In this thesis, to estimate geographic distribution pattern and discuss dispersal feature of intertidal sessile sponges, reproductive period, genetic structure and gene flow were investigated using the three commonest intertidal halichondrid sponges, Hymeniacidon sinapium (de Laubenfels, 1930), Hymeniacidon flavia Sim & Le, 2003, and Halichondria okadai (Kadoda, 1920) in Japan and Korea.

Sponge (Phylum Porifera) is primitive, evolutionary important and the group with highly diverse, though basic biological information of such as taxonomy, distribution ranges of each species, abundance, reproduction, and dispersal has not been sufficiently accumulated. Up until several years ago, sponges had been thought to be able to disperse widely across oceans during pelagic larval or gammule phase and many sponges, even in freshwater species, had been described as cosmopolitan species or with wide distribution. However, recently, genetic analyses have demonstrated cryptic divergence and genetic separation within species, species group and also more restricted region. From general estimation of short swimming larval period and low swimming ability of larvae, short dispersal distance and more restricted distribution has been expected. These indicate that wide distribution ranges were lead from lumping or miss-identification because of paucity of morphological character and difficulty of identification.

Three studied species inhabit sympatrically in upper intertidal zone, though distribution ranges differ remarkably. Hymeniacidon flavia and Hall. okadai distribute only western North Pacific coast, while Hym. sinapium distributes both eastern and western North Pacific coast. It was also suggested that Hym. sinapium and Hym. heliophila from Atlantic were genetically identical. These two species also share similar morphology. Since difference in distribution range might be caused by difference in dispersal ability, reproduction periods and genetic structures of three species were compared.

The larval morphology and reproductive period of Hym. sinapium and Hym. flavia were investigated by tissue observation and periodical sampling respectively. The reproductive period of Hym. sinapium and Hym. flavia were during wanner season of the year. Halichondria okadai was also known to reproduce during spring and summer. All three species had parenchymella larvae, which were entirely ciliated. The larvae of Hym. sinapium and Hali. okadai were elongated shape and that of Hym. flavia shaped more oval than the other two species. The larvae of two Hymeniacidon species have "style" type spicules that arrangements were different in each species. Because the reproductive periods are almost same, influences of ocean currents over larvae that are released to a water column might be similar. Concentration and arrangement of spicule in tiny larvae might have some impact on larval dispersal.

Geographic genetic structures of three species were investigated and several significant genetic structures were observed. The three species were analyzed using sequences of internal transcribed spacers (ITS 1 and 2) of nuclear rDNA and NADH dehydrogenase subunit 5 (nad5) gene of mitochondrial DNA. Several significant genetic structures were detected in Hym. flavia and Hall. okadai. In the both species, influences by historical geographical events after the last glacial phase and present sea current were supposed from the distribution patterns. While in Hym. sinapium, only a few genetic variations were detected in ITS sequence, and furthermore two Atlantic species, Hym. heliophila and Hym. perlevis had very similar sequence. It is highly likely that Hym. sinapium has high gene flow between distant localities and this species distribute wider range than expected. The results of the analysis using nad5 of two Hymeniacidon species agreed with the results from ITS analyses. Distribution patterns of nad5 haplotypes supported high dispersal ability in Hym. sinapium and stable population structure in Hym. flavia.

For Hym. sinapium, 8 microsatellite markers were developed and farther detailed genetic structure was examined using seven of eight loci. Significant genetic differentiations between collection localities were detected by population genetic analyses. From a result of mantel test indicated that level of genetic differentiation had positive relation with geographic distance between localities. However, high differentiation level was also observed even between adjacent localities and the level changed slightly with distance. The result of population assignment analysis using structure program suggested that most of collection localities constitute each reproductive population. Significant genetic differentiations between collection localities suggested that natural dispersal was more likely than human mediated dispersal around Japan and Korea. From these results, we concluded that most individuals disperse short distance like general expectation for sponges, however, there are individuals that could disperse to distant localities. Further analysis including related Atlantic and European Hymeniacidon species will be required to estimate the distribution origin and detailed historical demography.

In all three species, regional genetic differentiations were detected, suggesting individuals in restricted region constitute each reproductive population in the region. Influences by historical geographical events after the last glacial phase and present sea current were suggested by geographic genetic structures of these sponges. Higher dispersal ability of Hymeniacidon sinapium than the other species was also supported by the results. However, significant genetic differentiations were shown in "Hym. sinapium, natural dispersal was more likely than human mediated dispersal. Further analysis including Atlantic and European Hymeniacidon species will be required to estimate the distribution origin and detailed historical demography.

