Mangrove forests form conspicuous wetland ecosystems fringing extensive areas of coastlines of tropical and subtropical regions. Approximately 80 species from twenty plant families have been recognized, primarily consisting of trees and shrubs that normally grow above mean sea level in the intertidal zone of marine coastal environments or estuarine margins. Mangrove forests are extremely productive ecosystems that provide numerous good and services both to the marine environment and people including fisheries, timber and plant products, coastal protection, and tourism. Despite their ecological and economic importance, mangroves are being destroyed worldwide at an alarming rate due to a variety of destructive human practices, such as farmland conversion, embankment for aquaculture ponds, overharvesting for firewood and charcoal production, and recently rapid urban developments. The exploitation of mangroves has resulted in the loss of genetic diversity in mangrove ecosystems, as well as the loss of valuable forest resources.

In China, twenty-six (including one variety) mangrove species representing 15 genera and 12 families were recorded. Mangrove forests, occupying approximately 17,885 ha, naturally occur along the southeast coastline of four provinces: Hainan, Guangdong, Guangxi, and Fujian in addition to Hong Kong SAR, Macau SAR and Taiwan, intermittently extending from 18°N to 27°N. Nearly two-thirds of mangroves have been lost during the past 40 years. More than 80% of mangroves are secondary forests with 1-m-high trees and most populations are fragmented and scattered along the southeast coastline of China. Nowadays, the high priority of governments in China has been given to the conservation and sustainable management of mangrove forests. A total of 25 mangrove nature reserves including seven at the national level and six at the provincial level have been designated since 1980s, of which three belong to Ramsar international important wetlands. Surveys of genetic diversity of natural populations and mating system of mangrove species are necessary for effective mangrove forest management.

Seven mangrove species, Acanthus ilicifolius (Acanthaceae), Aegiceras corniculatum (Myrsinaceae), Avicennia marina (Avicenniaceae), Bruguiera gymnorrhiza (Rhizophoraceae), Kandelia candel (Rhizophoraceae), Lumnitzera racemosa (Combretaceae), and Rhizophora stylosa (Rhizophoraceae) are dominantly distributed in the coastline of South China. In the present study, the genetic diversity and population genetic structure of these seven mangrove species in South China were estimated by polymorphic nuclear SSR and cpSSR markers. Moreover, the mating system, pollen and propagule dispersals and spatial genetic structure of K. candel in a natural population were also analyzed to reveal its reproduction characteristics.

1.Isolation and characterization of nuclear and chloroplast microsatellite markers in four mangrove species, Acanthus ilicifolius, Aegiceras corniculatum, Avicennia marina and Lumnitzera racemosa

I employed two techniques to isolate nuclear microsatellite regions. One is dual-suppression polymerase chain reaction (PCR) technique and the other was an improved technique for isolating codominant compound microsatellite markers. For A. ilicifolius and A. corniculatum, both two techniques were used and for A. marina and L. racemosa, only the improved technique was used. In A. ilicifolius, eight polymorphic nuclear SSR markers were developed. These markers showed polymorphism of two to eight alleles per locus, the observed and expected heterozygosities of these markers ranged from 0.200 to 0.875 and from 0.227 to 0.798, respectively. In A. corniculatum, 18 polymorphic nuclear SSR loci were isolated. These loci provided nuclear SSR markers with polymorphism of two to eight alleles per locus. The observed and expected heterozygosities ranged from 0.050 to 0.550 and from 0.097 to 0.736, respectively. From A. marina, 10 polymorphic nuclear SSR loci were isolated. These isolated loci provided compound SSR markers with polymorphism of two to six alleles per locus. The observed and expected heterozygosities ranged from 0.025 to 0.625 and from 0.096 to 0.767, respectively. From L. racemosa, nine polymorphic SSR loci were isolated. The number of alleles per locus ranged from two to nine. The observed and expected heterozygosities ranged from 0.025 to 0.350 and from 0.073 to 0.792, respectively.

Chloroplast microsatellite (cpSSR) markers were developed for the four mangrove species by two methods. First, the cpSSR regions were isolated by sequencing the non-coding regions of chloroplast DNA (cpDNA). Second, the six universal cpSSR loci (ccmp2, ccmp3, ccmp5, ccmp6, ccmp7 and ccmp10) were also used to screen their polymorphism in four species. One, three, seven and six polymorphic cpSSR loci in A. ilicifolius, A. corniculatum, A. marina, and L. racemosa, respectively were developed. Characterization of 229, 509, 369, and 216 individuals of A. ilicifolius, A. corniculatum, A. marina, and L. racemosa, collected from different natural mangrove populations (A. ilicifolius, 6; A. corniculatum, 14; A. marina, 10; L. racemosa, 6) in South China showed that these loci provide cpSSR markers with polymorphisms ranging from two to four alleles per locus and gene diversity between 0.005 and 0.675. Combining these cpSSR loci, three, five, 11 and four cpSSR haplotypes were detected in populations of A. ilicifolius, A. corniculatum, A. marina, and L. racemosa in the southern coastline of China, respectively.

