards or bridges have been observed in the investigated samples (Figure 3A–H) as reported 142 in the previous study of Orobanche (Schneeweiss et al 2014a). During meiosis, all chromosome pairs 143 formed regular bivalents, each usually possessing two chiasmata (Figure 3D–E). In diakinesis, usually 144 a set of bivalents are associated with the nucleolus, suggesting the presence of at least one pairs of 145 active 35S rDNA loci (Figure 3B), and this was also confirmed in the mitosis and the karyotype of 146 Orobanche filicicola (Figure 2A–B). In O. coerulescens and its morphotype (syn. O. coerulescens for. 147 alba), all homologous chromosome pairs have formed regular bivalents as seen in O. filicicola (Figure 148 4A–B). 149 Several previous taxonomic studies have shown that many Korean Orobanche species are 150 characterized by slight morphological differences in plant size (i.e., plant height, and bract, calyx, and 151 corolla size), bract shape, and corolla color (Hyun et al 2003; Hong 2007; Lee et al 2006). However, 152 due to the loss of color during desiccation of herbarium specimens as well as the samples preserved in 153 formalin-acetic acid-alcohol (FAA) in field, Orobanche is one of the most difficult genera to study 154 taxonomic classification (Schneeweiss et al 2009). Additionally, Orobanche filicicola and O. 155 coerulescens have never been studied together to date (e.g. Hyun et al 2003; Hong 2007; Lee et al 156 2016). Therefore, more expanded samplings of the Orobanche populations are still required to clarify 157 whether the incidence of polyploidization exists in O. filicicola and O. coerulescens. Additionally, 158 further analyses including molecular barcoding, molecular cytogenetic approach involving FISH 159 (fluorescence in situ hybridization) using 3S and 35S rDNA probes as well as morphometrics are 160 required to re-evaluate the clear taxonomic position of the Korean Orobanche taxa. 161 162 Conclusion 163 The chromosome number of the Orobanche filicicola was reported in the present study for 164 the first time with 2n = 2x = 38, and the same chromosome number was confirmed for Korean 165 populations of O. coerulescens and its morphotype (syn. O. coerulescens for. alba). Investigations of 166 meiotic configuration of the of the Korean Orobanche taxa were constantly stable. The chromosomal 167 information of the Korean Orobanche species contributes towards a better understanding of taxonomy 168 and chromosomal evolution of the genus Orobanche. Further insight into the phylogeny, 169 morphological variation, cytological stabilization and molecular cytology of the Korean Orobanche 170 species and their closely related taxa are required to better understand the evolution of the genus 171 Orbanche. 172 173 174 175 Conflict of interest The authors declare that there is no conflict of interest. 176 177 Acknowledgments 178 The authors thank to Tae Hwan Kim for providing the detailed photographs of the 179 investigated taxa in fields and members at the Herbarium of Chungnam National University (CNUK) 180 for their help in various ways. This study was supported by grants on project as “The Genetic 181 Evaluation of Parasitic plant (I)” from the National Institute of Biological Resources of Ministry of 182 Environment (NIBR201905101) and the National Research Foundation of Korea (NRF) funded by the 183 Korea government (NRF-2018R1C1B6003170). 184 185 References 186 Arista M, Talavera M, Berjano R, et al. 2013. Abiotic factors may explain the geographical 187 distribution of flower colour morphs and the maintenance of colour polymorphism in the scarlet 188 pimpernel. Journal of Ecology 101 (6):1613–1622. 189 190 191 192 193 194 195 196 197 198 Choi B, Yang S, Song J-H, et al. 2019. Karyotype and genome size variation in Ajuga L. (AjugoideaeLamiaceae). Nordic Journal of Botany 37:e02337. Eaton DA, Fenster CB, Hereford J, et al. 2012. Floral diversity and community structure in Pedicularis (Orobanchaceae). Ecology 93 (sp8):S182–S194. Fedorov ANA. 1969. Chromosome Number of Flowering Plants. 