論文の内容の要旨

水圏生物科学専攻
平成２２年度博士課程 入学
氏名 アタチャイ カンタチュンポー
指導教員名 小松 輝久

論文題目 Studies on phylogeography of Sargassum polycystum C. Agardh in waters

of South East Asia and Japan
（東南アジアおよび日本の海域におけるコバモクの系統地理学に関する研究）

The genus Sargassum is the most widely distributed brown seaweed, which occurs in temperate

and tropical regions, especially in Indo-west Pacific region and Australia. This genus plays an important

role in marine ecological system that forms a dense submarine forest as an essential habitat for numerous

marine organisms and biosorption for improving environmental conditions. More than 400 species of the

genus Sargasum have been described based on morphological and anatomical structures; however, this

genus shows high levels of morphological variations that may lead to misidentification problem and

misunderstanding concept of taxonomic scenarios. In addition, this genus has been used as a model to

determine whether the distribution and connectivity of marine population that have been affected by

historical events, environmental condition or ocean circulation.

Molecular genetics have been applied as a useful tool for addressing some biological questions

regarding to taxonomy and distribution pattern of seaweed. With respect to the genus Sargassum,

molecular techniques combined with morphological data have been extensively applied to verify the

phylogenetic relationship and species boundaries, and also elucidate the perspicuous historical pattern of

population movement. Recently, most studies of phylogeny and phylogeography of this genus have been

successfully done in temperate region, while very few studies have been carried out in tropical region,
especially in Southeast Asia. As a result, species diversity, population structure and distribution pattern of

Sargassum in Southeast Asian region are yet to be completely explored. .

In order to gain a clear picture of phylogenetic diversity and phylogeographic pattern of the

genus Sargassum in Southeast Asian region, my PhD researches attempt (1) to investigate the

phylogenetic relationship of common species of Sargassum from Thailand based on morphological and

molecular data and,(2) to elucidate phylogeographic pattern of Sargassum polycystum C. Agardh, a

widely distributed species in South Asia, in more detail using three different DNA regions: cox1 and cox3

from mitochondrial DNA and ITS2 from nuclear DNA and then verify what factors influence such

patterns.

Taxonomic research of the genus Sargassum in Thailand was investigated mainly on the basis of

gross morphology and anatomical structures, and roughly, twelve species were reported to date. Due to

a high amount of morphological plasticity in this genus, some closely related species are morphologically

indistinguishable that easily lead to misidentification, especially when these species occur in the same

area. This study aimed to determine the taxonomic framework and phylogenetic relationship of some

common species of Sargassum from Thailand using morphological and molecular data. Twenty

specimens recently collected from both in the Gulf of Thailand and Andaman Sea were morphologically

identifiable to nine species: Sargassum baccularia (Mertens) C. Agardh, S. binderi Sorder, S. cinereum J.

Agardh, S. crassifolium J Agardh, S. cristaefolium C. Agardh, S. polycystum C. Agardh, S. oligocystum

Montagne, S. stolonifolium Phang et Yoshida and S. swartzii (Turuner) C. Agardh. Only one species, S.

polycystum, was found in both sides, while S. baccularia, S. binderi, S.cinereum, S. crassifolium, S.

oligocystum and S. swartzii occurred in the Gulf of Thailand and S. cristaefolium and S.stolonifolium

were found in Andaman Sea.

For molecular analysis, internal transcribed spacer 2 (ITS2) was used to investigate the

phylogenetic relationship within these nine species. Interspecific genetic variation in Sargassum species
found in Thailand was relatively low. Three different methods (MP, ML and BI) of phylogenetic analyses

revealed that our specimens formed a monophyletic group of the subgenus Sargassum with six distinct

phylogenetic clades: S. oligocystum/S. baccularia clade, S. cinereum clade, S. crassifolium/S.

crisraefolium clade, S. polycystum clade, S. stolonifolium clade and S. binderi/S. swartzii clade.