2. Genetic diversity and differentiation of seven dominant mangrove species in southern Chinese populations revealed by nuclear microsatellite markers

In this study, 6, 10, 11, 8, 10, 9 and 10 polymorphic SSR markers of A. ilicifolius, A. corniculatum, A. marina, B. gymnorrhiza, K. candel, L. racemosa and R. stylosa, respectively, were employed to estimate wide range of genetic diversity, gene flow and population genetic structure. 6, 14, 10, 9, 10, 6, and 15 populations of A. ilicifolius, A. corniculatum, A. marina, B. gymnorrhiza, K. candel, L. racemosa and R. stylosa in the coastlines of the South China were investigated in present study. For each species in the seven mangroves, number of alleles and allelic richness per locus per population ranged from 1.8 to 7.2 and 1.6 to 6.3, respectively. The observed (HO) and expected (HE) heterozygosity and inbreeding coefficient (FIS) had a mean value ranging from 0.088 to 0.544, 0.160 to 0.587, and 0.104 to 0.637, respectively. The genetic diversity was lowest in the R. stylosa but highest in the K. candel populations. These results indicate there are significant differences of genetic diversities among those seven mangrove species and most of the mangrove populations in coastlines of the South China may be reproduced by high inbreeding.

Significant genetic differentiations between the natural populations of all of these seven species were identified from the analysis. Overall genetic differentiation (FST) for populations of each species ranged from 0.165 to 0.629, of which the highest FST was detected in A. ilicifolius and the lowest in B. gymnorrhiza. The hierarchical AMOVA showed that most of genetic variations were found among populations in A. ilicifolius (62.9%), L. racemosa (50.7%) and R. stylosa (58.0%) but the other four species within populations (A. corniculatum, 72.9%; A. marina, 75.1%; B. gymnorrhiza, 83.5%; K. candel, 79.9%). The population genetic structure also showed significant genetic differentiation between populations of each seven mangrove species. These results indicated that limited gene flow via pollen and propagate dispersal between populations may occur between studied populations of seven mangrove species in South China. Furthermore, in six mangrove species except A. corniculatum (P<0.001; Mantel test), no significant relationship between geographical and genetic distance (FST/(1- FST)) was observed (P=0.10 to 0.65; Mantel test). The results indicate that there might be larger gene flow in A. corniculatum than in the other six species.

3.Diversity and genetic structure in populations of seven dominant mangrove species in southern China as revealed by cpSSR analysis

Total seven, five, 13, 11, eight, 16, 8 cpSSR markers were used in the populations of A. ilicifolius, A. corniculatum, A. marina, B. gymnorrhiza, K. candel, L. racemosa, and R. stylosa, respectively. The cpSSR analysis showed that genetic diversity was also low within natural populations of seven dominant mangrove species in South China. Number of polymorphic loci per population ranged 0.3 to 2.1. The number of haplotypes per population ranged from 1.2 to 3.2. Gene diversity per population ranged from 0.050 to 0.263. Three, five, 11, five, ten, four and four chloroplast haplotypes were identified from all samples of A. ilicifolius, A. corniculatum, A. marina, B. gymnorrhiza, K. candel, L. racemosa, and R. stylosa, respectively. Among these chloroplast haplotypes, two, one, three, one, one, two, one haplotypes were dominantly distributed in most of the populations of A. ilicifolius, A. corniculatum, A. marina, B. gymnorrhiza, K. candel, L. racemosa, and R. stylosa, respectively. The cpSSR analysis indicated that pioneer haplotypes of seven species were created by the long-distance dispersal of seedlings/seeds originating from few mother haplotypes.

The overall GST values assessed by cpSSR DNA ranged from 0.173 to 0.923, indicating high genetic differentiation between populations of these seven mangrove species in South China. The low migration number (Nm) values ranged from 0.042 to 2.391 in seven species. These results indicated limited propagate dispersal between populations may occur between studied populations of seven species.

Significant correlation between geographical distance and genetic distance (GST) was observed in A. marina and K. candel (P < 0.01; Mantel test), no significant relationship was found in other five species (P = 0.11 to 0.47; Mantel test). 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 A. marina and K. candel, but not in the other five species.

