Introduction

Mangroves form the most unique and conspicuous wetland ecosystem worldwide with an approximate area of 240, 000 km2 of sheltered coastlines. It servers as a protective barriers along the coast and also provides safe sties for many organism in costal-mangrove ecosystem. But for a last couple of decades, heavy exploitation of mangroves for excessive wood gathering, fishpond operations, mining, and development of coastal areas make a great threat to the world's mangroves. Conservation and ecological management of mangroves, therefore, now become the most important issues in the coastal areas of many countries.

　Bruguiera gymnorrhiza (L.)　Lamk, Kandelia candel (L.) Druce and Rhizophora stylosa (Griff.) are ecologically and somewhat economically important mangrove tree species of family Rhizophoraceae. In Japan, these three species are mainly found in the mangrove forests mostly distributed in the Ryukyu archipelago islands. Iriomote Island is an ecologically diverse one in the Ryukyu archipelago. About 73% of the total mangrove forests in Japan are distributed in this Island where B. gymnorrhiza, R. stylosa and K. candel are the most extensive and dominant tree species.

　Information about the genetic structure and reproduction characteristics of these three mangrove trees in Ryukyu archipelago islands can be inferred by investigating the populations in Iriomote Island. Precise analysis of genetic diversity, gene flow, genetic structure, maternal lineage and reproduction characteristics could provide important information for finding appropriate strategies for the conservation and ecological management of the whole coastal-mangrove ecosystem.

　The use of molecular data in plant ecological studies has increased dramatically during the last decade since molecular methods are now widely recognized as important tools for identifying appropriate population sources. Recently, microsatellites from nuclear DNA have been widely used in testing relatedness between　individuals, detecting population genetic structure, and assessing genetic diversity and recentpopulation history. Moreover, some　chloroplast DNA microsatellite regions were also found to be polymorphic and useful for inference of maternal lineage and seed dispersal manner.

　In this study, genetic diversity population genetic structure, and reproduction characteristics of three mangrove tree species, i.e. B. gymnorrhiza, K. candel and R. stylosa in their natural habitat have been investigated using polymorphic nuclear and chloroplast microsatellite markers.

1. Development and characterization of nuclear microsatellite markers in Bruguiera gymnorrhiza, Kandelia candel and Rhizophora stylsoa

Ten polymorphic　microsatellite markers were isolated from B. gymnorrhiza using a dual-suppression polymerase chain reaction (PCR) technique. These markers showed polymorphism of two to five alleles per locus among 216 individuals from nine B. gymnorrhiza natural populations in Iriomote Island. From K. candel, eight codominant compound microsatellite markers were isolated. The isolated compound SSR markers had polymorphism of three to 11 alleles per locus among 24 individuals in natural mangrove forests located in four provinces in the southern part of China. From R. stylosa, total 10 polymorphic microsatellite markers were isolated using a dual-suppression PCR technique. These markers had polymorphism of two to six alleles in each locus within 375 individuals from 19 natural populations of R. stylosa on three of the Sakishima Islands: Iriomote, Ishigaki and Miyako.

　2. Population genetic structure of Bruguiera gymnorrhiza, Kandelia candel and Rhizophora stylosa in Iriomote Island, Japan, revealed by nuclear microsatellite markers-In this study, 10, 10 and 9 polymorphic microsatellite markers of B. gymnorrhiza, R. stylosa and K candel, respectively, were employed to assess genetic diversity, population genetic structure and gene flow in the whole Iriomote Island. Nine populations from each of B. gymnorrhiza and R. stylosa and seven populations in K candel were investigated in the present study. The nuclear SSR analysis of natural populations in Iriomote Island showed that the average number of alleles (mean, 2.6, 1.7, 2.3 for B. gymnorrhiza, K candel and R. stylosa, respectively), allelic richness (mean, 2.6, 1.7, 2.3) and expected heterozygosity (mean, 0.408, 0.252, 0.266) per locus per population were low in all three species. Mean inbreeding coefficient (Fis) per locus within the natural populations of B. gymnorrhiza, K. candel and R. stylosa was 0.233, 0.506 and 0.576, respectively, indicating high inbreeding within the populations.

