M. Y. Chung et al. Effect of historical factor on genetic variation in three Cephalanthera species 1
1 Effect of historical factors on genetic variation in the three terrestrial 2 orchids Cephalanthera erecta, Cephalanthera falcata, and Cephalanthera 3 longibracteata on the Korean Peninsula differing in breeding systems 4 5 Mi Yoon Chung, Nhan Thien Lu, Jordi López-Pujol, Sonia Herrando-Moraira, Jae Min 6 Chung, Huai Zhen Tian, Kenji Suetsugu, Takayuki Kawahara, Tomohisa Yukawa, 7 Masayuki Maki, Pankaj Kumar, Young-Dong Kim, and Myong Gi Chung 8 9 M.Y. Chung, Research Institute of Natural Science, Gyeongsang National University, 10 Jinju 52828, Republic of Korea. – N. T. Lu and M. G. Chung (http://orcid.org/0000- 11 0002-1283-3574) ([email protected]), Division of Life Science and the Research 12 Institute of Natural Science, Gyeongsang National University, Jinju 52828, Republic of 13 Korea. – J. López-Pujol and S. Herrando-Moraira, Botanic Institute of de Barcelona 14 (IBB, CSIC-ICUB), Passeig del Migdia s/n, Barcelona 08038, Spain. – J. M. Chung, 15 Plant Conservation Division, Korea National Arboretum, Pocheon 11186, Republic of 16 Korea. – H. Z. Tian, School of Life Sciences, East China Normal University, Shanghai 17 200241, China. – K. Suetsugu, Department of Biology, Graduate School of Science, 18 Kobe University, Kobe, 657-8501, Japan. –T. Kawahara, Hokkaido Research Center, 19 Forestry and Forest Products Research Institute, Sapporo, Hokkaido, Japan. –T. 20 Yukawa, Tsukuba Botanical Garden, National Science Museum, Tsukuba, Ibaraki, 21 Japan. –M. Maki, Botanic Gardens, Tohoku University, Kawauchi 12-2, Sendai 12-2, 22 Sendai 980-0862, Japan. – P. Kumar, Kadoorie Farm & Botanic Garden, Lam Kam Rd., 23 Lam Tsuen, Tai Po, New Territories, Hong Kong SAR, China. – Y.-D. Kim, Department 24 of Life Sciences, Hallym University, Chuncheon 24252, Republic of Korea. 25 26 Corresponding author: Myong Gi Chung, Division of Life Science and the Research 27 Institute of Natural Science, Gyeongsang National University, Jinju 52828, Republic of 28 Korea. Email: [email protected] 29 M. Y. Chung et al. Effect of historical factor on genetic variation in three Cephalanthera species 2
30 Previous studies have shown that levels of genetic diversity in species of the genus 31 Cephalanthera vary depending on breeding systems. In the southern part of the Korean 32 Peninsula, the three self-compatible, terrestrial orchids Cephalanthera erecta, C. falcata, 33 and C. longibracteata flower synchronously in sympatric populations. The food- 34 deceptive C. falcata, with bright yellow flowers, is predominantly outcrossing, whereas 35 autogamy is the dominant strategy in both C. erecta and C. longibracteata, whose white 36 flowers do not fully open. Given this, we expect that populations of C. falcata will 37 harbor considerably higher levels of genetic variation than C. erecta and C. 38 longibracteata. We examined genetic diversity (by means of allozymes) of the three 39 species in sympatric populations (600 600-m area) in Yeonwhasan Provincial Park 40 (YPP) and in non-sympatric populations outside YPP, South Korea. Thirteen out of 21 41 putative loci were variable across the three species, but unexpectedly we found a 42 complete lack of allozyme variation within each species; in addition, the three species 43 showed several diagnostic or unique alleles. Cephalanthera erecta and C. 44 longibracteata are obligate autogamous species, with little chance to hybridize between 45 them. Consistent with this, we did not detect allozyme-based hybrids within sympatric 46 populations in YPP. Our results suggest that historical factors (i.e., the Quaternary 47 climate oscillations) played a major role in determining levels of genetic diversity of the 48 three Cephalanthera species. The Korean populations of C. erecta (a warm- 49 temperate/temperate element) and C. falcata (a warm-temperate element) could have 50 been established by a single introduction from a genetically depauperate ancestral 51 population, likely located outside the Korean Peninsula. On the other hand, since C. 52 longibracteata is a boreal/temperate element, it probably survived the Last Glacial 53 Maximum in microrefugia located at low elevation regions within the Peninsula. 54 55 Keywords: Allozymes, allogamy, autogamy, Cephalanthera, genetic diversity, 56 historical factors, hybridization 57 M. Y. Chung et al. Effect of historical factor on genetic variation in three Cephalanthera species 3
