Effect of Historical Factors on Genetic Variation in the Three Terrestrial Orchids Cephalanthera Erecta, Cephalanthera Falcata
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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.