4.Mating system, pollen and propagule dispersal, and spatial genetic structure in a high-density population of the mangrove tree Kandelia candel

We surveyed the mating system, pollen and propagule dispersal, and spatial genetic structure in a 0.55-ha high density natural population of K. candel in South China using nine and three polymorphic nuclear and cpSSR markers, respectively. In the study stand, all adult trees (n=2062) of K. candel were mapped and genotyped. 177 seedlings were arbitrarily selected from four plots to analyze the parentage of seedlings. Total 378 propagules were also arbitrarily chosen from 11 mother trees within the stand for paternity analysis.

High genetic diversity (HE = 0.738) were estimated by nuclear SSR markers in adults. Combining three cpSSR loci, 12 different chloroplast haplotypes were detected in the adults and six different chloroplast haplotypes were detected in the seedlings, of which one was specific to seedlings.

The mean value of Multilocus outcrossing rates (tm) was 0.905, which was significantly tm less than 1, suggesting that selfing occurred in this stand. The mean value of biparental inbreeding rates (tm- ts) was 0.058, which departed significantly from 0, indicating amount of biparental inbreeding. The estimated correlation of paternity (rp) was -0.021, which indicated that there must be a large number of individuals contributing to a well-mixed pollen pool in our study stand of K. candel.

For paternity analysis, the distance of pollen movement ranged from 0 to 74.0 m, with an average of 15.3 ± 15.0 m (SD), with 97.3% of the assigned pollen traveled less than 50 m. Modeling for pollen dispersal by the two-component normal model, a short-distance (σ1 = 4.5) and long-distance dispersal (σ2 = 35.1) occupied a distribution frequency of 23.7% and 76.3%, respectively. The average pollen dispersal distance was 34.8 m. The distance of propagule dispersal ranged from 0.03 to 59.6 m, with a mean of 9.4 ± 13.8 m (SD), with 82.1% of the assigned propagules traveled less than 20 m. Calculation by the two-component normal model revealed that 39.4% and the remaining 60.6% of the total pollen dispersal were of the short-distance type with σ1 = 1.1 and long-distance type with σ2 = 24.0, respectively. The average propagule dispersal distance was 18.8 m. Paternity and parentage analysis and modeling estimations revealed the presence of an extremely short-distance component of pollen and propagule dispersal.

Adults of K. candel in the study stand showed significant spatial genetic structure at distances up to 32 m. A clumped distribution of cpSSR haplotypes was seen in K. candel adults. These results indicate that the K. candel population was initiated by limited propagule founders from outside by long-distance dispersal followed by limited propagule dispersal from the founders, resulting in a half-sib family structure.

Conclusion

From the analysis of both nuclear and chloroplast microsatellite markers, it can be concluded that genetic diversity of seven dominant mangrove species in South China is very low except K. candel and the populations of these seven species may have been established mainly from few founders. Historical factors, such as repeated bottleneck effects, may also have played a role. The high level of genetic differentiation was observed in present populations of seven species, indicating the limited pollen and propagule dispersal distance. After initial establishment, there might be limited distance of propagule disperse from the founders. The study of pollen and propagule dispersals of K. candel approved the above hypothesis.

Moreover, the patterns of genetic diversity and population genetic structures of seven species are unique to each others. The differences reflect migratory potentials, extinction rates, adaptation, population sizes and other factors of respective species. Additionally, the present studied seven mangrove species have different habitats in the mangrove forests in South China.

This study addresses the urgent need for conservation and proper management of mangroves in China. Comparative studies of selected mangrove species were conducted in order to fill some of the large gaps in our knowledge of mangrove genetic resources, with emphases on their conservation genetics and reproductive biology.

マングローブ林は、熱帯・亜熱帯の河口や海岸沿い汽水域に形成される湿地生態系で、高い生産性を持つ。沿岸海洋環境の保護機能を持つマングローブ林は、薪炭林や観光、漁業資源としても重要な役割を果たしている。しかし、近年、大規模な養殖地の開発、農地や塩田への転用が進んでおり、マングローブ林の面積は世界規模で急激に減少している。これに伴い、マングローブ林における遺伝的多様性もまた急速に失われているものと考えられている。

世界で約80種あるマングローブ植物のうち、中国には26種が知られており、主に海南省、広東省、広西省、福建省の沿岸域に分布している。世界のマングローブ林と同様に、中国のマングローブ林もこの40年間で3分の2の面積が失われ、危機的状況にある。現存する17,885haのマングローブ林の80パーセントは二次林で、いずれも断片化が進んでいる。こうしたマングローブ林を保全・維持するためには現存する林分の遺伝的多様性やそれぞれのマングローブ植物の繁殖様式を調べる必要がある。本研究では中国南沿岸域に分布する、代表的なマングローブ7種(Acanthus ilicifolius、ツノヤブコウジ、ヒルギダマシ、オヒルギ、メヒルギ、ヒルギモドキ、ヤエヤマヒルギ)を対象に、核マイクロサテライト(SSR)マーカーと葉緑体SSR(cpSSR)マーカーを用いて、中国の分布域全体の集団遺伝構造や遺伝的多様性を調べた。さらに、マングローブの詳細な繁殖特性を解明するため、メヒルギ集団内の空間遺伝構造、交配様式、花粉・胎生種子の散布様式を調べた。