58 Introduction 59 Life-history traits influence levels and distributions of genetic diversity found within 60 species and populations (Hamrick and Godt 1989, Gray 1996). For example, breeding 61 system is an important factor affecting levels of genetic diversity of plant species. The 62 effects of breeding systems on levels of genetic diversity are particularly well known in 63 terrestrial orchid species of the genus Cephalanthera (Scacchi et al. 1991, Micheneau et 64 al. 2010). This genus provides a good study system to determine a causal relationship 65 between levels of genetic diversity and mating systems, because it has autogamous (e.g. 66 C. damasonium), allogamous (e.g. C. longifolia), and mixed-mating species (e.g. C. 67 rubra). Scacchi et al. (1991) examined levels of genetic diversity in the three- 68 abovementioned species from central Italy and found contrasting levels of genetic 69 diversity. The authors found no allozyme variation across 13 populations of C. 70 damasonium, but moderate levels (compared to other orchids; Table 1 in Chung et al. 71 2018) of genetic variation in three populations of C. longifolia and in seven populations 72 of C. rubra (Table 1). Micheneau et al. (2010), using plastid microsatellite loci, 73 examined genetic variability in the three Cephalanthera species across Europe and 74 obtained similar results: only one haplotype in C. damasonium, eight haplotypes in C. 75 longifolia, and nine in C. rubra. Recently, Brzosko and Wróblewska (2013) found low 76 levels of genetic variation in nine Polish populations of C. rubra (Table 1). All these 77 studies stress that the patterns of genetic variation in the three orchids were apparently 78 related to differences in their breeding systems. 79 As a replicate, it may be of interest to revisit this pattern in self-compatible, 80 non-clonal, terrestrial Cephalanthera species in East Asia. Cephalanthera erecta 81 (Thunb.) Blume occurs in warm-temperate and temperate regions in central and 82 southern China, central and southern Korea, and Japan (Chen et al. 2009). Similarly, C. 83 falcata (Thunb.) Blume mainly occurs in warm-temperate regions in central and 84 southern China, southern and southwestern Korea, and central and southern Japan 85 (Chen et al. 2009). In contrast to these two species, C. longibracteata Blume is a 86 boreal/temperate species in East Asia and occurs in northeastern China (Liaoning 87 Province and south of Jilin Province), Russian Far East, Korea, and Japan (Chen et al. 88 2009). On the southern part of the Korean Peninsula, C. erecta and C. falcata largely 89 occur in low elevation mountain hills, whereas C. longibracteata occurs in low to mid 90 elevations. The three species often flower synchronously in sympatric populations on 91 the peninsula. Autogamy is the dominant strategy in C. erecta and C. longibracteata, 92 whose white flowers do not fully open, whereas C. falcata, with bright yellow flowers, M. Y. Chung et al. Effect of historical factor on genetic variation in three Cephalanthera species 4
93 is a food-deceptive, predominantly outcrossing species (Tanaka 1965, Suetsugu et al. 94 2015, Ito et al. 2016). 95 A recent review on orchid allozyme based-genetic diversity revealed that most 96 orchid species examined so far exhibit ‘diagnostic’ or unique alleles at several loci 97 (Chung and Chung 2012). This finding suggests that allozyme markers can be useful for 98 delimiting species boundaries and identifying hybrids (e.g. Crawford 1989, Arduino et 99 al. 1996, Harris and Abbott 1997, Hedrén 1996, 2001, Chung et al. 2005, López-Pujol et 100 al. 2012). For example, Arduino et al. (1996) detected 12 out of 25 allozyme loci 101 showing alternative alleles (diagnostic loci) between populations of Orchis laxiflora and 102 of O. palustris in Europe. Similarly, Chung et al. (2005), using 11 polymorphic 103 allozyme loci, identified 22 unique alleles to Liparis makinoana, three unique alleles to 104 L. kumokiri, and found several alleles in putative hybrids that were unique to one or the 105 other parental species in two sympatric populations in central Korea. 106 Given these different breeding systems and, as previously found in populations 107 of the three Cephalanthera species in Europe, we expect that populations of the 108 allogamous species (C. falcata) will harbor considerably higher levels of genetic 109 variation than those of the two autogamous species (C. erecta and C. longibracteata). 110 Since C. erecta and C. longibracteata are obligate autogamous species, we expect no 111 hybridization between Cephalanthera species in sympatry. To test these predictions, we 112 examined genetic diversity (by means of allozymes) of the three species in sympatric 113 populations (600 600-m area) in Yeonwhasan Provincial Park (YPP), located in 114 Gyeongsangnam Province of South Korea, and in non-sympatric populations outside 115 YPP (Fig. 1). 