本論文は,第一章のイントロダクション,第二章の繁殖と幼生,第三章の地理的な遺伝構造,第四章のディスカッションの四部から構成されている.

第一章のイントロダクションでは,材料としている海綿動物の生物学的特性とこれまでの研究の現状,および海洋生物の個体群の地理的な遺伝構造の特徴とそれに関する研究の歴史を的確にまとめており,本研究で目指すところについて述べられている.

海綿動物は,系統的には後生動物の起源に位置し動物界の進化を研究する上で重要な位置を占める分類群であるとともに,近年は二次代謝物に関する数多くの研究がなされ応用面でも大きな注目を浴びている.しかし,海綿動物の分類や生態といった基礎的な生物学的特性については未だ不明なことが多く,これらを扱った本研究は,海綿動物の応用に向けての基本情報を提供するという点においても大きく貢献するものと言える.生物はそれぞれの分布範囲を広げるために様々な方法で分散を行うが,固着性の底生生物である海綿動物は,生活史の一部である浮遊幼生期に分散を行うと考えられる.海洋生物の地理的分布やその成り立ちを理解する上で,浮遊幼生による分散の実態を明らかにすることは非常に重要であるが,本研究では遺伝的な方法を用いることにより分散の程度を推測した.海綿動物では,これまで遺伝的な解析が適用された種は非常に少なく,本研究は遺伝子マーカーの開発などの技術的な困難を乗り越えて新たな研究に取り組んでおり,高い評価に値する.研究対象として選んだイソカイメン類3種,Hymeniacidon sinapium,Hy.flavia,Halichondria okadaiは,日本国内の岩礁潮間帯において最も普通に見られる種であるにも関わらず,それらの生物学的特性はこれまでほとんどわかっていないままであった.また,このように系統的にも近縁で,かつ同所的に生息し同じ生態的地位を占めている3種を用いて比較することで,地理的な遺伝構造の差を浮き彫りにした議論を可能にしたことは本研究のオリジナリティーの一つと言えるであろう.

第二章では定期的に採集を行った季節標本に基づき,繁殖時期と幼生形態の比較を行った.その結果,3種の繁殖時期はほぼ同じで,初夏~初秋の水温が高い時期となっていた.繁殖時期がほぼ一緒であることから,ほぼ同じ海洋環境の時に幼生が放出されることが推定された.幼生の形態を比較したところ,幼生の外形や,幼生骨片の体内での分布が異なることが示され,このような幼生の形態の違いによって,分散の程度に差が生じる可能性が示唆された.繁殖や幼生は各種の分散や分布を決定する上で非常に重要な特徴であるにもかかわらず,これまで海綿類ではこれらの比較研究はほとんど行われておらず,本研究は繁殖や幼生を考慮しての種の分散や分布の研究の先駆けとなるものである.

第三章では,日本各地および韓国から採集された標本を用いて,核のITS領域(約600~700bp),ミトコンドリアのnad5領域(約1000bp)のDNA塩基配列を分析し3種の遺伝的解析を行ったところ,種によって大きく結果が異なることが明らかとなった.特にITSの分析結果から,Hy.flavia,Ha.okadaiの2種では遺伝的変異が大きく,分散による遺伝子流動は非常に限られていることが示された.これら2種の分布が西太平洋に限られているのに対して,遺伝的変異が非常に小さかったHy.sinapiumは太平洋に広く分布しており,さらに大西洋産の別種とされてきたHy.heliophila及びHy.perlevisとも種内変異を越えるような塩基配列の差が無いことが示され,大洋に渡って広く遺伝子流動があることを示唆するとともに,これまでのあいまいな形態形質のみに基づく海綿動物の分類に警鐘を鳴らしている.さらに,Hy.sinapium初についてはより詳しく日本周辺での遺伝構造を解析するためにマイクロサテライトマーカーの開発を行った.その解析により,広く分散している本種においても瀬戸内海の2地点といった海洋条件や距離スケールでは同一の繁殖集団を形成している場合があることが示唆された.

第三章の一部および第四章のディスカッションにおいては,以上の研究成果をふまえ,現在のイソカイメン類3種の分布が,過去の地史イベントや海流などの現在の海洋環境との関係でどのようにできあがっていったかについて論じている.本研究で採用した,遺伝だけでなく繁殖,幼生の情報を含めて,実際の分散や分布を研究する方法は,今後の海産底生動物の系統地理学的研究を進める上での指針となっていくものであると考えられる.

なお,本論文第三章の一部は藤田敏彦・斉藤大地との共同研究であるが,論文提出者が主体となって分析及び検証を行ったもので,論文提出者の寄与が十分であると判断する.

以上のことから,博士(理学)の学位を授与できると認める.