序論では世界のマングローブの分布、形態的・生理的特性、保全の状況および集団遺伝学的研究の状況などについて詳述した。

詳細な集団遺伝解析などには、多型性が高く共優性でしかも再現性が高いSSRマーカーの利用が不可欠であるが、今回研究対象とした7種の中にはSSRマーカーが開発されていない樹種も含まれる。そこで、第一章では、Acanthus ilicifolius、ツノヤブコウジ、ヒルギモドキとヒルギダマシの計4種から、47の核SSRマーカーと17のcpSSRマーカーを新たに作成し、その特性を記載した。

第二章では、第一章で作製したSSRマーカーと既存のSSRマーカーを用い、中国のマングローブ生息域のほぼ全域において、マングローブ7種の集団遺伝構造を調べた。解析集団数はAcanthus ilicifolius 6、ツノヤブコウジ 14、ヒルギダマシ 10、オヒルギ 9、メヒルギ 10、ヒルギモドキ 6、ヤエヤマヒルギ 15である。cpSSRで識別可能な葉緑体ハプロタイプは種毎に3-11タイプあり、メヒルギ(10)とヒルギダマシ(11)以外は5ハプロタイプ以下しか認められなかった。また、いずれの種でも限られたハプロタイプが各集団で優占していたことから、各集団は限られた祖先によって成立した可能性が考えられる。核SSRマーカーでの解析の結果、7種のマングローブはいずれも集団内の遺伝的多様性が低いことがわかった。集団内の近交係数はすべての種で有意に高かったことから、近親交配が集団内の多様性低下を加速している可能性が考えられる。いずれの種でも集団間の遺伝的分化は有意であり、花粉や種子の散布を通しての集団間の遺伝的交流が極めて少ないことが示唆された。これまで、マングローブにおける集団遺伝研究は一種のみを扱ったものばかりであったが、本研究は同所的に生育するマングローブ複数種を同時に解析し、共通する遺伝構造を初めて明らかにした。

第三章では、マングローブの集団内の繁殖特性を詳細に明らかにするため、代表的なメヒルギ集団を対象にして、空間遺伝構造、交配様式、花粉・胎生種子の散布様式を調べた。0.55haの調査区を設定し、その中に分布する全ての成木(2062本)と調査区内に設定された4つの実生プロット内の実生(177本)から葉サンプルを採取した。また、花粉散布パターンを調べるため、調査区内の11母樹から378個の胎生種子サンプルも採取した。各サンプルについて、核SSRとcpSSRマーカーを用いて遺伝子型を決定し、空間遺伝構造と花粉・胎生種子の散布パターンを明らかにした。その結果、調査区内の成木集団では12葉緑体ハプロタイプが同定されたものの、上位5タイプによって成木個体の98.7%が占められていた。成木の分布には、cpSSRと核SSRマーカーのいずれの解析においても空間遺伝構造が存在することがわかった。花粉と胎生種子の散布距離が非常に短かかったことが(花粉平均15.2m、胎生種子平均9.4m)、集団内の空間遺伝構造の要因と考えられる。以上の結果から、この集団は限られた祖先が長距離散布によって定着し、その後集団内で成長した母樹が近距離の種子散布を繰り返すことで成立したものと推測される。

総合考察では、二章と三章で得られたマングローブの集団遺伝的構造と繁殖特性をまとめ、中国の南沿岸域に生育するマングローブ林の保全に関する考察を述べている。

以上のように本研究は、中国南沿岸域のマングローブで優占する7種について、それらの個体群間の地理的遺伝分化や遺伝的多様性などを分子生態学的解析法により明らかにした。これまで、同所的に生育する複数種を同時に解析し、個々の種の遺伝的構造だけでなく群集全体としての遺伝的多様性を把握する分子生態学の解析は皆無である。また、マングローブ林において親子解析から花粉と種子の散布パターンを明らかにした例もこれまで無く、本研究の先駆的意義は大きい。さらに、本研究によって得られたマングローブの遺伝的多様性と繁殖特性に関する基礎的情報は、中国南沿岸域のマングローブ林保全を考える上で有益な情報となるであろう。従って、本研究は、学術と応用上の貢献が極めて大きく、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。