116 117 Material and Methods 118 Plant species 119 Cephalanthera erecta is 20–40 cm high, with 3–10 flowers per inflorescence. In South 120 Korea, C. erecta grows in humus-rich soil under broad-leaved or deciduous pine-oak 121 forests mainly at low elevations in the southeastern corner of the country (M. Y. Chung, 122 and M. G. Chung, personal observations). White flowers bloom during May and June. 123 The first bract length is variable within species (1.5–7.0 cm long; Lee and Kim 1986). 124 The pollinia of C. erecta are in contact with the upper margin of the stigma situated 125 below them (Tanaka 1965). Thus, C. erecta is also highly self-compatible, and 126 autogamy is the dominant mating strategy (M. Y. Chung and M. G. Chung, personal 127 observations; K. Suetsugu, personal observation). High fruit set (92–95%) for C. erecta 128 in dense forest understories may reflect capability for autonomous self-pollination (M. M. Y. Chung et al. Effect of historical factor on genetic variation in three Cephalanthera species 5
129 Y. Chung and M. G. Chung, personal observations). Flowers (without odor) only open 130 partially (from one-third to half; Lee and Kim 1986). The chromosome number is 2n = 131 34 (Lee and Kim 1986). 132 Cephalanthera falcata is 40–70 cm high, with 3–12 bright yellow flowers per 133 raceme that bloom from late April to late June. In South Korea, C. falcata grows 134 sparsely at the edges or on the understory of broadleaved or pine-oak forests at low 135 elevations (M. Y. Chung and M. G. Chung, personal observations). The first bracts 136 (0.2–0.8 cm long; Lee and Kim 1986) are significantly shorter than those of C. erecta 137 and C. longibracteata. As the species is food-deceptive (non-rewarding), flower’s 138 coloration could function in mimicry of specific rewarding plants (Suetsugu et al. 2015). 139 Flowers, which have faint but sweet scent, open fully (Lee and Kim 1986, Suetsugu et 140 al. 2015). Tanaka (1965) suggested the location of the pollinia bounded by the upper 141 margin of stigma to be an adaptation for preventing autonomous attachment to the 142 stigmatic surface of the same flower. Previous pollination experiments conducted in 143 Sanbu City (a warm temperate area of eastern Japan) demonstrated that C. falcata is 144 neither autogamous nor apogamous, but is strongly pollinator (the andrenid bee 145 Andrena aburana: Andrenidae) dependent (Suetsugu et al. 2015, Ito et al. 2016). The 146 chromosome number is 2n = 34 (Lee and Kim 1986, Kitamura et al. 1986). 147 Cephalanthera longibracteata is 30–50 cm tall, with 3–12 flowers per inflorescence. 148 In South Korea, C. longibracteata commonly grows in humus-rich soil under deciduous 149 pine-oak forests at low to mid elevations (M. Y. Chung, and M. G. Chung, personal 150 observations). Like C. erecta, the length of the first bracts is also variable within species 151 (4–12 cm long; Lee and Kim 1986). The relatively small (ca. 1.0 cm long) white 152 flowers bloom in May and June. Like C. erecta, flowers, without odor, do not fully 153 open (from one-third to half; Lee and Kim 1986). In YPP, we only observed a small bee 154 (Lasioglossum sp.: Halictidae) visiting flowers (but the bee did not enter inside the 155 labellum) of C. longibracteata. Like C. erecta, the pollinia of C. longibracteata are in 156 contact with the upper edge of the stigma (Tanaka 1965). Cephalanthera longibracteata 157 is also highly self-compatible and predominantly autogamous (K. Suetsugu, personal 158 observation). We have observed high fruit-set (ca. 98%) in a pollinator-free screened 159 greenhouse (M. Y. Chung and M. G. Chung, unpublished data). The chromosome 160 number is 2n = 32 (Lee and Kim 1986). 161 In southern Korea, C. erecta grows together with C. falcata and C. 162 longibracteata in a few sympatric populations (including Geojae Island, Namhae Island, 163 and YPP), where the density of C. erecta is often low (number of individuals are often < 164 n = 50) compared to the other two species. M. Y. Chung et al. Effect of historical factor on genetic variation in three Cephalanthera species 6