In the section level of subgenus Sargassum, morphological data and ITS2 sequences presented

the controversy classification schemes, which found in section Binderianae. Morphologically, the section

Binderianea consisted of four species found in Thailand: S. baccularia, S. binderi, S. oligocystum and S.

swartzii, while phylogenetic analyses showed two well-separated clades within this section: Binderianae I

clade (S. oligocystum and S. baccularia) and Binderianae II clade (S. binderi and S. swartzii). This result

indicated that section Binderianae is possibly divided into two distinct sections; however additional

samples of all members within this section and further studies in morphology and different molecular

markers are required. In the species level, closely related species of S. binderi and S. swartzii yielded

nearly identical sequences of ITS2, indicating possible species complex within these two species.

This study attempted to use a combined morphological and molecular approach for better

understanding of taxonomy of Thai Sargassum, yet the phylogenetic relationship and species boundaries

remained ambiguous. Further studies using multidisciplinary approaches, namely morphometric,

multi-markers phylogeny and coalescent strategies of species delimitation, are needed to gain more

insight into systematics and evolutionary affinities of this genus.

Figure1 Bayesian tree based on ITS2 gene sequences. The bootstrap values shown at each node were

MP/ML/BI (Bayesian analysis). Scale bar = 0.03 substitutions per site.

Climate changes may have affected historical or contemporary geographic distribution,

abundance and genetic structure of marine organisms. Sargassum polycystum C. Agardh is the most

abundant species, which is likely to be an excellent model for studying distribution pattern and population

connectivity. In the study, phylogenetic patterns of S. polycystum were investigated using different

genetic markers of mitochondrial DNA (cox1 and cox3) and nuclear DNA (ITS2). Eleven populations

(141samples) from cox1, thirteen populations (141 samples) from cox3 and ten populations (127 samples)

form ITS2 were successfully carried out. All samples were randomly collected from Japan to Indonesia

(Two localities in Japan, one locality in Cambodia, four to seven localities in Thailand, one locality in

Singapore and one or two localities in Indonesia), emphasizing in the area of the Gulf of Thailand. In

mitochondrial DNA, 10 haplotypes (H1-H10) was found in cox1, and 6 haplotypes (S1-S6) was found in
cox3, while nuclear DNA ITS2 yielded 12 haplotypes (A1-A12). The results showed that H1, S1 and A1

were likely to be an ancestral haplotype as indicated by consisting these haplotype from almost all

populations. Differences in haplotypes diversity and haplotype sharing among population suggested two

distinct geographical areas, representing two main groups of population: northern group (Japan,

Cambodia and the Gulf of Thailand) and southern group (western of Thailand, Singapore and Indonesia)

High haplotype diversity was found in southern part of South East Asia (Bali, Indonesia and

Phuket, Thailand), while low haplotype diversity was encountered in between the Gulf of Thailand and

Japan. This suggested that the southern area of Southeast Asia was likely to be an origin of S. polycystum,

and distribution pattern of this species seemed to expand southward to the northern areas of Southeast

Asia.

Distribution pattern and population connectivity of S. polycystum corresponded to historical

events of the last glacial maximum (LGM). In last glacial period, Southeast Asia region was connected by

land bridge between Java Sea and the Gulf of Thailand as called Sundaland. Since the LGM period, sea

levels in Sundaland have risen and affected coastal habitats, and this has an important impact on

population structure change in S. polycystum as found in population connectivity between southern and

northern population. Results also indicated that the northern population (the Gulf of Thailand to Japan)

has more recently expanded than southern population (Phuket to Bali).

Figure 2 Geographical distribution of haplotypes in Sargassum polycystum based on mtDNA cox1. Size

of the circle is proportional to the sample size of each populations, and each pie-graph shows the

frequency of haplotype in the population

Figure 3 Geographical distribution of haplotypes in Sargassum polycystum based on mtDNA cox3. Size

of the circle is proportional to the sample size of each populations, and each pie-graph shows the

frequency of haplotype in the population.

Figure 4 Geographical distribution of haplotypes in Sargassum polycystum based on nrDNA ITS2. Size

of the circle is proportional to the sample size of each populations, and each pie-graph shows the

frequency of haplotype in the population