Abstracts

Molecular dissection of translation elongation factor 2, a key molecule to resolve the relationship between red algae and green plants.

Global relationship among major eukaryotic groups remains to be one of the biggest contentious issues in biology. Particularly, in order to elucidate the origin and evolution of photosynthetic eukaryotes, the phylogenetic relationship among glaucophytes, red algae, and green plants-organisms containing the "primary" plastids-is extremely important. Although the molecular data analyses of elongation factor 2 (EF-2) strongly unite red algae and green plants, Stiller, Riley and Hall (2006 J Mol Evol 52:527-39) claimed that (i) red algae and green plants are not direct sisters, and (ii) the robust clade of red algae plus green plant ("R+G clade") recovered in EF-2 phylogenies is the result of gene recombination (GR). In this study, we precisely investigated the phylogenetic signal in EF-2 sequences to examine whether the signal of potential recombination between the red algal and green plant genes could be recovered by using a maximum-likelihood method. In our investigation, the strong signal for the R+G clade was detected in the outside of the putative GR region proposed by Stiller, Riley and Hall (2006). Thus, there is no strong rationale to hypothesize the GR between red algae and green plants in EF-2 phylogeny. In addition, we generated a large multi-gene data set with ?10,000 alignment positions to assess whether non-EF-2 genes supported the monophyly of red algae and green plants. Our multi-gene analyses clearly indicated that the data without the EF-2 gene supported the R+G clade. Considering these results, we conclude that (i) the monophyly of red algae and green plants is genuine, and (ii) the EF-2 genes in the two eukaryotic lineages most probably experienced no recombination as proposed by Stiller, Riley and Hall (2006).

真核生物メタジェノミクスは可能か？

これまでの研究により、地球上に生息する真核生物グループのほとんどが同定されていると考えられない。したがって、真核生物進化の全容解明のためには新奇真核生物グループを発見・解析する必要があることは当然である。しかし、現時点での培養技術には限界があることは周知の事実であり、研究対象として興味深い細胞があっても、実験室内で培養できず分子生物学的アプローチが不可能となっている場合が多い。原核生物研究では、メタジェノミクスが大きな進展を見せており、培養せずとも完全ゲノム配列が決定できる時代になっている。このようなメタジェノミクスアプローチが難培養性真核生物種の研究に応用可能となれば、真核生物の進化研究に大きなインパクトを与えることは必至である。本発表では、真核生物を対象としたメタジェノミクスが可能なのか、もし可能ならばどのようなアウトプットが期待されるか議論したい。参考ウェブはこちら。

Eukaryotic phylogeny inferred from a 24-gene data set: Impact of the aberrant phylogenetic "signal" from tubulins.

Global relationship among major eukaryotic groups remains to be one of the biggest contentious issues in biology. Particularly, in order to elucidate the origin and evolution of photosynthetic eukaryotes, the phylogenetic relationship among glaucophytes, red algae, and green plants-organisms containing the "primary" plastids-is extremely important. Although the molecular data analyses of elongation factor 2 (EF-2) strongly unite red algae and green plants, Stiller, Riley and Hall (2006 J Mol Evol 52:527-39) claimed that (i) red algae and green plants are not direct sisters, and (ii) the robust clade of red algae plus green plant ("R+G clade") recovered in EF-2 phylogenies is the result of gene recombination (GR). In this study, we precisely investigated the phylogenetic signal in EF-2 sequences to examine whether the signal of potential recombination between the red algal and green plant genes could be recovered by using a maximum-likelihood method. In our investigation, the strong signal for the R+G clade was detected in the outside of the putative GR region proposed by Stiller, Riley and Hall (2006). Thus, there is no strong rationale to hypothesize the GR between red algae and green plants in EF-2 phylogeny. In addition, we generated a large multi-gene data set with ?10,000 alignment positions to assess whether non-EF-2 genes supported the monophyly of red algae and green plants. Our multi-gene analyses clearly indicated that the data without the EF-2 gene supported the R+G clade. Considering these results, we conclude that (i) the monophyly of red algae and green plants is genuine, and (ii) the EF-2 genes in the two eukaryotic lineages most probably experienced no recombination as proposed by Stiller, Riley and Hall (2006).