165 166 Studied sites and sampling procedure 167 To survey allozyme variation within and among populations and to screen unique 168 (diagnostic) alleles to each species, we collected 1 cm2 of leaf samples from ‘pure’ 169 populations of each species (that is, from locations where only one species grows). Up 170 to 139 samples were collected from three populations, located at low elevation (< 300 m 171 asl), representing each species (C. erecta, ERE, n = 18; C. falcata, FAL, n = 31; C. 172 longibracteata, LON, n = 90; Fig. 1 and Table 2). 173 To estimate levels of genetic diversity and to determine the presence of 174 interspecific hybridization, we collected samples from all the individuals that were 175 present within a series of small sympatric populations at the landscape level in YPP 176 (600 600-m area [36 ha]; altitude, 230 m asl; Fig. 1 and Table 2). The collected 177 samples varied depending on locations: YPP-1 (C. falcata, n = 50); YPP-2 (C. falcata, n 178 = 49; C. erecta, n =1); YPP-3 (C. falcata, n = 19; C. erecta, n = 20; C. longibracteata, n 179 = 13); and YPP-4 (C. falcata, n = 23; C. longibracteata, n = 14). Although YPP-1 180 consisted only of C. falcata, we included it because of spatial proximity with the other 181 three populations (Fig. 1). To facilitate allele designation and to see allelic variation 182 patterns of other congeners, we included 12 samples of C. longifolia (L.) Fritsch from 183 Ulleung Island (designated as ‘lf’ in the legend of Fig. 2) and 11 samples of C. 184 subaphylla Miyabe & Kudô from YPP (‘s’ in the legend of Fig. 2). 185 186 Starch-gel electrophoresis 187 We transported leaf samples on ice after collection to the laboratory, where we finely 188 cut and crushed them within 24 h with a precooled mortar and pestle in a phosphate 189 polyvinylpyrrolidone extraction buffer (Mitton et al. 1979). We absorbed enzyme 190 extracts onto 4 6-mm wicks cut from Whatman 3 MM chromatography paper 191 (Whatman International, Maidstone, UK), which were then stored at –70°C until needed. 192 We determined allozyme variation via horizontal starch-gel electrophoresis. We 193 prepared 13% gels about 12 hours before gel running. We used a Poulik system (Poulik 194 1957) to resolve alcohol dehydrogenase (Adh; dimer, E.C. 1.1.1.1), cathodal peroxidase 195 (Cpx; monomer, E.C. 1.11.1.7), diaphorase (Dia-1, Dia-2; monomer, E.C. 1.6.99.-), 196 fluorescent esterase (Fe-1, Fe-2; monomer, E.C. 3.1.1.-), leucine aminopeptidase (Lap; 197 monomer, E.C. 3.4.11.1), malic enzyme (Me; dimer, E.C. 1.1.1.40), 198 phosphoglucoisomerase (Pgi-1, Pgi-2; dimer, E.C. 5.3.1.9), phosphoglucomutase (Pgm- 199 1, Pgm-2; monomer, E.C. 5.4.2.2), and triosephosphate isomerase (Tpi-1, Tpi-2; dimer, 200 E.C. 5.3.1.1). We also used the morpholine citrate buffer system (pH 6.1) of Clayton M. Y. Chung et al. Effect of historical factor on genetic variation in three Cephalanthera species 7
201 and Tretiak (1972) to resolve isocitrate dehydrogenase (Idh-1, Idh-2; dimer, E.C. 202 1.1.1.42), malate dehydrogenase (Mdh-1, Mdh-2; dimer, E.C. 1.1.1.37), 6- 203 phosphogluconate dehydrogenase (6Pgd-2; dimer, E.C. 1.1.1.44), and shikimic acid 204 dehydrogenase (Skdh-1, Skdh-2; monomer, E.C. 1.1.1.25). Following Soltis et al. (1983) 205 stain recipes (except for diaphorase; Cheliak and Pitel 1984), we stained starch gels for 206 the 13 enzyme systems, which produced 21 putative loci. We designated putative loci 207 sequentially, with the most anodally migrating isozyme designated ‘1,’ the next ‘2,’ and 208 so on. Likewise, we further designated alleles sequentially with the most anodally 209 migrating allele designated ‘a’, ‘b’, ‘c’, and ‘d’. 210 211 Data analysis 212 Since no allozyme variation was found within each species (see Results), standard 213 genetic diversity parameters and Wright’s F-statistics (Wright 1965) were not used. 214 Instead, we calculated allele frequencies for all populations to determine diagnostic 215 alleles among species. With the raw genotypic data, we estimated genetic divergence 216 among populations/species by calculating Nei’s (1978) genetic identity (I) for all pair of 217 populations using the program POPGENE (Yeh et al. 1999). 218 219 Results 220 Genetic diversity
221 There was no allozyme variation within each species (expected heterozygosity, He, was 222 0) because all the populations belonging to each species were fixed for one allele at 223 each locus. 224 Average Nei’s genetic identities I between orchid species pairs were within the 225 range of values found for many congeneric pairs (mean I = of 0.453 from 190 allozyme- 226 based studies; Chung and Chung 2012): 0.476 for C. erecta vs. C. falcata; 0.429 for C. 227 erecta vs. C. longibracteata; and 0.619 for C. falcata vs. C. longibracteata. 228 229 Allele profiles between the three Cephalanthera species 230 Among 21 putative loci, eight were monomorphic (Dia-2, Idh-1, Idh-2, Me, Pgi-1, Tpi- 231 1, Tpi-2, and Skdh-2) across all three species while the remaining 13 loci were useful for 232 Cephalanthera species delimitation (Table 2). Of the 31 alleles harbored by these 13 233 loci, each of the three alleles at five loci (Lap, 6Pgd-2, Pgi-2, Pgm-2, and Skdh-1) were 234 unique to a single species: Lapb, 6Pgd-2a, Pgi-2a, Pgm-2b, and Skdh-1c for C. erecta; 235 Lapc, 6Pgd-2c, Pgi-2c, Pgm-2c, and Skdh-1a for C. falcata; Lapa, 6Pgd-2b, Pgi-2b, Pgm- 236 2a, and Skdh-1b for C. longibracteata (Table 2). In addition, five alleles (Adhb, Fe-1b, M. Y. Chung et al. Effect of historical factor on genetic variation in three Cephalanthera species 8