　Significant genetic differentiations among the natural populations of all three species were identified from the analysis of molecular variances and overall F(ST). Low migration number (Nm) values, i.e. 2.235, 0.717 and 1.238, were detected, in B. gymnorrhiza, K. candel and R. stylosa respectively. These results indicated low gene flow, i.e. limited pollen and propagule dispersal among the populations in all three species. Pairwise genetic differentiations　(F(ST)/(1-F(ST))) were not significantly correlated with geographic distances in B. gymnorrhiza and K candel, but in R. stylosa. Since the correlation means the presence of some gene flow between populations, there might be relatively larger gene flow in R. stylosa than that found in other two species.

　3. Development of chloroplast microsatellite (cpSSR) markers for maternal lineage analysis in Bruguiera gymnorrhiza, Kandelia candel and Rhizophora stylosa.

Chloroplast microsatellite markers were developed from B. gymnorrhiza, K. candel and R. stylosa, respectively. Chloroplast DNA fragments of each species were amplified by PCR using universal chloroplast primer pairs. From the noncoding regions, 11, 11 and eight cpSSR markers in B. gymnorrhiza, K candel and R. stylosa were isolated, respectively. When individuals from different natural populations in Iriomote Island were analysed by these markers, 6, 2 and 3 markers in B. gymnorrhiza, K. candel and R. stylosa showed polymorphism, respectively.

　4. Genetic structure and maternal lineages of Bruguiera gymnorrhiza, Kandelia candel and Rhizophora stylosa in Iriomote Island, Japan revealed by chloroplast microsatellite markers

The cpSSR analysis also showed low genetic diversity within the natural populations of all three dominant mangrove species in Iriomote Island. The number of alleles at each locus ranged from two to four in B. gymnorrhiza. In K. candel and R. stylosa, only two alleles at each locus were identified throughout the populations. Haplotypes of cpSSR, i.e., combinations of alleles at different polymorphic cpSSR loci, were five, three and two in B. gymnorrhiza, K. candel and R. stylosa, respectively. Although five haplotypes and three haplotypes in B. gymnorrhiza and K candel were detected, dominant two types for each species were distributed within the populations throughout the entire island. These results indicated that populations of these three mangrove trees in Iriomote Island may be originated from only a few founders.

　The overall G(ST) values assessed by cpDNA were estimated 0.310, 0.446 and 0.257 in B. gymnorrhiza, K. candel and R. stylosa, respectively, indicating high genetic differentiations among populations. Significant correlation between cpDNA genetic distances and geographic distances was observed in R. stylosa but not in B. gymnorrhiza and K candel. Since chloroplast DNA of the mangrove species may be maternally inherited, the result indicated that some gene flow via propagule dispersal may be present in R. stylosa.

　5. Fine scale genetic structure and reproduction characteristics in Bruguiera gymnorrhiza, Kandelia candel and Rhizophora stylosa in Iriomote Island, Japan

The study was conducted in a natural mangrove plot (200 x 120 m) on the shore at the river mouth of the river Urauchi, Iriomote Island. The positions of all adult trees of B. gymnorrhiza (147), K candel (242), and R. stylosa (192), and sampled seedlings of B. gymnorrhiza (44), K candel (190), and R. stylosa (188) within the plot were measured and leaf samples were collected from all of them. The propagules from three mother trees of each species were also collected. Both nuclear SSR and cpSSR markers were used to identify the genotypes of all samples.

　Low genetic diversities within the adult tree cohorts and seedling cohorts for all the species within the plot were detected in nuclear microsatellite analysis. The average number of alleles within the adult tree cohorts and seedling cohorts were 3.2 and 2.8; 2.6 and 2.3; and, 2.8 and 2.4 in B. gymnorrhiza, K. candel and R. stylosa. Combination of alleles at polymorphic cpSSR loci constituted only two haplotypes within the adult tree and seedling cohorts for each species.

　The high inbreeding rates in the three species were confirmed. The mean F(IS) values within the adult tree and seedling cohorts were 0.216 and 0.321, 0.261 and 0.333, and 0.621 and 0.633 in B. gymnorrhiza, K. candel and R. stylosa, respectively. The paternal analysis of propagules from mother trees showed that the high outcrossing could occur in B. gymnorrhiza. Both pollen and propagule dispersal patterns within the plot were assumed from the spatial distributions of rare alleles at nuclear SSR loci and cpSSR haplotypes. The seedlings from the propagules migrating from outside of the plot were observed. Moreover, seedlings under a mother tree were not always the offspring of the corresponding mother tree. The results indicated that propagule dispersals occurred within the plot and some propagules could be dispersed by relatively long distance.