Fragmentation of mitochondrial large subunit rRNA in dinoflagellates.

Extensive fragmentations of apicomplexa mitochondria revealed that ribosomal RNAs (rRNAs) are fragmented into multiple short pieces, and oligo-A are attached to the 3' termini of these rRNA fragments. In this study, we report the "Plasmodium-like" architecture of mitochondrial (mt) rRNA in a dinoflagellate Alexandrium catenella. A piece of large subunit of rRNA (LSU rRNA), which is most probably homologous to P. falciparum LSU rRNA E fragment, was identified in a 1.7 kbp DNA clone. We further confirmed that (i) A. catenella "E-like" fragment could form appropriate secondary structures, and (ii) is transcriptionally active. In addition, the expressions of two extra rRNA fragments, which correspond to P. falciparum F and G fragments, were confirmed. Our data clearly suggest that these rRNA fragments are separately encoded in A. catenella mt genome. Since apicomplexa and dinoflagellates are the closest relatives in eukaryotic (organismal) phylogeny, we propose here that the extensive mt rRNA fragmentation was established prior to the divergent of the two groups.

Phylogenetic position of the centroheliozoan Raphidiophrys contractilis based on six genes.

The Centrohelida are one of the majore groups of Heliozoa, and they had been classified into Sarcodina. Recent phylogenetic studies have shown that the Sarcodina split into eukarotic super-groups such as Amoebozoa, Rhizaria, and Opithokonta. The heliozoan groups are also divided into different eukaryotic major groups, i.e., Actionphrydia fall within Stramenopiles, and Desmothoracida and Taxopodida belong to Rhizaria. The Centrohelida, however, are not closely related to other heliozoan groups, or to any eukaryotic super-groups. To clarify the phylogenetic position of the Centrohelida in the global eukaryotic phylogeny by concatenated molecular data analyses, we sequenced elongation factor 2 (EF2), heat shock protein 70 (HSP70) and HSP90 genes from a centroheliozoan Raphidiophrys contractilis. Maximum likelihood (ML) analyses of a six-protein data set (actin, α-, β-tubulin, EF2, HSP70 and HSP90) showed the Centrohelida branched after Amoebozoa, and were placed at the basal position within bikonts. However, the placement of the Centrohelida received weak bootstrap proportion supports, indicating that the current data size i insufficient to resolve the phylogenetic position of the Centrohelida. We further discuss the results of concatenated ML analyses considering six proteins with/without a SSU rRNA data set.

Ultrastructure and phylogeny of a new excavate, Dysnectes brevis gen. et sp. nov.

The excavates comprise ten groups of protists: jakobids, Malawimonas, Heterolobosea, Euglenozoa, Trimastix, oxymonads, Carpediemonas, retortamonads, diplomonads and parabasalids. Both morphological and molecular data suggest that the excavates are key organisms for better understanding of early evolution of eukaryotes. Unfortunately, many of the excavates studied so far are parasitic members, and their ultrastructure and genes are likely biased by parasitic life form. It is necessary to hunt for free-living excavates, which would be free from parasitic bias. However, only a few such excabates have been acquired. We isolated a previously undescribed flagellate, Dysnectes brevis gen. et sp. nov. with micro-pipettes from an enrichment culture of sediment taken from seafloor of Kagoshima bay, and established uniprotistan cultures. This organism possesses a conspicuous feeding groove on the ventral side of the cell, suggesting that this flagellate is a member of excavates. Our detailed observations using the electron microscope revealed that this flagellate shares all the common ultrastructural features of the flagellar apparatus found in the typical excavates. In particular, D. brevis is very similar to the members of Fornicata (Carpediemonas, retortamonas and diplomonads) in prossessing an arched B fiber, a fibrous component that covers the right root, posterior basal body adn left root. Phylogenetic analyses using 18S rRNA sqeuences also showed that D. brevis is closely realted to the Fornicata and branched prior to the divergence of the Fornicata, though precise phylogenetic position within the Fornicata is uncertain.