237 Fe-2a, Mdh-1a, and Mdh-2b) were unique to C. erecta, one allele (Pgm-1b) to C. falcata, 238 and two alleles (Cpxb and Dia-1b) to C. longibracteata (Table 2). In total, ten, six, and 239 seven alleles were unique to C. erecta, C. falcata, and C. longibracteata, respectively 240 (Table 2). On the other hand, C. erecta and C. falcata shared two alleles (Cpxa and Dia- 241 1a), C. falcata and C. longibracteata five (Adha, Fe-1a, Fe-2b, Mdh-1b, and Mdh-2a), and 242 C. erecta and C. longibracteata only one (Pgm-1a) (Table 2). Since each species had 243 enough unique alleles, we used these alleles to detect putative hybrids in sympatric 244 populations in YPP. We did not find any hybrids in the three populations outside YPP 245 because we did not observe any combination of different alleles at any loci (Table 2). 246 The allelic composition in YPP for the three species was the same as found in the three 247 pure populations of these three species (Table 2). Therefore, although two species co- 248 occur in YPP-2 (15 15-m) and in YPP 4 (10 10-m), and three species co-occur in 249 YPP-3 (25 25-m), each species showed the same allelic composition as that found in 250 the pure populations (Table 2), thus finding no evidence of putative hybrids. 251 252 Discussion 253 Lack of allozyme diversity in Cephalanthera erecta (temperate/warm-temperate 254 species) and Cephalanthera falcata (warm-temperate species): inference of 255 population history 256 The present results do not support our first prediction that populations of C. falcata will 257 harbor substantially greater levels of genetic variation than C. erecta and C. 258 longibracteata. The lack of genetic polymorphism in C. erecta, C. falcata, and C. 259 longibracteata would be attributable to a common trait of the whole genus; in other 260 words, that the genus Cephalanthera originally had low genetic diversity. We strongly 261 believe that this is not the case, because C. longifolia and C. rubra in Europe harbor
262 moderate levels of genetic diversity (HeS = 0.188 for C. longifolia; HeS = 0.125 and 263 0.180 for C. rubra; Table 1). Given this, it would be conservative to ascribe our results 264 to historical factors, which may leave a distinctive genetic signature within and among 265 recently established plant populations (e.g. latitudinal decreases of genetic variation as a 266 consequence of postglacial recolonization processes; for examples in the Japanese 267 Archipelago, see Maki et al. 2008, 2010). On the Korean Peninsula, all the 268 palaeoecological evidences (reviewed in Chung et al. 2017a) suggest that the warm- 269 temperate and subtropical vegetation (typically broad-leaved evergreen/warm mixed 270 forests) would have vanished from the peninsula during the Last Glacial Maximum 271 (LGM). This vegetation, currently forming a narrow strip on the southern and 272 southwestern coast, should be considered, thus, of post-glacial origin, and could have M. Y. Chung et al. Effect of historical factor on genetic variation in three Cephalanthera species 9
273 colonized its current range in Korea through migrations from southern glacial refugia 274 (likely located in southern Japan, southern China, or even in some areas of the exposed 275 East China Sea; e.g. Harrison et al. 2001). Such migrations could frequently take place 276 in orchids, as they produce tiny seeds, which are potentially capable of long-distance 277 dispersal (LDD) by wind or storms (Dressler 1981, Arditti and Ghani 2000, Trapnell 278 and Hamrick 2004, Yukawa et al. 2012, Takashima et al. 2016). 279 The genetic monomorphism detected for C. erecta and C. falcata suggest that 280 contemporary populations would have been established by a single introduction event 281 from an ancestral population, perhaps located in southern Japan or in southern China, 282 with low (or no) genetic variability. A very similar scenario was found in other warm- 283 temperate plant species on the southern part of the Korean Peninsula, including six 284 orchid species (reviewed in Chung et al. 2018), the sundew Drosera peltata Thunb. var. 285 nipponica (Masam.) Ohwi ex E. H. Walker (Chung et al. 2013), and the two terrestrial 286 bladderworts Utricularia bifida L. and U. caerulea L. (Chung et al. 2017b). Alternative 287 scenarios, including the occurrence of multiple introduction events and/or the existence 288 of multiple glacial refugia seem to be unlikely, as the lack of genetic variation at both C. 289 erecta and C. falcata would have required all the propagules and/or all the source 290 populations to be genetically identical and homozygous. 