　From the analysis of both nuclear and chloroplast microsatellite markers, it can be concluded that genetic diversity of three dominant mangrove species in Iriomote Island is very low and each species may be regenerated in Iriomote Island mainly from two different mother types, probably after the last seismic uplift occurred ca. 1000 years. After initial establishment, there may be a little gene flow between populations in different watersheds. The populations in each watershed of Iriomote Island may be reproduced by high inbreeding. The high genetic differentiation among populations in different watersheds indicates that each population contains different genetic composition and is considered as a unique unit for conservation and management of mangrove forests in Iriomote Island. Moreover, populations that have the　significant distinct haplotypes need to be carefully protected. In my knowledge, this is the first report of microsatellite analysis of mangrove tree populations on a scale of a whole Island.

　マングローブ林は、熱帯、亜熱帯地域の河口を中心とした沿岸域に形成されている。マングローブ林は、沿岸部を覆うように分布しているため、高潮、津波など、海からの災害や土壌浸食などを防ぐ護岸的役割を果たしている。一方で、とりわけバングラデシュのような発展途上国では、隣接する村落の住民の食糧を始め生活に必要な生物資源の供給源として、重要な役割を果たしている。

　マングローブ林は、世界でおよそ24万km2あるが、近年、養殖場の設置や近代化の波にもまれて、その面積は、年々少なくなっている。マングローブ林の保全は、世界的な環境保全の課題の一つとなっている。こうした背景のもとで、本研究では、マングローブ林保全の基礎となる、構成樹木の繁殖特性を、西表島を試験地として、分子生態学的なアプローチによって明らかにした。

　序論では、本研究の目的と背景を述べ、合わせて、マングローブ林および西表島のマングローブ林優占樹種であるオヒルギ、メヒルギ、ヤエヤマヒルギの三種の特徴を述べている。

　第1章では、分子系統学的解析の前提となるマイクロサテライトマーカーの作成方法と、得られたマーカーの特徴を記述している。オヒルギで10、メヒルギで8、ヤエヤマヒルギでは10の多型性マーカーを作製した。

　第2章では、第1章で作製したマイクロサテライトマーカーを用いて、西表島の9河川流域の個体群から葉を採集し、多型解析を行った。その結果、三種とも、流域毎に遺伝的に分化していることがわかった。これは、各流域間では、花粉散布や種子散布を通しての遺伝的交流が、極めて少ないことを示している。加えて、これら三種の個体群では、近親交配の比率が高いことも推定された。

　第3章では、母系を通して種子散布を考察するために、母性遺伝すると考えられる葉緑体遺伝子を用いて、多型マーカーを作製した。

　第4章では、第3章で作製した葉緑体SSRマーカーを用いて、西表島の9流域について、母系解析を行った。その結果、第3章と同じように、三種とも、流域毎に遺伝的に分化していることがわかった。これは、各流域間では、種子散布を通しての遺伝的交流が、極めて少ないことを示している。

　第5章では、より細かいスケールでの繁殖特性を把握するために、1ヘクタール程度の試験地を設定し、成木、実生、母樹についている繁殖体のSSR多型解析を行った。その結果、母樹の下に定着している実生が必ずしもその母樹由来ではないこと、言い換えると母樹から離れた繁殖体の移動範囲はかなり広いことがわかった。

　総合考察では、上記のマングローブ樹種の繁殖特性をまとめ、マングローブ林の保全に関する考察を述べている。

　以上より、本研究は、西表島のマングローブで優占する、オヒルギ、メヒルギ、ヤエヤマヒルギの三樹種に関し、それらの繁殖特性を分子生態学的解析法により明らかにした。マングローブ樹種に関しては、世界的分布範囲の中で数地点を調べた、広範囲なスケールでの遺伝地理学的研究はあるが、一つの島の中、あるいは流域間、さらには個体群内といった、比較的小さなスケールでのマングローブ林の分子生態学的解析は皆無であり、本研究の先駆的意義は大きい。また、本研究によって得られたマングローブ樹種の繁殖特性に関する情報は、マングローブ林保全を考える上で、極めて重要なものといえよう。したがって、本研究は、学術上、応用上貢献するところが大きく、審査委員一同は本論文が博士(農学)の学位論文として価値あるものと認めた。