紅藻と緑色植物は真の姉妹群か？： 24遺伝子配列連結データによる解析

真核生物系統中において，紅藻類と緑色植物の関係はいまだに確定されていない。翻訳伸長因子2（EF-2）配列の系統解析は紅藻類と緑色植物の近縁性が強く支持される（「紅藻類＋緑色植物」説）。ところが，EF1α ＋tubulin＋actin配列に基づく複数遺伝子データ解析は紅藻類と緑色植物の近縁性を支持せず，オピストコンタ（多細胞動物＋菌類）・アメーバ類との近縁性を示唆している。我々は，24遺伝子・10,000アミノ酸座位以上を含む巨大アライメントデータを作成し，最尤法により紅藻類の進化的位置を探索した。紅藻類と緑色植物をふくむ7つの真核生物大グループ間で生成可能な全945樹形の尤度を網羅的に計算した結果，「紅藻類＋緑色植物」説が強く支持された。各遺伝子の進化情報を詳細に解析したところ，「紅藻類＋緑色植物」説を極めて強く支持するEF-2に加え，αチューブリン（Tba），βチューブリン（Tbb）の進化シグナルが他の遺伝子にくらべ大きく離れていることが判明した。そこで3つの遺伝子を排除し解析を行ったが，再度「紅藻類＋緑色植物」説を支持する結果となった。また，Tba・Tbb配列はオピストコンタ類とディプロモナス類・パラバサリア類を強く結びつけるアーティファクトを引き起こすことが判明した。本解析の結果は，一貫して紅藻類と緑色植物との単系統性を示唆するものである。今後，真核生物大系統を精度良く探索するには，(i)より大きな連結データを作成すること，(ii)｢ハズレ者｣遺伝子をシステマティックに検出・排除することが必須であると考えられる。

微細構造と分子系統解析に基づく新規エクスカベート類Dysnectes brevisの研究

エクスカベート類は細胞腹部に大きな溝を有する生物群であり、Giardia等の寄生性原生生物、jakoba類などの自由生活性・従属栄養性原生生物、Euglenaなどの独立栄養性原生生物を含む真核生物における大きなグループである。貧酸素環境に適応した種が多く、ミトコンドリアを持たないグループも知られている。エクスカベート類は多様な生活様式を示すが、微細構造形質では非常に共通性が高い。しかし分子形質からはその単系統性は示されていない。

2005年3月鹿児島湾より細胞腹部に大きな溝を有する自由生活性・従属栄養性原生生物を単離し、培養株とした。この原生生物は微好気条件下で生育し、バクテリアを捕食し栄養摂取を行う。電子顕微鏡観察の結果、この原生生物はミトコンドリアを欠きハイドロジェノソーム様の細胞小器官をもつこと、また典型的なエクスカベートと共通する複雑な鞭毛装置構造をもつことが分かった。微小管に付随する繊維構造の特徴はシアルデイアや自由生活性のCarpediemonasを含むエクスカベート類Fornicataの特徴と一致した。18S rDNAによる分子系統解析の結果、本原生生物はFornicataに含まれることが示されたが、Fornicata内での系統的位置は明らかにできなかった。以上の結果から本原生生物をFornicataの新属・新種Dysnectes brevisとして記載予定である今回の結果を含め、エクスカベート類とそのほかの原生生物との形態形質比較を行ったところ、様々なグループとの共通点が見られた。このような事実はエクスカベート類における微細構造的な共通性が真核生物の原始的な形態を残している結果であることを示唆している。