291 How the two terrestrial orchids recolonized to the Korean Peninsula after LGM 292 from their glacial refugia remains a controversial issue, but it may have occurred 293 through LDD. Seeds of most orchids are tiny and dust-like, and feature features that are 294 nown to promote LDD (Dressler 1981, Arditti and Ghani 2000, Trapnell and Hamrick 295 2004, Eriksson and Kainulainen 2011). Given these traits, seeds would have transported 296 from southern Japan or southern China by strong gusts of wind (e.g. typhoons, which 297 are very common in East Asia) from early summer to autumn, the flowering and fruiting 298 time of both C. erecta and C. falcata. 299 300 Lack of allozyme diversity in Cephalanthera longibracteata (temperate/boreal 301 species): inference of population history 302 In contrast to the warm-temperate and subtropical vegetation (which is of post-glacial 303 origin), the boreal and temperate plants probably survived the LGM on the continental 304 Korean Peninsula (Chung et al. 2017a and references therein), although there is no 305 consensus whether species enjoyed large refugial areas (“macrorefugia” sensu Rull 306 2009) or they were confined to small patches (“microrefugia”; Rull 2009). According to 307 Chung et al. (2016, 2017a), the largest and most significant Korean glacial refugia 308 (macrorefugia sensu Rull 2009) would have been located within or near the main M. Y. Chung et al. Effect of historical factor on genetic variation in three Cephalanthera species 10
309 mountain range of the peninsula (the so-called the “Baekdudaegan”, hereafter BDDG). 310 In many cases, this scenario has been demonstrated to be true for many boreal or 311 temperate plants growing on the BDDG and its mountain branches (e.g. the 312 “Nakdongjeongmaek”, hereafter ‘NDJM’), as they harbor high/moderate within- 313 population and low or moderate between-population genetic variation (Chung et al. 314 2017a). In addition, the possibility of altitudinal migration in mountainous areas would 315 have granted the BDDG and the NDJM to harbor large refugial areas. 316 Chung et al. (2004) examined allozyme variation in three populations, all 317 occurring on the BDDG and the NDJM, of the boreal/temperate C. longibracteata and
318 found very low levels of within-population genetic variation (HeP = 0.036; Table 1), 319 with one of the populations having no polymorphism. Considering this, it is not 320 surprising to find that LON, YPP-3, and YPP-4 exhibit no allozyme variation. We argue 321 that populations of C. longibracteata that currently occur at low-elevation areas would 322 have descended from the BDDG (and its branches) to the surrounding lowlands during 323 the LGM. Lowlands would have been more arid than mountains (which were benefited 324 from the orographic precipitation) whereas they also lacked of the sheltered areas of the 325 mountains (e.g. closed valleys, ravines, and gorges); therefore, C. longibracteata would 326 have survived the LGM mainly in lowland small refuge areas (microrefugia), where 327 populations probably suffered from bottlenecks. 328 329 No hybrids between Cephalanthera species in YPP 330 As predicted, we could not find any evidence of putative hybrids both in the three 331 presumed pure populations outside YPP and in the sympatric populations (YPP-2, YPP- 332 3, and YPP-4). Although the flowering phenology of the three Cephalanthera species 333 overlaps (the three species flower from May to June in YPP), flowers of C. 334 longibracteata and C. erecta do not fully open and are predominantly autogamous, 335 whereas the bright yellow flowers of C. falcata open fully (with faint but sweet scent) 336 and are predominantly outcrossing. Potentially different breeding systems between the 337 three Cephalanthera species would be a main factor involved in the maintenance of 338 species boundaries, even in sympatry. 339 To determine whether there is a relationship between fine-scale distribution of 340 congeneric individuals and the absence of hybridization in Cephalanthera, Chung et al. 341 (2017c) conducted bivariate analysis in YPP-3 and YPP-4 and found spatial 342 independence in the interspecific spatial distribution (i.e. they neither detected ‘spatial 343 repulsion’—the expected outcome under the no hybridization scenario—nor ‘spatial 344 attraction’). The authors hypothesized that the random spatial associations among M. Y. Chung et al. Effect of historical factor on genetic variation in three Cephalanthera species 11