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Zhang Z, Tzvelev NN. 1998. Orobanchaceae. In: Wu Z-Y, Raven PH. editors. Flora of China, Vol. 18. Beijing: Beijing; St. Louis: Missouri Botanical Garden Press. pp. 229–243. 246 Table 1. Information on the plant material used for the chromosome number in Orobanche filicicola, 247 O. coerulescens and its morphotype (syn. O. coerulescens for. alba). Collector Taxon & locality* Chromosome number collection number Orobanche filicicola Nakai ex Hyun, H.C. Lim & Shin J-043 TS, JM, JH, DK, TH, SH,YC 2n = 2x = 38 J-044 TS, JM, JH, DK, TH, SH,YC 2n = 2x = 38 J-045 TS, JM, JH, DK, TH, SH, YC 2n = 2x = 38 J-046 TS, JM, JH, DK, TH, SH, YC 2n = 2x = 38 J-052 TS, JM, JH, DK, TH, SH, YC 2n = 2x = 38 J-024 TS, YC 2n = 2x = 38 J-034 TS, JM, JH, DK, TH, SH, YC 2n = 2x = 38 O. coerulescens Stephan J-022 TS, JM, JH, DK, TH, SH, YC 2n = 2x = 38 J-S24 TS, JM, JH, DK, TH, SH, YC 2n = 2x = 38 J-0016 TS, YC 2n = 2x = 38 090517 JM, MS 2n = 2x = 38 SK19-p013-1 MS, NR 2n = 2x = 38 Morphotype of O. coerulescens Stephan (syn. O. coerulescens Stephan f. alba Y.N. Lee) J-013 TS, JM, JH, DK, TH, SH, YC 2n = 2x = 38 248 Collectors: TS = Tae-Soo Jang; JM = Jeong-Mi Park; MS = Minsu Park; NR = Nuree Na; JH = Ji 249 Hoon Kim; DK = Do Keun Lee; TH = Tae Hwan Kim; SH = Sunhee Sim; YC =Young-Min Choi. 250 *The information of localities was not indicated in this Table for protection purpose of the endangered 251 taxa (see Appendix A). 252 253 Legend of Figures 254 255 Figure 1. Habit and floral morphology of the Korean Orobanche species.: A, Orobanche filicicola; B- 256 C, floral color variation in O. coerulescence; B, typical color type; C, whitish yellow morphotype of 257 O. coerulescence (syn. O. coerulescens for. alba). 258 259 Figure 2. Chromosomes (A) and karyotype (B) of Orobanche filicicola: 2n = 2x = 38 (metaphase of 260 mitotic cell division). <scale bar: 5 µm> 261 262 Figure 3. Meiotic behavior in Orobanche filicicola.: A, Pachytene; B, Diakinesis; C, Metaphase I; D, 263 Anaphase I; E, Later anaphase I; F, Telophase I; G, Tetrad; H, Young pollen grains. <scale bar: 5 µm> 264 265 Figure 4. PMC (pollen mother cell) of two different flower color types (typical purple: A, whitish 266 yellow: B) in Orobanche coerulescence at diakinesis showing n = 19 chromosome number with 267 regular bivalents paring during meiosis.: A, Orobanche coerulescence; B, morphotype of O. 268 coerulescence (syn. O. coerulescens for. alba). <scale bar: 5 µm> 269 270 Appendix A. Voucher specimens examined in the present study. The detailed information of the 271 collection sites was not indicated for protection purpose of the all investigated populations. 272 Orobanche filicicola Nakai ex Hyun, H.C. Lim & Shin: Korea, Jeju-do, 26 Apr 2019, JM Park et al., 273 J-043 (CNUK00001197); Korea, Jeju-do, 26 Apr 2019, JM Park et al., J-044 (CNUK00001198); 274 Korea, Jeju-do, 26 Apr 2019, JM Park et al., J-045 (CNUK00001199); Korea, Jeju-do, 26 Apr 2019, 275 JM Park et al., J-046 (CNUK00001200); Korea, Jeju-do, 26 Apr 2019, JM Park et al., J-052 276 (CNUK00002098); Korea, Jeju-do, 26 Apr 2019, TS Jang & YM Choi, J-024 (CNUK00001178); 277 Korea, Jeju-do, 26 Apr 2019, JM Park et al., J-034 (CNUK00001188). 278 O. coerulescens Stephan: Korea, Jeju-do, 26 Apr 2019, JM Park et al., J-022 (CNUK00001176); 279 Korea, Jeju-do, 26 Apr 2019, JM Park et al., J-S24 (CNUK00002097); Korea, Jeju-do, 26 Apr 2019, 280 TS Jang & YM Choi, J-0016 (CNUK00001170); Korea, Jeolla-do, 26 Apr 2019, JM Park & MS Park, 281 090517 (CNUK00002096); Korea, Gyeongsang-do, 26 Apr 2019, MS Park & NR Na, SK19-p013-1 282 (CNUK00002095). 283 Morphotype of O. coerulescens Stephan (syn. O. coerulescens for. alba): Korea, Jeju-do, 26 Apr 284 2019, TS Jang & YM Choi, J-013 (CNUK00001167). Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: