Kobe University Repository : Thesis Hybrid origin of potamogeton anguillanus and its genetic and 学位論文題目 ecological diversification(オオササエビモの雑種起源と遺伝的および生 Title 態的多様化) 氏名 飯田, 聡子 Author 専攻分野 博士（理学） Degree 学位授与の日付 2003-03-31 Date of Degree 資源タイプ Thesis or Dissertation / 学位論文 Resource Type 報告番号 甲2880 Report Number 権利 Rights JaLCDOI URL http://www.lib.kobe-u.ac.jp/handle_kernel/D1002880 ※当コンテンツは神戸大学の学術成果です。無断複製・不正使用等を禁じます。著作権法で認められている範囲内で、適切にご利用ください。 PDF issue: 2021-10-05 博 士 論 文 Hybrid origin of Potamogeton anguillanus and its genetic and ecological diversification オオササエビモの雑種起源と遺伝的および生態的多様化 平成 15 年 2 月 神戸大学大学院自然科学研究科 飯 田 聡 子 Contents Pages Summary 1 - 4 Chapter One: Molecular phylogeny and evolution of heterophylly in Japanese Potamogeton 5 - 16 Chapter Two: Origin of genetic diversity of Potamogeton anguillanus, a putative hybrid between P. mal aianus and P. perfoliatus in Lake Biwa, Japan 17 - 37 Chapter Three: Population genetic structure of Potamogeton anguillanus in Lake Shnji, Japan 38 - 42 Chapter Four: Ecological and genetic differentiations in Potamogeton anguillanus with special reference to phenotypic plasticity 43 - 55 Acknowlegements 55 - 56 References 57 - 69 19 Tables 24 Figures 2 Appendixes SUMMARY The genus Potamogeton (Potamogetonaceae) is one of the representative aquatic macrophytes consisting of over 80 species worldwide and 19 species in Japan. The genus Potamogeton shows the remarkable diversification of its morphology and ecology. In the first chapter, molecular phylogenetic analyses of inter-genic spacer region of chloroplast DNA were carried out to examine the relationships among Japanese Potamogeton species. Phylogenetic analyses revealed that Japanese Potamogeton was divided into the species of subgenus Potamogeton and subgenus Coleogeton (Potamogeton pectinatus). Three distinct groups were further recognized within the subgenus Potamogeton, Group A: P. distinctus, P. malaianus, P. perfoliatus, P. gramineus, P. dentatus, P. macckianus, P. crispus and P. alpinus, Group B: P. natans, P. fryeri and P. praelongus and Group C: P. pusillus, P. panormitanus, P. oxyphyllus, P. obtusifolius, P. compressus, P. octandrus and P. cristatus. Species with different growth forms, namely floating-leaved form and submerged form, belonged to each of the three groups and floating–leaved form has evolved in parallel in different lineages of subgenus Potamogeton. In Group A, both diploid and polyploid species were included. The species in Group B were only polyploids and those in Group C were only diploids. - 1 - Either genetic changes of the small number of genes related to the development of floating-leaved form or the combination of diploid genome with large plasticity followed by polyploidization may have the relationship with the observed differentiation in growth form. Inter-specific hybridization occurred frequently in Potamogeton and over 200 hybrids have been reported so far. Potamogeton anguillanus is one of the presumed hybrids between P. malaianus and P. perfoliatus based on its low seed setting and the intermediate external morphology. In Chapters Two and Three, genetic markers of the nuclear and chloroplast genome were characterized for P. malaianus and P. perfoliatus and the origin of P. anguillanus was examined. Analyses of nuclear DNA by allozyme electrophoresis revealed that different alleles at four loci characterized P. malaianus and P. perfoliatus. Potamogeton anguillanus possessed both characteristic alleles of P. malaianus and P. perfoliatus, respectively. Among 28 genotypes in P. anguillanus, fifteen were genotypes expected in F1 hybrids between P. malaianus and P. perfoliatus (F1 type) and they were concluded to be F1 hybrids. The remaining 13 genotypes were either genotype that could have resulted from segregation of parental alleles in later generation hybrids (segregated type) or genotypes that include allele unique to P. anguillanus - 2 - (orphan type). Analyses of chloroplast DNA clarified the nucleotide substitutions to distinguish P. malaianus from P. perfoliatus in inter-genic spacer region between coding gene for tRNA-Thr (UGU) (trn T) and tRNA-Leu (UAA) (trn L) 5’exon. Both chloroplast haplotypes identical to P. malaianus and to P. perfoliatus were found in P. anguillanus. In conclusion, the allozyme analyses support the view that P. anguillanus has been originated by inter-specific hybridization between P. malaianus and P. perfoliatus. The genetic diversity of P. anguillanus in Lake Biwa is considered to have originated through multiple and reciprocal hybridizations between P. malaianus and P. perfoliatus and maintained through hybrid segregation and vegetative reproduction in the later generations. The closely related P. malaianus and P. perfoliatus show the contrasting plasticity in growth form. When the water level decreases, the growth form of P. malaianus changes from submerged to floating-leaved and further to terrestrial forms, but not in P. perfoliatus. In Chapter Four, the plasticity in growth form has been examined in P. anguillanus, which is inter-specific hybrid between P. malaianus and P. perfoliatus. The degree of plasticity for developing terrestrial shoots (% of number of shoots produced under - 3 - terrestrial condition / number of shoots produced under submerged condition), an indicator of the degree of plasticity, were 0% in P. perfoliatus, 0% - 69% in P. anguillanus and 36% - 150 % in P. malaianus. The terrestrial leaves of P. malaianus and P. anguillanus were differentiated from submerged leaves in external morphology and in anatomical structure. In P. anguillanus, the degree of plasticity for developing terrestrial shoots tended to be higher in strains with the chloroplast haplotype identical to P. malaianus than strains with the chloroplast haplotype identical to P. perfoliatus. Different combination of genome inherited from parental species may be resulted in the ecological differentiation as expressed in phenotypic plasticity in P. anguillanus. The chloroplast haplotype of P. malaianus can easily be distinguished from that of P. perfoliatus. Analyses of gene coding large subunit of ribulose-bisphosphate carboxylase / oxygenase (rbcL) revealed amino acid substitutions at the position which was one of structurally important positions in this enzyme. As ribulose-bisphosphate carboxylase/oxygenase is the photosynthetic enzyme, the different chloroplast haplotype of P. anguillanus may be related to the differentiation in the degree phenotypic plasticity for the tolerance to terrestrial condition. This is a future subject to be studied. - 4 - CHAPTER ONE MOLECULAR PHYLOGENY AND EVOLUTION OF HETEROPHYLLY IN JAPANESE POTAMOGETON Introduction Potamogeton (Potamogetonaceae) is an aquatic plant and distributes in various kinds of waters, such as rivers, freshwater and brackish lakes, marshes, and artificial ponds. So far 80 to 90 species have been described worldwide (Wiegleb, 1988). Potamogetonaceae consists of two genera, Groenlandia and Potamogeton. The genus Ruppia has sometimes been included in Potamogetonaceae but Les et al. (1997) has recently clarified that this genus is closely related to Cymodoceaceae, one of the lineages of seagrasses. In Potamogetonaceae, Groenlandia densa (L.) Fourr. is a monotypic species in the genus Groenlandia and is clearly distinguished from Potamogeton species by basic chromosome number, leaf arrangement (Ascherson and Graebner, 1907), number of flowers in an inflorescence - 5 - (Guo and Cook, 1990) and pollen morphology (Sorsa, 1988). Potamogeton pectinatus and its allied species are also distinguished from the remaining species in the genus Potamogeton. Thus they have sometimes been treated as either separate subgenera or genera (e.g. Holub, 1997; Les and Haynes, 1996). Although most of students hold the same view for the phylogenetic relationships at generic/subgeneric level, the lower level taxonomy within the subgenus Potamogeton is rather confusing. Species in Potamogeton are characterized by a high degree of variability of many characters. Many small species groups corresponding to sections/subsections have been proposed by a number of researchers (Ascherson and Graebner, 1907; Hagstrom, 1916 etc.). Two contrasting growth forms, i.e., floating-leaved and submerged forms, were recognized in Potamogeton and have conveniently been used to discriminate Potamogeton species. Variation in characters other than external morphology has also been reported such as anatomical characters (Raunkiær, 1903; Hagström, 1916; Miki, 1937; Ogden, 1943), chromosomes numbers (reviewed in Hollingsworth et al., 1998), seed morphology (Jessen, 1955; Aalto, 1970), pollen morphology (Sorsa, 1988), flavonoids (Les and Sheridan, 1990) and allozymes (Hettiarachchi and Triest, 1991). Based on these characters, 15 species groups have been recognized in the genus - 6 - Potamogeton and these groups show a complex pattern of mutual similarity (Wiegleb, 1988). Then, the exact pictures of species relationships in Potamogeton cannot be given, so far. Molecular analyses have clarified the phylogenetic relationships in land plants at higher taxonomic levels (e.g. Chase, et al., 1993). However, the resolution of analyses has not been high enough to clarify the relationship at lower taxonomic level. The intra-genic relationships using the nucleotide sequences of non-coding regions of chroloplast DNA has been recently revealed in several plant groups (Taberlet et al., 1991; Small et al., 1998). In this chapter, the comparison of nucleotide sequences in this spacer region