345 Cephalanthera species in YPP might be due to the sharing of the same or similar 346 mycorrhizal fungi. 347 348 Conclusions 349 The Korean populations of C. erecta (a warm-temperate/temperate element) and C. 350 falcata (a warm-temperate element) are highly likely of post-glacial origin, given that 351 warm-temperate vegetation was absent from the peninsula during the LGM. The lack of 352 genetic variation in the studied populations suggests that southern Korean populations 353 of C. erecta and C. falcata might have established by a single post-glacial introduction 354 from a genetically depauperate ancestral population. On the other hand, since C. 355 longibracteata is a boreal and temperate element, it probably survived the LGM within 356 the peninsula. The absence of genetic variation might indicate that such survival was in 357 microrefugia instead of macrorefugia, and that such small refugia would have been 358 situated at low elevation regions. The Quaternary climate oscillations would have 359 played, therefore, a major role in determining levels of genetic diversity of the three 360 Cephalanthera species on the Korean Peninsula; the lack of polymorphism would have 361 occurred through founder effects (e.g. post-glacial migration as inferred in C. erecta and 362 C. falcata) and bottlenecks (e.g. survival in microrefugia as in C. longibracteata). 363 364 Acknowledgements – The authors thank M. S. Park for assistance with gel 365 electrophoresis and Hoa Thi Quynh Le for searching references. 366 Funding – This research was supported by a Korea Research Foundation grants; NRF- 367 2013R1A1A2063524 to M.Y. C. and NRF-2013R1A1A3010892 and NRF- 368 2017R1A2B4012215 to M. G. C. 369 M. Y. Chung et al. Effect of historical factor on genetic variation in three Cephalanthera species 12
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487 Table 1. Allozyme-based genetic diversity and genetic differentiation in Cephalanthera 488 species. Species (country Ecol. NPc Genetic parametersd Refe
a b sampled) (R/C) affinity %PS %PP AS AP HeS HeP GST
Cephalanthera erecta WT, T 3 0.0 0.0 1.00 1.00 0.000 0.000 na 1
(South Korea) (R)
C. falcata (South Korea) (R) WT 5 0.0 0.0 1.00 1.00 0.000 0.000 na 1 C. longibracteata B, T 3 0.0 0.0 1.00 1.00 0.000 0.000 na 1
(South Korea) (C)
C. longibracteata B, T 3 30.0 18.0 1.45 1.27 0.097 0.036 0.247 2
(South Korea) (C)
C. subaphylla (South Korea) (R) B, T 2 0.0 0.0 1.00 1.00 0.000 0.000 na 3 C. damasonium (Central Italy) (C) T, WT 13 0.0 0.0 1.00 1.00 0.000 0.000 na 4 C. longifolia (Central Italy) (C) B, T 3 55.6 48.1 1.67 1.59 0.188 0.168 0.104 4 C. rubra (Central Italy) (C) T, WT 7 66.7 33.3 1.67 1.33 0.180 0.127 0.247 4 C. rubra (Northeast Poland) (C) T 9 53.9 13.9 1.54 1.14 0.125 0.059 0.267 5 489 a R/C, rare or common in countries sampled for allozyme studies. 490 b Ecological affinity: B, boreal; T, temperate; WT, warm temperate (or subtropical). 491 c NP, number of populations examined. d 492 %P, percentage of polymorphic loci; A, mean number of alleles per locus; He, genetic 493 diversity. The subscript “S” denotes species’ (or pooled samples) values, while the
494 subscript “P” indicates population means. GST (FST), measures of among-population 495 differentiation; na, not applicable. 496 e Source references: 1. Present study; 2, Chung et al. (2004); 3, Chung et al. (2018); 4, 497 Scacchi et al. (1991); 5, Brzosko and Wróblewska (2013). 498 M. Y. Chung et al. Effect of historical factor on genetic variation in three Cephalanthera species 17
499 Table 2. Allele frequencies at 13 polymorphic loci in three putatively pure populations 500 and in four populations (YPP-1 toYPP-4) in Yeonwhasan Provincial Park (YPP) of the 501 three Cephalanthera species in southern Korea. Boxes identify shared alleles between 502 species. Each of the three alleles at five loci in bold are unique to single species. ERE, 503 C. erecta in Changryeong County; FAL, C. falcata in Buan County; LON, C. 504 longibracteata in Danyang County.
Cephalanthera erecta Cephalanthera falcata Cephalanthera longibracteata
ERE YPP-2 YPP-3 FAL YPP-1 YPP-2 YPP-3 YPP-4 LON YPP-3 YPP-4
Locus Allele (n = 18) (n = 1) (n = 20) (n = 31) (n = 50) (n = 49) (n = 19) (n = 23) (n = 90) (n = 13) (n = 14)
Adh a 0 0 0 1 1 1 1 1 1 1 1
b 1 1 1 0 0 0 0 0 0 0 0
Cpx a 1 1 1 1 1 1 1 1 0 0 0
b 0 0 0 0 0 0 0 0 1 1 1
Dia-1 a 1 1 1 1 1 1 1 1 0 0 0
b 0 0 0 0 0 0 0 0 1 1 1
Fe-1 a 0 0 0 1 1 1 1 1 1 1 1
b 1 1 1 0 0 0 0 0 0 0 0
Fe-2 a 1 1 1 0 0 0 0 0 0 0 0
b 0 0 0 1 1 1 1 1 1 1 1
Lap a 1 1 1 0 0 0 0 0 0 0 0
b 0 0 0 0 0 0 0 0 1 1 1
c 0 0 0 1 1 1 1 1 0 0 0
Mdh-1 a 1 1 1 0 0 0 0 0 0 0 0
b 0 0 0 1 1 1 1 1 1 1 1
Mdh-2 a 0 0 0 1 1 1 1 1 1 1 1
b 1 1 1 0 0 0 0 0 0 0 0
6Pgd-2 b 1 1 1 0 0 0 0 0 0 0 0
c 0 0 0 0 0 0 0 0 1 1 1
d 0 0 0 1 1 1 1 1 0 0 0
Pgi-2 a 1 1 1 0 0 0 0 0 0 0 0
b 0 0 0 0 0 0 0 0 1 1 1
d 0 0 0 1 1 1 1 1 0 0 0 505 Table 2. Continued.
Cephalanthera erecta Cephalanthera falcata Cephalanthera longibracteata
ERE YPP-2 YPP-3 FAL YPP-1 YPP-2 YPP-3 YPP-4 LON YPP-3 YPP-4 M. Y. Chung et al. Effect of historical factor on genetic variation in three Cephalanthera species 18
Locus Allele (n = 18) (n = 1) (n = 20) (n = 31) (n = 50) (n = 49) (n = 19) (n = 23) (n = 90) (n = 13) (n = 14)
Pgm-1 a 1 1 1 0 0 0 0 0 1 1 1
b 0 0 0 1 1 1 1 1 0 0 0
Pgm-2 a 0 0 0 0 0 0 0 0 1 1 1
b 1 1 1 0 0 0 0 0 0 0 0
c 0 0 0 1 1 1 1 1 0 0 0
Skdh-1 a 0 0 0 1 1 1 1 1 0 0 0
b 0 0 0 0 0 0 0 0 1 1 1
c 1 1 1 0 0 0 0 0 0 0 0 506 507 M. Y. Chung et al. Effect of historical factor on genetic variation in three Cephalanthera species 19
508 Figure legends 509 510 Figure 1. Collection sites of Cephalanthera erecta (ERE), C. falcata (FAL), and C. 511 longibracteata (LON); ERE, FAL, and LON were collected from Changryeong County, 512 Buan County, and Danyang County, respectively. Relative locations of four populations 513 of Cephalanthera species (YPP-1 toYPP-4) within a 600 600-m area (36 ha). 514 515 Figure 2. Representative zymograms for the three Cephalanthera species studied herein, 516 and two congeneric species that were included to facilitate genotype scoring; C. erecta 517 (e), C. falcata (f), C. longibracteata (lb), C. longifolia (lf), and C. subaphylla (s). Arrow 518 designates the standard (‘ST’, one individual from C. falcata, except Fe-2 at which 519 Epipactis thunbergii was used). Note the very faint ST at Adh, Dia-1, and 6Pgd-2. All 520 samples were from YPP-3, except Fe-2 (C. erecta from ERE and C. falcata from YPP- 521 1) and Skdh-1 (C. erecta from ERE and C. falcata from FAL; Table 2) (A) Adh, allele 522 ‘a’ at the lanes 1–6 (lf), 7–10 (lb), 19–22 (s), 23–26 (f) and allele ‘b’ at 11, 12 and 14– 523 18 (e); (B) Cpx, allele ‘a’ at the lanes 5–11 and 16, 17 (lf), 19–22 (f) and allele ‘b’ at 1– 524 4 (lb), 12, 14, 15, 18 (e), 23–26 (s); (C) Dia-1, allele ‘a’ at the lanes 1–6 (f), 24–26 (e) 525 and allele ‘b’ at 7–12, 14–23 (lb); (D) Fe-2, allele ‘a’ at the lanes 9–11 (e) and allele ‘b’ 526 at 1–8, 12, 14–26 (f); (E) Lap, allele ‘a’ at the lanes 11–12, 14–18 (e), allele ‘b’ at 1–6 527 (lf), 7–10 (lb), and allele ‘c’ at 19–22 (s), 23–26 (f); (F) Mdh-1, allele ‘a’ at the lanes 12, 528 14, 15, 18 (e), allele ‘b’ at 1–4 (lb), 19–22 (f), and allele ‘c’ at 5–11, 16, 17 (lf), 23–26 529 (s); (G) 6Pgd-2, allele ‘a’ at lanes 5–7 (s), 20–26 (lf), allele ‘b’ at 8–13 (e), allele ‘c’ at 530 15–19 (lb), and allele ‘d’ at 1–4 (f); (H) Pgi-2, allele ‘a’ at the lanes 14–19 (e), allele ‘b’ 531 at 8–12 (lb), allele ‘c’ at 20–22 (s), and allele ‘d’ at 1–7 (lf), 23–26 (f); (I) Pgm-1, allele 532 ‘a’ at the lanes 7–10 (lb), 11, 12, 14–18 (e), 19–22 (s) and allele ‘b’ at 1–6 (lf), 23–26 533 (f); (J) Skdh-1, allele ‘a’ at the lanes 1–7 (f), 11, 12, 14–17 (f), 25, 26 (f) and allele ‘b’ at 534 8–10 (e), 18–24 (e). 535 M. Y. Chung et al. Effect of historical factor on genetic variation in three Cephalanthera species 20
536
537 538 539 540 Figure 1 541 M. Y. Chung et al. Effect of historical factor on genetic variation in three Cephalanthera species 21
542
543